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Sample records for i-deficient cmsii mutant

  1. The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion.

    PubMed

    Priault, P; Tcherkez, G; Cornic, G; De Paepe, R; Naik, R; Ghashghaie, J; Streb, P

    2006-01-01

    The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.

  2. Respiratory complex I deficiency induces drought tolerance by impacting leaf stomatal and hydraulic conductances.

    PubMed

    Djebbar, Reda; Rzigui, Touhami; Pétriacq, Pierre; Mauve, Caroline; Priault, Pierrick; Fresneau, Chantal; De Paepe, Marianne; Florez-Sarasa, Igor; Benhassaine-Kesri, Ghouziel; Streb, Peter; Gakière, Bertrand; Cornic, Gabriel; De Paepe, Rosine

    2012-03-01

    To investigate the role of plant mitochondria in drought tolerance, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant, which has low-efficient respiration and photosynthesis, high levels of amino acids and pyridine nucleotides, and increased antioxidant capacity. We show that the delayed decrease in relative water content after water withholding in CMSII, as compared to WT leaves, is due to a lower stomatal conductance. The stomatal index and the abscisic acid (ABA) content were unaffected in well-watered mutant leaves, but the ABA/stomatal conductance relation was altered during drought, indicating that specific factors interact with ABA signalling. Leaf hydraulic conductance was lower in mutant leaves when compared to WT leaves and the role of oxidative aquaporin gating in attaining a maximum stomatal conductance is discussed. In addition, differences in leaf metabolic status between the mutant and the WT might contribute to the low stomatal conductance, as reported for TCA cycle-deficient plants. After withholding watering, TCA cycle derived organic acids declined more in CMSII leaves than in the WT, and ATP content decreased only in the CMSII. Moreover, in contrast to the WT, total free amino acid levels declined whilst soluble protein content increased in CMSII leaves, suggesting an accelerated amino acid remobilisation. We propose that oxidative and metabolic disturbances resulting from remodelled respiration in the absence of Complex I activity could be involved in bringing about the lower stomatal and hydraulic conductances.

  3. Genetics Home Reference: complement factor I deficiency

    MedlinePlus

    ... factor I deficiency can also be associated with autoimmune disorders such as rheumatoid arthritis or systemic lupus erythematosus (SLE). Autoimmune disorders occur when the immune system malfunctions and attacks ...

  4. A novel mitochondrial MTND5 frameshift mutation causing isolated complex I deficiency, renal failure and myopathy.

    PubMed

    Alston, Charlotte L; Morak, Monika; Reid, Christopher; Hargreaves, Iain P; Pope, Simon A S; Land, John M; Heales, Simon J; Horvath, Rita; Mundy, Helen; Taylor, Robert W

    2010-02-01

    Isolated complex I deficiency is the most commonly reported enzyme defect in paediatric mitochondrial disorders, and may arise due to mutations in nuclear-encoded structural or assembly genes, or the mitochondrial genome. We present the clinical, biochemical and molecular genetic data in a young girl whose clinical picture is dominated by chronic renal failure, myopathy and persistent lactic acidosis. An isolated complex I deficiency in muscle was identified due to a novel mutation (m.12425delA) in the MTND5 gene. This single nucleotide deletion is heteroplasmic and detectable in several tissues from the proband but not her mother, suggesting a de novo mutation event. The description of the first frameshift mutation in a mitochondrial complex I gene affirms mitochondrial DNA mutations as an important cause of isolated complex I deficiency in children and the importance of whole mitochondrial genome sequencing in the diagnostic work-up to elucidate the underlying molecular genetic abnormality and provide important genetic advice.

  5. Characterization of high density lipoprotein particles in familial apolipoprotein A-I deficiency

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our aim was to characterize HDL subspecies and fat-soluble vitamin levels in a kindred with familial apolipoprotein A-I (apoA-I) deficiency. Sequencing of the APOA1 gene revealed a nonsense mutation at codon 22, Q[22]X, with two documented homozygotes, eight heterozygotes, and two normal subjects in...

  6. Allospecific rejection of MHC class I-deficient bone marrow by CD8 T cells

    PubMed Central

    Haspot, Fabienne; Li, Hao Wei; Lucas, Carrie L.; Fehr, Thomas; Beyaz, Semir; Sykes, Megan

    2014-01-01

    Avoidance of long-term immunosuppression is a desired goal in organ transplantation. Mixed chimerism offers a promising approach to tolerance induction, and we have aimed to develop low-toxicity, non-immunodepleting approaches to achieve this outcome. In a mouse model achieving fully MHC-mismatched allogeneic bone marrow engraftment with minimal conditioning (3 Gy total body irradiation followed by anti-CD154 and T cell-depleted allogeneic bone marrow cells), CD4 T cells in the recipient are required to promote tolerance of pre-existing alloreactive recipient CD8 T cells and thereby permit chimerism induction. We now demonstrate that mice devoid of CD4 T cells and NK cells reject MHC class-I deficient and class I/class II-deficient marrow in a CD8 T cell-dependent manner. This rejection is specific for donor alloantigens, since recipient hematopoiesis is not affected by donor marrow rejection and MHC class-I deficient bone marrow that is syngeneic to the recipient is not rejected. Recipient CD8 T cells are activated and develop cytotoxicity against MHC class I-deficient donor cells in association with rejection. These data implicate a novel CD8 T cell-dependent bone marrow rejection pathway, wherein recipient CD8 T cells indirectly activated by donor alloantigens promote direct killing, in a TCR-independent manner, of class I-deficient donor cells. PMID:24304495

  7. Human conditions of insulin-like growth factor-I (IGF-I) deficiency

    PubMed Central

    2012-01-01

    Insulin-like growth factor I (IGF-I) is a polypeptide hormone produced mainly by the liver in response to the endocrine GH stimulus, but it is also secreted by multiple tissues for autocrine/paracrine purposes. IGF-I is partly responsible for systemic GH activities although it possesses a wide number of own properties (anabolic, antioxidant, anti-inflammatory and cytoprotective actions). IGF-I is a closely regulated hormone. Consequently, its logical therapeutical applications seems to be limited to restore physiological circulating levels in order to recover the clinical consequences of IGF-I deficiency, conditions where, despite continuous discrepancies, IGF-I treatment has never been related to oncogenesis. Currently the best characterized conditions of IGF-I deficiency are Laron Syndrome, in children; liver cirrhosis, in adults; aging including age-related-cardiovascular and neurological diseases; and more recently, intrauterine growth restriction. The aim of this review is to summarize the increasing list of roles of IGF-I, both in physiological and pathological conditions, underlying that its potential therapeutical options seem to be limited to those proven states of local or systemic IGF-I deficiency as a replacement treatment, rather than increasing its level upper the normal range. PMID:23148873

  8. Biallelic Mutations in TMEM126B Cause Severe Complex I Deficiency with a Variable Clinical Phenotype.

    PubMed

    Alston, Charlotte L; Compton, Alison G; Formosa, Luke E; Strecker, Valentina; Oláhová, Monika; Haack, Tobias B; Smet, Joél; Stouffs, Katrien; Diakumis, Peter; Ciara, Elżbieta; Cassiman, David; Romain, Nadine; Yarham, John W; He, Langping; De Paepe, Boel; Vanlander, Arnaud V; Seneca, Sara; Feichtinger, René G; Płoski, Rafal; Rokicki, Dariusz; Pronicka, Ewa; Haller, Ronald G; Van Hove, Johan L K; Bahlo, Melanie; Mayr, Johannes A; Van Coster, Rudy; Prokisch, Holger; Wittig, Ilka; Ryan, Michael T; Thorburn, David R; Taylor, Robert W

    2016-07-07

    Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs(∗)2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.

  9. Mesencephalic complex I deficiency does not correlate with parkinsonism in mitochondrial DNA maintenance disorders.

    PubMed

    Palin, Eino J H; Paetau, Anders; Suomalainen, Anu

    2013-08-01

    Genetic evidence from recessively inherited Parkinson's disease has indicated a clear causative role for mitochondrial dysfunction in Parkinson's disease. This role has long been discussed based on findings that toxic inhibition of mitochondrial respiratory complex I caused parkinsonism and that tissues of patients with Parkinson's disease show complex I deficiency. Disorders of mitochondrial DNA maintenance are a common cause of inherited neurodegenerative disorders, and lead to mitochondrial DNA deletions or depletion and respiratory chain defect, including complex I deficiency. However, parkinsonism associates typically with defects of catalytic domain of mitochondrial DNA polymerase gamma. Surprisingly, however, not all mutations affecting DNA polymerase gamma manifest as parkinsonism, but, for example, spacer region mutations lead to spinocerebellar ataxia and/or severe epilepsy. Furthermore, defective Twinkle helicase, a close functional companion of DNA polymerase gamma in mitochondrial DNA replication, results in infantile-onset spinocerebellar ataxia, epilepsy or adult-onset mitochondrial myopathy, but not typically parkinsonism. Here we sought for clues for this specificity in the neurological manifestations of mitochondrial DNA maintenance disorders by studying mesencephalic neuropathology of patients with DNA polymerase gamma or Twinkle defects, with or without parkinsonism. We show here that all patients with mitochondrial DNA maintenance disorders had neuronopathy in substantia nigra, most severe in DNA polymerase gamma-associated parkinsonism. The oculomotor nucleus was also affected, but less severely. In substantia nigra, all patients had a considerable decrease of respiratory chain complex I, but other respiratory chain enzymes were not affected. Complex I deficiency did not correlate with parkinsonism, age, affected gene or inheritance. We conclude that the cell number in substantia nigra correlated well with parkinsonism in DNA polymerase gamma

  10. Neurofibromatosis Type 1: A Novel NF1 Mutation Associated with Mitochondrial Complex I Deficiency

    PubMed Central

    Isidoro, Lara; Rocha, Dalila

    2014-01-01

    Background. Neurofibromatosis type 1 is a multisystemic, progressive disease, with an estimated incidence of 1/3500-2500. Mitochondrial diseases are generally multisystemic and may be present at any age, and the global prevalence is 1/8500. The diagnosis of these disorders is complex because of its clinical and genetic heterogeneity. Case Report. We present a rare case of the association of these two different genetic diseases, in which a heterozygous missense mutation in the NF1 gene was identified which had not yet been described (p.M1149 V). Additionally, the patient is suspected of carrying an unspecified mutation causing respiratory chain complex I deficiency. Clinical presentation included hypotonia, global development delay, reduced growth rate, progressive microcephaly, and numerous café-au-lait spots. Discussion. To the best of our knowledge this is the first report of complex I deficiency in a patient with neurofibromatosis type 1. It is very important to maintain a high index of suspicion for the diagnosis of mitochondrial disorders. In this patient, both the laboratory screening and muscle histology were normal and only the biochemical study of muscle allowed us to confirm the diagnosis. PMID:24711935

  11. L-dihydroxyphenylalanine and complex I deficiency in Parkinson's disease brain.

    PubMed

    Cooper, J M; Daniel, S E; Marsden, C D; Schapira, A H

    1995-05-01

    There is evidence for a 37% deficiency of complex I activity in Parkinson's disease (PD), which appears to be specific for PD amongst parkinsonian syndromes and selective for the substantia nigra within the central nervous system. Rat studies have shown that, in the context of a normal nigrostriatal dopaminergic cell population, L-dihydroxyphenylalanine (L-dopa) causes a reversible 25% defect of complex I activity in nigral and striatal tissue. Analysis of striatal tissue from PD patients after prolonged exposure to high-dose L-dopa does not show such a defect. Results of these and other studies suggest that L-dopa therapy does not cause complex I deficiency in PD striatum. However, it cannot be excluded that, in the particular environment of the PD substantia nigra, L-dopa may enhance a preexisting complex I defect.

  12. Investigating complex I deficiency in Purkinje cells and synapses in patients with mitochondrial disease

    PubMed Central

    Chrysostomou, Alexia; Grady, John P.; Laude, Alex; Taylor, Robert W.; Turnbull, Doug M.

    2015-01-01

    Aims Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus. Methods We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age‐matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses. Results Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses. Conclusions This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia. PMID:26337858

  13. The skeletal structure of insulin-like growth factor I-deficient mice

    NASA Technical Reports Server (NTRS)

    Bikle, D.; Majumdar, S.; Laib, A.; Powell-Braxton, L.; Rosen, C.; Beamer, W.; Nauman, E.; Leary, C.; Halloran, B.

    2001-01-01

    The importance of insulin-like growth factor I (IGF-I) for growth is well established. However, the lack of IGF-I on the skeleton has not been examined thoroughly. Therefore, we analyzed the structural properties of bone from mice rendered IGF-I deficient by homologous recombination (knockout [k/o]) using histomorphometry, peripheral quantitative computerized tomography (pQCT), and microcomputerized tomography (muCT). The k/o mice were 24% the size of their wild-type littermates at the time of study (4 months). The k/o tibias were 28% and L1 vertebrae were 26% the size of wild-type bones. Bone formation rates (BFR) of k/o tibias were 27% that of the wild-type littermates. The k/o bones responded normally to growth hormone (GH; 1.7-fold increase) and supranormally to IGF-I (5.2-fold increase) with respect to BFR. Cortical thickness of the proximal tibia was reduced 17% in the k/o mouse. However, trabecular bone volume (bone volume/total volume [BV/TV]) was increased 23% (male mice) and 88% (female mice) in the k/o mice compared with wild-type controls as a result of increased connectivity, increased number, and decreased spacing of the trabeculae. These changes were either less or not found in L1. Thus, lack of IGF-I leads to the development of a bone structure, which, although smaller, appears more compact.

  14. Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

    PubMed Central

    Karamanlidis, Georgios; Lee, Chi Fung; Garcia-Menendez, Lorena; Kolwicz, Stephen C.; Suthammarak, Wichit; Gong, Guohua; Sedensky, Margaret M.; Morgan, Philip G.; Wang, Wang; Tian, Rong

    2013-01-01

    Summary Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases including heart failure but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by inactivation of the Ndufs4 gene, a protein critical for Complex I (C-I) assembly, in the mouse heart (cKO). While C-I supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD+/NADH ratio by C-I deficiency inhibited Sirt3 activity, leading to increase in protein acetylation, and sensitization of the permeability transition in mitochondria (mPTP). NAD+ precursor supplementation to cKO mice partially normalized the NAD+/NADH ratio, protein acetylation and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target. PMID:23931755

  15. Peroxiredoxin I deficiency attenuates phagocytic capacity of macrophage in clearance of the red blood cells damaged by oxidative stress.

    PubMed

    Han, Ying-Hao; Kwon, Taeho; Kim, Sun-Uk; Ha, Hye-Lin; Lee, Tae-Hoon; Kim, Jin-Man; Jo, Eun-Kyeong; Kim, Bo Yeon; Yoon, Do Young; Yu, Dae-Yeul

    2012-10-01

    The role of peroxiredoxin (Prx) I as an erythrocyte antioxidant defense in red blood cells (RBCs) is controversial. Here we investigated the function of Prx I by using Prx I(-/-) and Prx I/II(-/-) mice. Prx I(-/-) mice exhibited a normal blood profile. However, Prx I/II(-/-) mice showed more significantly increased Heinz body formation as compared with Prx II(-/-) mice. The clearance rate of Heinz body-containing RBCs in Prx I(-/-) mice decreased significantly through the treatment of aniline hydrochloride (AH) compared with wild-type mice. Prx I deficiency decreased the phagocytic capacity of macrophage in clearing Heinz body-containing RBCs. Our data demonstrate that Prx I deficiency did not cause hemolytic anemia, but showed that further increased hemolytic anemia symptoms in Prx II(-/-) mice by attenuating phagocytic capacity of macrophage in oxidative stress damaged RBCs, suggesting a novel role of Prx I in phagocytosis of macrophage.

  16. Analysis of the mitochondrial encoded subunits of complex I in 20 patients with a complex I deficiency.

    PubMed

    Meulemans, Ann; Lissens, Willy; Van Coster, Rudy; De Meirleir, Linda; Smet, Joél; Nassogne, Marie-Cécile; Liebaers, Inge; Seneca, Sara

    2004-01-01

    NADH-ubiquinone oxidoreductase or complex I deficiency is a frequently diagnosed enzyme defect of the oxidative phosphorylation (OXPHOS) system in humans. However, in many patients, with complex I deficiency and clinical symptoms suggestive of mitochondrial disease, often no genetic defect can be found after investigation of the most common mitochondrial DNA (mtDNA) mutations. In this study, 20 patients were selected with a biochemically documented complex I defect and no common mtDNA mutation. We used the Denaturing Gradient Gel Electrophoresis (DGGE) method with primers encompassing all mitochondrial encoded fragments, to search in a systematic manner for mutations in the mitochondrial genome of complex I. In our group of patients, we were able to detect a total of 96 nucleotide changes. We were not able to find any disease causing mutation in the mitochondrial encoded subunits of complex I. These results suggested that the complex I deficiency in this group of patients is most probably caused by a defect in one of the nuclear encoded structural genes of complex I, or in one of the genes involved in proper assembly of the enzyme.

  17. Absence of neurological deficits following extensive demyelination in a class I-deficient murine model of multiple sclerosis

    PubMed Central

    Schmelzer, James D.; Hunter, Samuel F.; Low, Phillip A.; Rodriguez, Moses

    2017-01-01

    Demyelination alone has been considered sufficient for development of neurological deficits following central nervous system (CNS) disease. However, extensive demyelination is not always associated with clinical deficits in patients with multiple sclerosis (MS), the most common primary demyelinating disease in humans. We used the Theiler’s murine encephalomyelitis virus model of demyelination to investigate the role of major histocompatibility complex (MHC) class I and class II gene products in the development of functional and neurophysiological deficits following demyelination. We measured spontaneous clinical activity by two independent assays and recorded hind-limb motor-evoked potentials in infected class I-deficient and class II-deficient mice of an identical genetic background as well as in highly susceptible SJL/J mice. The results show that despite a similar distribution and extent of demyelinated lesions in all mice, only class I-deficient mice were functionally normal. We propose that the mechanism by which demyelinated class I-deficient mice maintain neurologic function results from increased sodium channel densities and the relative preservation of axons. These findings are the first to implicate a role for MHC class I in the development of neurological deficits following demyelination. PMID:9461192

  18. Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency.

    PubMed

    Koopman, Werner J H; Visch, Henk-Jan; Verkaart, Sjoerd; van den Heuvel, Lambertus W P J; Smeitink, Jan A M; Willems, Peter H G M

    2005-10-01

    Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

  19. The p.M292T NDUFS2 mutation causes complex I-deficient Leigh syndrome in multiple families

    PubMed Central

    Tuppen, Helen A. L.; Hogan, Vanessa E.; He, Langping; Blakely, Emma L.; Worgan, Lisa; Al-Dosary, Mazhor; Saretzki, Gabriele; Alston, Charlotte L.; Morris, Andrew A.; Clarke, Michael; Jones, Simon; Devlin, Anita M.; Mansour, Sahar; Chrzanowska-Lightowlers, Zofia M. A.; Thorburn, David R.; McFarland, Robert

    2010-01-01

    Isolated complex I deficiency is the most frequently observed oxidative phosphorylation defect in children with mitochondrial disease, leading to a diverse range of clinical presentations, including Leigh syndrome. For most patients the genetic cause of the biochemical defect remains unknown due to incomplete understanding of the complex I assembly process. Nonetheless, a plethora of pathogenic mutations have been described to date in the seven mitochondrial-encoded subunits of complex I as well as in 12 of the nuclear-encoded subunits and in six assembly factors. Whilst several mitochondrial DNA mutations are recurrent, the majority of these mutations are reported in single families. We have sequenced core structural and functional nuclear-encoded subunits of complex I in a cohort of 34 paediatric patients with isolated complex I deficiency, identifying pathogenic mutations in 6 patients. These included a novel homozygous NDUFS1 mutation in an Asian child with Leigh syndrome, a previously identified NDUFS8 mutation (c.236C>T, p.P79L) in a second Asian child with Leigh-like syndrome and six novel, compound heterozygous NDUFS2 mutations in four white Caucasian patients with Leigh or Leigh-like syndrome. Three of these children harboured an identical NDUFS2 mutation (c.875T>C, p.M292T), which was also identified in conjunction with a novel NDUFS2 splice site mutation (c.866+4A>G) in a fourth Caucasian child who presented to a different diagnostic centre, with microsatellite and single nucleotide polymorphism analyses indicating that this was due to an ancient common founder event. Our results confirm that NDUFS2 is a mutational hotspot in Caucasian children with isolated complex I deficiency and recommend the routine diagnostic investigation of this gene in patients with Leigh or Leigh-like phenotypes. PMID:20819849

  20. Development and biological function of the female gonads and genitalia in IGF-I deficiency -- Laron syndrome as a model.

    PubMed

    Laron, Zvi

    2006-01-01

    Laron syndrome (LS) or primary GH insensitivity is a unique human model to study the effects of congenital IGF-I deficiency. Within our cohort of 63 patients with LS, 15 female patients were regularly followed since birth or infancy, throughout puberty. We observed that they were short at birth, with small genitalia and gonads -- during puberty, developed delayed puberty but eventually reached between 16 and 19 1/2 years full sexual development. Reproduction is unaffected at a young adult age. It is concluded that IGF-I in concert with the sex hormones has a modulatory but not essential function on female sexual development and maturation.

  1. Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6.

    PubMed

    Hartmannová, Hana; Piherová, Lenka; Tauchmannová, Kateřina; Kidd, Kendrah; Acott, Philip D; Crocker, John F S; Oussedik, Youcef; Mallet, Marcel; Hodaňová, Kateřina; Stránecký, Viktor; Přistoupilová, Anna; Barešová, Veronika; Jedličková, Ivana; Živná, Martina; Sovová, Jana; Hůlková, Helena; Robins, Vicki; Vrbacký, Marek; Pecina, Petr; Kaplanová, Vilma; Houštěk, Josef; Mráček, Tomáš; Thibeault, Yves; Bleyer, Anthony J; Kmoch, Stanislav

    2016-09-15

    The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.

  2. Therapeutic applications of the TAT-mediated protein transduction system for complex I deficiency and other mitochondrial diseases.

    PubMed

    Lin, Bo-Yu; Kao, Mou-Chieh

    2015-09-01

    Among the five enzyme complexes in the oxidative phosphorylation system, NADH-coenzyme Q oxidoreductase (also called complex I) is the largest, most intricate, and least understood. This enzyme complex spans the inner mitochondrial membrane and catalyzes the first step of electron transfer by the oxidation of NADH, and thereby provides two electrons for the reduction of quinone to quinol. Complex I deficiency is associated with many severe mitochondrial diseases, including Leber hereditary optic neuropathy and Leigh syndrome. However, to date, conventional treatments for the majority of genetic mitochondrial diseases are only palliative. Developing a reliable and convenient therapeutic approach is therefore considered to be an urgent need. Targeted proteins fused with the protein transduction domain of human immunodeficiency virus 1 transactivator of transcription (TAT) have been shown to enter cells by crossing plasma membranes while retaining their biological activities. Recent developments show that, in fusion with mitochondrial targeting sequences (MTSs), TAT-MTS-bound cargo can be correctly transported into mitochondria and restore the missing function of the cargo protein in patients' cells. The available evidence suggests that the TAT-mediated protein transduction system holds great promise as a potential therapeutic approach to treat complex I deficiency, as well as other mitochondrial diseases.

  3. Effects of TAT-conjugated platinum nanoparticles on lifespan of mitochondrial electron transport complex I-deficient Caenorhabditis elegans, nuo-1.

    PubMed

    Sakaue, Yuri; Kim, Juewon; Miyamoto, Yusei

    2010-09-20

    Platinum nanoparticle (Pt-np) species are superoxide dismutase/catalase mimetics and also have an activity similar to that of mitochondrial electron transport complex I. To examine if this complex I-like activity functions in vivo, we studied the effects of Pt-nps on the lifespan of a mitochondrial complex I-deficient Caenorhabditis elegans mutant, nuo-1 (LB25) compared with wild-type N2. We synthesized a fusion protein of a cell-penetrating peptide, human immunodeficiency virus-1 TAT (48-60), C-terminally linked to a peptide with a high affinity to platinum (GRKKRRQRRRPPQ-DRTSTWR). Pt-nps were functionalized by conjugation with this fusion protein at a 1:1 ratio of TAT-PtBP to Pt atoms. Adult worms were treated with conjugated Pt-nps for 10 days. The mean lifespan of untreated N2 and LB25 was 19.6 ± 0.4 and 11.8 ± 0.3 days, respectively. Using 5 μM of conjugated Pt-nps, the lifespan of N2 and LB25 was maximally extended. This maximal lifespan extension of LB25 was 31.9 ± 2.6%, which was significantly greater than that of N2 (21.1 ± 1.7%, P < 0.05 by Student's t-test). Internalization of Pt into the whole body and mitochondria was similar between these two strains. Excessive accumulation of reactive oxygen species was not observed in the cytosol or mitochondria of untreated LB25. Treatment for five days with 5 μM conjugated Pt-nps decreased cytosolic and mitochondrial reactive oxygen species in N2 and LB25 to a similar extent. The ratio of [NAD(+)]/[NADH] was very low in the whole body and mitochondria of control LB25. After five days of treatment with 5 μM conjugated Pt-nps, the ratio of [NAD(+)]/[NADH] was increased in N2 and LB25. However, the degree of the increase was much higher in LB25 than in N2. Pt-nps function as NADH oxidase and recover the [NAD(+)]/[NADH] ratio in LB25, leading to effective extension of the lifespan of LB25.

  4. Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm.

    PubMed

    Satoh, Hikaru; Nishi, Aiko; Yamashita, Kazuhiro; Takemoto, Yoko; Tanaka, Yasumasa; Hosaka, Yuko; Sakurai, Aya; Fujita, Naoko; Nakamura, Yasunori

    2003-11-01

    We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) > or = 37 and short chains with DP 12 to 21, marked increase in short chains with DP < or = 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2-3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.

  5. Characterization of High Density Lipoprotein Particles in Familial Apolipoprotein A-I Deficiency With Premature Coronary Atherosclerosis, Corneal Arcus and Opacification, and Tubo-Eruptive and Planar Xanthomas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe two male siblings with homozygous familial apolipoprotein (apo) A-I deficiency, markedly decreased high density lipoprotein (HDL) cholesterol levels, undetectable plasma apoA-1, tubo-eruptive and planar xanthomas, and mild corneal arcus and opacification. Sequencing of the apoA-I gene re...

  6. Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia.

    PubMed Central

    Ng, D S; Leiter, L A; Vezina, C; Connelly, P W; Hegele, R A

    1994-01-01

    We report a Canadian kindred with a novel mutation in the apolipoprotein (apo) A-I gene causing analphalipoproteinemia. The 34-yr-old proband, product of a consanguineous marriage, had bilateral retinopathy, bilateral cataracts, spinocerebellar ataxia, and tendon xanthomata. High density lipoprotein cholesterol (HDL-C) was < 0.1 mM and apoA-I was undetectable. Genomic DNA sequencing of the proband's apoA-I gene identified a nonsense mutation at codon [-2], which we designate as Q[-2]X. This mutation causes a loss of endonuclease digestion sites for both BbvI and Fnu4HI. Genotyping identified four additional homozygotes, four heterozygotes, and two unaffected subjects among the first-degree relatives. Q[-2]X homozygosity causes a selective failure to produce any portion of mature apoA-I, resulting in very low plasma level of HDL. Heterozygosity results in approximately half-normal apoA-I and HDL. Gradient gel electrophoresis and differential electroimmunodiffusion assay revealed that the HDL particles of the homozygotes had peak Stokes diameter of 7.9 nm and contained apoA-II without apoA-I (Lp-AII). Heterozygotes had an additional fraction of HDL3-like particles. Two of the proband's affected sisters had documented premature coronary heart disease. This kindred, the third reported apoA-I gene mutation causing isolated complete apoA-I deficiency, appears to be at significantly increased risk for atherosclerosis. Images PMID:8282791

  7. Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus.

    PubMed

    Chungopast, Sirinapa; Thapanapongworakul, Pilunthana; Matsuura, Hiroyuki; Van Dao, Tan; Asahi, Toshimasa; Tada, Kuninao; Tajima, Shigeyuki; Nomura, Mika

    2014-03-01

    In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume-rhizobia symbiosis by modifying nodule development and metabolic processes.

  8. Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency.

    PubMed

    Visch, Henk-Jan; Koopman, Werner J H; Leusink, Anouk; van Emst-de Vries, Sjenet E; van den Heuvel, Lambertus W P J; Willems, Peter H G M; Smeitink, Jan A M

    2006-01-01

    Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.

  9. Treatment with N- and C-Terminal Peptides of Parathyroid Hormone-Related Protein Partly Compensate the Skeletal Abnormalities in IGF-I Deficient Mice

    PubMed Central

    Portal-Núñez, Sergio; Murillo-Cuesta, Silvia; Lozano, Daniel; Cediel, Rafael; Esbrit, Pedro

    2014-01-01

    Insulin-like growth factor-I (IGF-I) deficiency causes growth delay, and IGF-I has been shown to partially mediate bone anabolism by parathyroid hormone (PTH). PTH-related protein (PTHrP) is abundant in bone, and has osteogenic features by poorly defined mechanisms. We here examined the capacity of PTHrP (1–36) and PTHrP (107–111) (osteostatin) to reverse the skeletal alterations associated with IGF-I deficiency. Igf1-null mice and their wild type littermates were treated with each PTHrP peptide (80 µg/Kg/every other day/2 weeks; 2 males and 4 females for each genotype) or saline vehicle (3 males and 3 females for each genotype). We found that treatment with either PTHrP peptide ameliorated trabecular structure in the femur in both genotypes. However, these peptides were ineffective in normalizing the altered cortical structure at this bone site in Igf1-null mice. An aberrant gene expression of factors associated with osteoblast differentiation and function, namely runx2, osteoprotegerin/receptor activator of NF-κB ligand ratio, Wnt3a, cyclin D1, connexin 43, catalase and Gadd45, as well as in osteocyte sclerostin, was found in the long bones of Igf1-null mice. These mice also displayed a lower amount of trabecular osteoblasts and osteoclasts in the tibial metaphysis than those in wild type mice. These alterations in Igf1-null mice were only partially corrected by each PTHrP peptide treatment. The skeletal expression of Igf2, Igf1 receptor and Irs2 was increased in Igf1-null mice, and this compensatory profile was further improved by treatment with each PTHrP peptide related to ERK1/2 and FoxM1 activation. In vitro, PTHrP (1–36) and osteostatin were effective in promoting bone marrow stromal cell mineralization in normal mice but not in IGF-I-deficient mice. Collectively, these findings indicate that PTHrP (1–36) and osteostatin can exert several osteogenic actions even in the absence of IGF-I in the mouse bone. PMID:24503961

  10. A novel mutation in TAP1 gene leading to MHC class I deficiency: Report of two cases and review of the literature.

    PubMed

    Hanalioglu, Damla; Ayvaz, Deniz Cagdas; Ozgur, Tuba Turul; van der Burg, Mirjam; Sanal, Ozden; Tezcan, Ilhan

    2017-02-02

    Major histocompatibility complex (MHC) class I deficiency syndrome is a rare primary immunodeficiency caused by mutations in the peptide transporter complex associated with antigen presentation (TAP) gene which plays a crucial role in intracellular peptide antigen presentation. A few cases have been reported to date. Recurrent sinopulmonary infections and skin ulcers are the main characteristics of the syndrome. Here we report two siblings diagnosed with TAP1 deficiency syndrome associated only with recurrent sinopulmonary infections with the description of a novel mutation leading to a premature stop codon in TAP1 gene and review of the relevant literature. Both of the siblings had recurrent sinopulmonary infections since childhood, responded to antibiotherapy well, neither of them had hospitalization history because of infections. One had chronic hepatitis B infection which may possibly be related to TAP1 gene defect.

  11. D-galactose induces a mitochondrial complex I deficiency in mouse skeletal muscle: potential benefits of nutrient combination in ameliorating muscle impairment.

    PubMed

    Chang, Liao; Liu, Xin; Liu, Jing; Li, Hua; Yang, Yanshen; Liu, Jia; Guo, Zihao; Xiao, Ke; Zhang, Chen; Liu, Jiankang; Zhao-Wilson, Xi; Long, Jiangang

    2014-03-01

    Accumulating research has shown that chronic D-galactose (D-gal) exposure induces symptoms similar to natural aging in animals. Therefore, rodents chronically exposed to D-gal are increasingly used as a model for aging and delay-of-aging pharmacological research. Mitochondrial dysfunction is thought to play a vital role in aging and age-related diseases; however, whether mitochondrial dysfunction plays a significant role in mice exposed to D-gal remains unknown. In the present study, we investigated cognitive dysfunction, locomotor activity, and mitochondrial dysfunction involved in D-gal exposure in mice. We found that D-gal exposure (125 mg/kg/day, 8 weeks) resulted in a serious impairment in grip strength in mice, whereas spatial memory and locomotor coordination remained intact. Interestingly, muscular mitochondrial complex I deficiency occurred in the skeletal muscle of mice exposed to D-gal. Mitochondrial ultrastructure abnormality was implicated as a contributing factor in D-gal-induced muscular impairment. Moreover, three combinations (A, B, and C) of nutrients applied in this study effectively reversed D-gal-induced muscular impairment. Nutrient formulas B and C were especially effective in reversing complex I dysfunction in both skeletal muscle and heart muscle. These findings suggest the following: (1) chronic exposure to D-gal first results in specific muscular impairment in mice, rather than causing general, premature aging; (2) poor skeletal muscle strength induced by D-gal might be due to the mitochondrial dysfunction caused by complex I deficiency; and (3) the nutrient complexes applied in the study attenuated the skeletal muscle impairment, most likely by improving mitochondrial function.

  12. Defective functionality of HDL particles in familial apoA-I deficiency: relevance of alterations in HDL lipidome and proteome[S

    PubMed Central

    Rached, Fabiana; Santos, Raul D.; Camont, Laurent; Miname, Marcio H.; Lhomme, Marie; Dauteuille, Carolane; Lecocq, Sora; Serrano, Carlos V.; Chapman, M. John; Kontush, Anatol

    2014-01-01

    To evaluate functional and compositional properties of HDL in subjects from a kindred of genetic apoA-I deficiency, two homozygotes and six heterozygotes, with a nonsense mutation at APOA1 codon -2, Q[-2]X, were recruited together with age- and sex-matched healthy controls (n = 11). Homozygotes displayed undetectable plasma levels of apoA-I and reduced levels of HDL-cholesterol (HDL-C) and apoC-III (5.4% and 42.6% of controls, respectively). Heterozygotes displayed low HDL-C (21 ± 9 mg/dl), low apoA-I (79 ± 24 mg/dl), normal LDL-cholesterol (132 ± 25 mg/dl), and elevated TG (130 ± 45 mg/dl) levels. Cholesterol efflux capacity of ultracentrifugally isolated HDL subpopulations was reduced (up to −25%, P < 0.01, on a glycerophospholipid [GP] basis) in heterozygotes versus controls. Small, dense HDL3 and total HDL from heterozygotes exhibited diminished antioxidative activity (up to −48%, P < 0.001 on a total mass basis) versus controls. HDL subpopulations from both homozygotes and heterozygotes displayed altered chemical composition, with depletion in apoA-I, GP, and cholesteryl ester; enrichment in apoA-II, free cholesterol, and TG; and altered phosphosphingolipidome. The defective atheroprotective activities of HDL were correlated with altered lipid and apo composition. These data reveal that atheroprotective activities of HDL particles are impaired in homozygous and heterozygous apoA-I deficiency and are intimately related to marked alterations in protein and lipid composition. PMID:25341944

  13. Saccharomyces cerevisiae aldolase mutants.

    PubMed Central

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo. PMID:6384192

  14. Neonatal multiorgan failure due to ACAD9 mutation and complex I deficiency with mitochondrial hyperplasia in liver, cardiac myocytes, skeletal muscle, and renal tubules.

    PubMed

    Leslie, Nancy; Wang, Xinjian; Peng, Yanyan; Valencia, C Alexander; Khuchua, Zaza; Hata, Jessica; Witte, David; Huang, Taosheng; Bove, Kevin E

    2016-03-01

    Complex I deficiency causes Leigh syndrome, fatal infant lactic acidosis, and neonatal cardiomyopathy. Mutations in more than 100 nuclear DNA and mitochondrial DNA genes miscode for complex I subunits or assembly factors. ACAD9 is an acyl-CoA dehydrogenase with a novel function in assembly of complex I; biallelic mutations cause progressive encephalomyopathy, recurrent Reye syndrome, and fatal cardiomyopathy. We describe the first autopsy in fatal neonatal lethal lactic acidosis due to mutations in ACAD9 that reduced complex I activity. We identified mitochondrial hyperplasia in cardiac myocytes, diaphragm muscle, and liver and renal tubules in formalin-fixed, paraffin-embedded tissue using immunohistochemistry for mitochondrial antigens. Whole-exome sequencing revealed compound heterozygous variants in the ACAD9 gene: c.187G>T (p.E63*) and c.941T>C (p.L314P). The nonsense mutation causes late infantile lethality; the missense variant is novel. Autopsy-derived fibroblasts had reduced complex I activity (53% of control) with normal activity in complexes II to IV, similar to reported cases of ACAD9 deficiency.

  15. Heptanoate as a neural fuel: energetic and neurotransmitter precursors in normal and glucose transporter I-deficient (G1D) brain

    PubMed Central

    Marin-Valencia, Isaac; Good, Levi B; Ma, Qian; Malloy, Craig R; Pascual, Juan M

    2013-01-01

    It has been postulated that triheptanoin can ameliorate seizures by supplying the tricarboxylic acid cycle with both acetyl-CoA for energy production and propionyl-CoA to replenish cycle intermediates. These potential effects may also be important in other disorders associated with impaired glucose metabolism because glucose supplies, in addition to acetyl-CoA, pyruvate, which fulfills biosynthetic demands via carboxylation. In patients with glucose transporter type I deficiency (G1D), ketogenic diet fat (a source only of acetyl-CoA) reduces seizures, but other symptoms persist, providing the motivation for studying heptanoate metabolism. In this work, metabolism of infused [5,6,7-13C3]heptanoate was examined in the normal mouse brain and in G1D by 13C-nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). In both groups, plasma glucose was enriched in 13C, confirming gluconeogenesis from heptanoate. Acetyl-CoA and glutamine levels became significantly higher in the brain of G1D mice relative to normal mice. In addition, brain glutamine concentration and 13C enrichment were also greater when compared with glutamate in both animal groups, suggesting that heptanoate and/or C5 ketones are primarily metabolized by glia. These results enlighten the mechanism of heptanoate metabolism in the normal and glucose-deficient brain and encourage further studies to elucidate its potential antiepileptic effects in disorders of energy metabolism. PMID:23072752

  16. PDGFRA-mutant syndrome.

    PubMed

    Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

    2015-07-01

    Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

  17. Carbon dioxide fixation and photoevolution of hydrogen and oxygen in a mutant of Chlamydomonas lacking Photosystem I

    SciTech Connect

    Greenbaum, E.; Lee, J.W.; Tevault, C.V.

    1995-09-01

    Sustained photoassimilation of atmospheric CO{sub 2} and simultaneous photoevolution of molecular hydrogen and oxygen has been observed in a Photosystem I deficient mutant B4 of Chlamydomonas reinhardtii that contains only Photosystem II. The data indicate that Photosystem II alone is capable of spanning the potential difference between water oxidation/oxygen evolution and ferredoxin reduction. The rates of both CO{sub 2} fixation and hydrogen and oxygen evolution are similar in the mutant to that of the wild-type C. reinhardtii 137c containing both photosystems. The wild-type had stable photosynthetic activity, measured as CO{sub 2} fixation, under both air and anaerobic conditions, while the mutant was stable only under anaerobic conditions. The results are discussed in terms of the fundamental mechanisms and energetics of photosynthesis and possible implications for the evolution of oxygenic photosynthesis.

  18. Glucose transporter type I deficiency (G1D) at 25 (1990–2015): Presumptions, facts and the lives of persons with this rare disease

    PubMed Central

    Pascual, Juan M.; Ronen, Gabriel M.

    2015-01-01

    As is often the case for rare diseases, the number of published reviews and case reports of Glucose transporter type I deficiency (G1D) approaches or exceeds that of original research. This can indicate medical interest, but also scientific stagnation. In assessing this state of affairs here, we focus not on what is peculiar or disparate about G1D, but on the assumptions that have reigned thus far undisputed, and critique them as a potential impediment to progress. To summarize the most common G1D phenotype, we trace the 25-year story of G1D in parallel with the natural history of one of two index patients, identified in 1990 by one of us (G.M.R.) and brought up to date by the other (J.M.P.) while later examining widely-repeated but little-scrutinized statements. Among them are those that pertain to assumptions about brain fuels; energy-failure; cerebrospinal glucose concentration; the purpose of ketogenic diet; the role of the defective blood brain barrier; genotype-phenotype correlations; a bewildering array of phenotypes; ictogenesis, seizures and the electroencephalogram; the use of mice to model the disorder; and what treatments may and may not be expected to accomplish. We reach the forgone conclusion that the proper study of mankind - and of one of its ailments (G1D) - is man itself (rather than mice, isolated cells or extrapolated inferences), and propose a framework for rigorous investigation that we hope will lead to a better understanding and to better treatments for this and for rare disorders in general. These considerations, together with experience drawn from other disorders, lead, as a logical consequence, to the nullification of the view that therapeutic development (i.e., trials) for rare diseases could or should be accelerated without the most vigorous scientific scrutiny: Trial and error constitute an inseparable couple, such that, at the present time, hastening the former is bound to precipitate the latter. PMID:26341673

  19. IFNγ producing CD8+ T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas

    PubMed Central

    Buferne, Michel; Chasson, Lionel; Grange, Magali; Mas, Amandine; Arnoux, Fanny; Bertuzzi, Mélanie; Naquet, Philippe; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

    2015-01-01

    Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients. PMID:25949872

  20. IFNγ producing CD8(+) T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas.

    PubMed

    Buferne, Michel; Chasson, Lionel; Grange, Magali; Mas, Amandine; Arnoux, Fanny; Bertuzzi, Mélanie; Naquet, Philippe; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

    2015-02-01

    Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8(+) T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8(+) T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8(+) T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8(+) T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8(+) T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8(+) T cells to inhibition both by PD-1/PDL-1 engagement and by TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.

  1. Differential effects of eNOS uncoupling on conduit and small arteries in GTP-cyclohydrolase I-deficient hph-1 mice

    PubMed Central

    d'Uscio, Livius V.; Smith, Leslie A.

    2011-01-01

    In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH4) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH4 levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH4-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser1177-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS. PMID:21963838

  2. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  3. Interstitial deletion of chromosome 2q32-34 associated with multiple congenital anomalies and a urea cycle defect (CPS I deficiency).

    PubMed

    Loscalzo, M L; Galczynski, R L; Hamosh, A; Summar, M; Chinsky, J M; Thomas, G H

    2004-07-30

    A de novo deletion of the long arm of chromosome 2 at 2q31-33 was observed in the fetal amniocyte G-banded karyotype performed because of possible multiple malformations identified by ultrasound at 23 weeks gestation. Two days after the uneventful term delivery of a 2.45 kg male, the neonate experienced cardiopulmonary decompensation and biochemical changes compatible with carbamoyl phosphate synthetase I (CPS I) deficiency (elevated ammonia with a peak of 948 micromol/L, deficiency of citrulline, and no increase in orotic acid). The child died on day 3 of life. Physical anomalies confirmed at autopsy included double superior vena cava, ectopic adrenal tissue, and metatarsus adductus. The autopsy also revealed histologic evidence consistent with CPS deficiency, most notably microvesicular steatosis of the liver and Alzheimer's Type II changes with hypertrophic astrocytes in the basal ganglia. A postnatal lymphocyte karyotype confirmed the chromosome 2q31-33 deletion. Enzyme analysis on postmortem liver tissue confirmed the diagnosis of CPS deficiency. CPS I is reported to be mapped to 2q35 by NCBI (http://www.ncbi.nlm.nih.gov/mapview/) and 2q34 by ENSEMBL (http://www.ensembl.org/). The UCSC Human Genome Browser July 2003 assembly also places the gene at 2q34 (http://genome.UCSC.edu/). Fluorescence in situ hybridization (FISH) analysis with a BAC clone (RP11-349G4) of CPS I demonstrated that one copy of the gene was deleted in this infant. Using additional probes corresponding to the bands in the region of deletion, we identified the deleted region as 2q32-2q34. Our observations support the CPS I map position (ENSEMBL, UCSC) at 2q34. Additionally, potential conditions associated with deletions narrowly defined by standard cytogenetic techniques merit consideration in prenatal counseling. As demonstrated here, deletions may not only result in malformations and mental retardation but also increase the likelihood of revealing mutated genes located in the undeleted

  4. Increased mitochondrial ATP production capacity in brain of healthy mice and a mouse model of isolated complex I deficiency after isoflurane anesthesia.

    PubMed

    Manjeri, Ganesh R; Rodenburg, Richard J; Blanchet, Lionel; Roelofs, Suzanne; Nijtmans, Leo G; Smeitink, Jan A; Driessen, Jacques J; Koopman, Werner J H; Willems, Peter H

    2016-01-01

    We reported before that the minimal alveolar concentration (MAC) of isoflurane is decreased in complex I-deficient mice lacking the NDUFS4 subunit of the respiratory chain (RC) (1.55 and 0.81% at postnatal (PN) 22-25 days and 1.68 and 0.65% at PN 31-34 days for wildtype (WT) and CI-deficient KO, respectively). A more severe respiratory depression was caused by 1.0 MAC isoflurane in KO mice (respiratory rate values of 86 and 45 at PN 22-25 days and 69 and 29 at PN 31-34 days for anesthetized WT and KO, respectively). Here, we address the idea that isoflurane anesthesia causes a much larger decrease in brain mitochondrial ATP production in KO mice thus explaining their increased sensitivity to this anesthetic. Brains from WT and KO mice of the above study were removed immediately after MAC determination at PN 31-34 days and a mitochondria-enriched fraction was prepared. Aliquots were used for measurement of maximal ATP production in the presence of pyruvate, malate, ADP and creatine and, after freeze-thawing, the maximal activity of the individual RC complexes in the presence of complex-specific substrates. CI activity was dramatically decreased in KO, whereas ATP production was decreased by only 26% (p < 0.05). The activities of CII, CIII, and CIV were the same for WT and KO. Isoflurane anesthesia decreased the activity of CI by 30% (p < 0.001) in WT. In sharp contrast, it increased the activity of CII by 37% (p < 0.001) and 50% (p < 0.001) and that of CIII by 37% (p < 0.001) and 40% (p < 0.001) in WT and KO, respectively, whereas it tended to increase that of CIV in both WT and KO. Isoflurane anesthesia increased ATP production by 52 and 69% in WT (p < 0.05) and KO (p < 0.01), respectively. Together these findings indicate that isoflurane anesthesia interferes positively rather than negatively with the ability of CI-deficient mice brain mitochondria to convert their main substrate pyruvate into ATP.

  5. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

  6. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  7. Mutant power: using mutant allele collections for yeast functional genomics.

    PubMed

    Norman, Kaitlyn L; Kumar, Anuj

    2016-03-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology.

  8. Genetics Home Reference: carnitine palmitoyltransferase I deficiency

    MedlinePlus

    ... in cells. A group of fats called long-chain fatty acids cannot enter mitochondria unless they are ... carnitine. Carnitine palmitoyltransferase 1A connects carnitine to long-chain fatty acids so they can enter mitochondria and ...

  9. Complex I deficiencies in neurological disorders.

    PubMed

    Papa, Sergio; De Rasmo, Domenico

    2013-01-01

    Complex I is the point of entry in the mitochondrial electron transport chain for NADH reducing equivalents, and it behaves as a regulatable pacemaker of respiratory ATP production in human cells. Defects in complex I are associated with several human neurological disorders, including primary mitochondrial diseases, Parkinson disease (PD), and Down syndrome, and understanding the activity and regulation of complex I may reveal aspects of the underlying pathogenic mechanisms. Complex I is regulated by cyclic AMP (cAMP) and the protein kinase A (PKA) signal transduction pathway, and elucidating the role of the cAMP/PKA system in regulating complex I and oxygen free radical production provides new perspectives for devising therapeutic strategies for neurological diseases.

  10. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  11. Selection of chemotaxis mutants of Dictyostelium discoideum

    PubMed Central

    1987-01-01

    A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant- degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described. PMID:3793759

  12. Targeting ESR1-Mutant Breast Cancer

    DTIC Science & Technology

    2015-09-01

    Award Number: W81XWH-14-1-0360 TITLE: Targeting ESR1- Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Geoffrey L. Greene, Ph.D. CONTRACTING...ADDRESS. 1. REPORT DATE September 2015 2. REPORT TYPE Annual 3. DATES COVERED 1 Sep 2014 - 31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1- Mutant ...approved hormonal therapies and that more potent, selective estrogen receptor degraders (SERDs) will enable complete inhibition of mutant ER signaling and

  13. Targeting ESR1-Mutant Breast Cancer

    DTIC Science & Technology

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1- Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1- Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...current FDA approved hormonal therapies and that more potent, selective estrogen receptor degraders (SERDs) will enable complete inhibition of mutant

  14. Electrophysiological study of Drosophila rhodopsin mutants

    PubMed Central

    1986-01-01

    Electrophysiological investigations were carried out on several independently isolated mutants of the ninaE gene, which encodes opsin in R1-6 photoreceptors, and a mutant of the ninaD gene, which is probably important in the formation of the rhodopsin chromophore. In these mutants, the rhodopsin content in R1-6 photoreceptors is reduced by 10(2)-10(6)-fold. Light-induced bumps recorded from even the most severely affected mutants are physiologically normal. Moreover, a detailed noise analysis shows that photoreceptor responses of both a ninaE mutant and a ninaD mutant follow the adapting bump model. Since any extensive rhodopsin-rhodopsin interactions are not likely in these mutants, the above results suggest that such interactions are not needed for the generation and adaptation of light-induced bumps. Mutant bumps are strikingly larger in amplitude than wild-type bumps. This difference is observed both in ninaD and ninaE mutants, which suggests that it is due to severe depletion of rhodopsin content, rather than to any specific alterations in the opsin protein. Lowering or buffering the intracellular calcium concentration by EGTA injection mimics the effects of the mutations on the bump amplitude, but, unlike the mutations, it also affects the latency and kinetics of light responses. PMID:3097245

  15. Identifying representative drug resistant mutants of HIV

    PubMed Central

    2015-01-01

    Background Drug resistance is one of the most important causes for failure of anti-AIDS treatment. During therapy, multiple mutations accumulate in the HIV genome, eventually rendering the drugs ineffective in blocking replication of the mutant virus. The huge number of possible mutants precludes experimental analysis to explore the molecular mechanisms of resistance and develop improved antiviral drugs. Results In order to solve this problem, we have developed a new algorithm to reveal the most representative mutants from the whole drug resistant mutant database based on our newly proposed unified protein sequence and 3D structure encoding method. Mean shift clustering and multiple regression analysis were applied on genotype-resistance data for mutants of HIV protease and reverse transcriptase. This approach successfully chooses less than 100 mutants with the highest resistance to each drug out of about 10K in the whole database. When considering high level resistance to multiple drugs, the numbers reduce to one or two representative mutants. Conclusion This approach for predicting the most representative mutants for each drug has major importance for experimental verification since the results provide a small number of representative sequences, which will be amenable for in vitro testing and characterization of the expressed mutant proteins. PMID:26678327

  16. Mutant calreticulin requires both its mutant C-terminus and the thrombopoietin receptor for oncogenic transformation

    PubMed Central

    Elf, Shannon; Abdelfattah, Nouran S.; Chen, Edwin; Perales-Patón, Javier; Rosen, Emily A.; Ko, Amy; Peisker, Fabian; Florescu, Natalie; Giannini, Silvia; Wolach, Ofir; Morgan, Elizabeth A.; Tothova, Zuzana; Losman, Julie-Aurore; Schneider, Rebekka K.; Al-Shahrour, Fatima; Mullally, Ann

    2016-01-01

    Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN) but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of CALR-mutant MPN patients. We further show that the thrombopoietin receptor, MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. PMID:26951227

  17. Enhancers of Conidiation Mutants in Aspergillus Nidulans

    PubMed Central

    Gems, D. H.; Clutterbuck, A. J.

    1994-01-01

    Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as ``Abacoid.'' sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies. PMID:8056325

  18. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  19. A halotolerant mutant of Saccharomyces cerevisiae.

    PubMed Central

    Gaxiola, R; Corona, M; Zinker, S

    1996-01-01

    FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. PMID:8631691

  20. crl mutants of Saccharomyces cerevisiae resemble both mutants affecting general control of amino acid biosynthesis and omnipotent translational suppressor mutants.

    PubMed

    McCusker, J H; Haber, J E

    1988-06-01

    Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.

  1. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  2. Salmonella typhimurium mutants lacking NAD pyrophosphatase.

    PubMed Central

    Park, U E; Roth, J R; Olivera, B M

    1988-01-01

    NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

  3. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

    Petrie-Hanson, Lora; Hohn, Claudia; Hanson, Larry

    2009-01-01

    Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

  4. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity.

    PubMed

    Lippincott, B B; Lippincott, J A

    1966-10-01

    Lippincott, Barbara B. (Northwestern University, Evanston, Ill.), and James A. Lippincott. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity. J. Bacteriol. 92:937-945. 166.-Mutants of Agrobacterium tumefaciens auxotrophic for adenine, methionine, or asparagine are less infectious than the wild-type strain B6 from which they were derived and show increased infectivity on pinto bean leaves when the specific compounds required for growth of the mutants are added to the infected leaf. Reversion to a prototrophic form of nutrition is accompanied by increased infectivity. Tumors initiated by these auxotrophic mutants are shown to arise only at large wound sites where nutritional conditions may be less restricting. The data indicate that, after inoculation, the bacteria pass through a phase in which host-supplied nutrients are utilized for the production of one or more factors necessary for successful tumor initiation.

  5. Mutant IDH1 and thrombosis in gliomas.

    PubMed

    Unruh, Dusten; Schwarze, Steven R; Khoury, Laith; Thomas, Cheddhi; Wu, Meijing; Chen, Li; Chen, Rui; Liu, Yinxing; Schwartz, Margaret A; Amidei, Christina; Kumthekar, Priya; Benjamin, Carolina G; Song, Kristine; Dawson, Caleb; Rispoli, Joanne M; Fatterpekar, Girish; Golfinos, John G; Kondziolka, Douglas; Karajannis, Matthias; Pacione, Donato; Zagzag, David; McIntyre, Thomas; Snuderl, Matija; Horbinski, Craig

    2016-12-01

    Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

  6. Isolation and characterization of unusual gin mutants.

    PubMed Central

    Klippel, A; Cloppenborg, K; Kahmann, R

    1988-01-01

    Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation. Images PMID:2974801

  7. Quantitative Analysis of Triple Mutant Genetic Interactions

    PubMed Central

    Braberg, Hannes; Alexander, Richard; Shales, Michael; Xu, Jiewei; Franks-Skiba, Kathleen E.; Wu, Qiuqin; Haber, James E.; Krogan, Nevan J.

    2014-01-01

    The quantitative analysis of genetic interactions between pairs of gene mutations has proven effective for characterizing cellular functions but can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed Triple Mutant Analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, that is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principle actors are deleted. TMA has also uncovered double mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete, and measures interactions for up to 30 double mutants against a library of 1536 single mutants. PMID:25010907

  8. Characterization of shrunken endosperm mutants in barley.

    PubMed

    Ma, Jian; Jiang, Qian-Tao; Wei, Long; Wang, Ji-Rui; Chen, Guo-Yue; Liu, Ya-Xi; Li, Wei; Wei, Yu-Ming; Liu, Chunji; Zheng, You-Liang

    2014-04-10

    Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1-seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87 kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27 days after anthesis (DAA) after reaching the peak at 15-21 DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.

  9. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  10. Computing border bases using mutant strategies

    NASA Astrophysics Data System (ADS)

    Ullah, E.; Abbas Khan, S.

    2014-01-01

    Border bases, a generalization of Gröbner bases, have actively been addressed during recent years due to their applicability to industrial problems. In cryptography and coding theory a useful application of border based is to solve zero-dimensional systems of polynomial equations over finite fields, which motivates us for developing optimizations of the algorithms that compute border bases. In 2006, Kehrein and Kreuzer formulated the Border Basis Algorithm (BBA), an algorithm which allows the computation of border bases that relate to a degree compatible term ordering. In 2007, J. Ding et al. introduced mutant strategies bases on finding special lower degree polynomials in the ideal. The mutant strategies aim to distinguish special lower degree polynomials (mutants) from the other polynomials and give them priority in the process of generating new polynomials in the ideal. In this paper we develop hybrid algorithms that use the ideas of J. Ding et al. involving the concept of mutants to optimize the Border Basis Algorithm for solving systems of polynomial equations over finite fields. In particular, we recall a version of the Border Basis Algorithm which is actually called the Improved Border Basis Algorithm and propose two hybrid algorithms, called MBBA and IMBBA. The new mutants variants provide us space efficiency as well as time efficiency. The efficiency of these newly developed hybrid algorithms is discussed using standard cryptographic examples.

  11. Isolation and characterization of transcription fidelity mutants.

    PubMed

    Strathern, Jeffrey N; Jin, Ding Jun; Court, Donald L; Kashlev, Mikhail

    2012-07-01

    Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

  12. Sleep restores behavioral plasticity to Drosophila mutants.

    PubMed

    Dissel, Stephane; Angadi, Veena; Kirszenblat, Leonie; Suzuki, Yasuko; Donlea, Jeff; Klose, Markus; Koch, Zachary; English, Denis; Winsky-Sommerer, Raphaelle; van Swinderen, Bruno; Shaw, Paul J

    2015-05-18

    Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

  13. Nonphotic phase shifting in hamster clock mutants.

    PubMed

    Mrosovsky, N; Salmon, P A; Menaker, M; Ralph, M R

    1992-01-01

    Golden hamsters with the tau mutation were kept in the dark and induced to become active through confinement to a novel running wheel for 3 hr. The response of the mutants to this nonphotic phase-shifting stimulus differed from that of wild-type hamsters. The mutants showed larger phase shifts, and their phase response curves differed in shape, with an advance portion at about circadian time 24, a phase at which wild types show delays. The results establish that the tau mutation, in addition to its already known effects, alters the response of the circadian system to nonphotic events.

  14. Fluoroquinolone-resistant mutants of Burkholderia cepacia.

    PubMed

    Pope, C F; Gillespie, S H; Pratten, J R; McHugh, T D

    2008-03-01

    Fluoroquinolone-resistant Burkholderia cepacia mutants were selected on ciprofloxacin. The rate of mutation in gyrA was estimated to be 9.6 x 10(-11) mutations per division. Mutations in gyrA conferred 12- to 64-fold increases in MIC, and an additional parC mutation conferred a large increase in MIC (>256-fold). Growth rate, biofilm formation, and survival in water and during drying were not impaired in strains containing single gyrA mutations. Double mutants were impaired only in growth rate (0.85, relative to the susceptible parent).

  15. Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response.

    PubMed

    Cove, David J; Quatrano, Ralph S

    2006-07-01

    New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.

  16. Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

    PubMed

    Araki, Marito; Yang, Yinjie; Masubuchi, Nami; Hironaka, Yumi; Takei, Hiraku; Morishita, Soji; Mizukami, Yoshihisa; Kan, Shin; Shirane, Shuichi; Edahiro, Yoko; Sunami, Yoshitaka; Ohsaka, Akimichi; Komatsu, Norio

    2016-03-10

    Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

  17. Targeting ESR1-Mutant Breast Cancer

    DTIC Science & Technology

    2015-09-01

    current FDA approved hormonal therapies and that more potent, selective estrogen receptor degraders (SERDs) will enable complete inhibition of mutant...resistance to current FDA approved ER antagonists, but that more potent and selective estrogen receptor antagonists will be sufficiently active to...Antagonist Selective Estrogen Receptor Modulator Selective Estrogen Receptor Degrader Tamoxifen Fulvestrant Bazedoxifene Raloxifene GDC-0810

  18. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    PubMed

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  19. Comprehensive transposon mutant library of Pseudomonas aeruginosa

    PubMed Central

    Jacobs, Michael A.; Alwood, Ashley; Thaipisuttikul, Iyarit; Spencer, David; Haugen, Eric; Ernst, Stephen; Will, Oliver; Kaul, Rajinder; Raymond, Christopher; Levy, Ruth; Chun-Rong, Liu; Guenthner, Donald; Bovee, Donald; Olson, Maynard V.; Manoil, Colin

    2003-01-01

    We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering. PMID:14617778

  20. Comprehensive transposon mutant library of Pseudomonas aeruginosa.

    PubMed

    Jacobs, Michael A; Alwood, Ashley; Thaipisuttikul, Iyarit; Spencer, David; Haugen, Eric; Ernst, Stephen; Will, Oliver; Kaul, Rajinder; Raymond, Christopher; Levy, Ruth; Chun-Rong, Liu; Guenthner, Donald; Bovee, Donald; Olson, Maynard V; Manoil, Colin

    2003-11-25

    We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

  1. Yeast mutants overproducing iso-cytochromes c

    SciTech Connect

    Sherman, F.; Cardillo, T.S.; Errede, B.; Friedman, L.; McKnight, G.; Stiles, J.I.

    1980-01-01

    For over 15 years, the iso-cytochrome c system in the yeast Saccharomyces cerevisiae has been used to investigate a multitude of problems in genetics and molecular biology. More recently, attention has been focused on using mutants for examining translation and transcriptional processes and for probing regulatory regions governing gene expression. In an effort to explore regulatory mechanisms and to investigate mutational alterations that lead to increased levels of gene products, we have isolated and characterized mutants that overproduce cytochrome c. In this paper we have briefly summarized background information of some essential features of the iso-cytochrome c system and we have described the types of mutants that overproduce iso-1-cytochrome c or iso-2-cytochrome c. Genetic procedures and recombinant DNA procedures were used to demonstrate that abnormally high amounts of gene products occur in mutants as result of duplications of gene copies or of extended alteration of regulatory regions. The results summarized in this paper point out the requirements of gross mutational changes or rearrangements of chromosomal segments for augmenting gene products.

  2. Ethanol production using engineered mutant E. coli

    DOEpatents

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

    The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

  3. Genotyping-by-sequencing of glossy mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glossy mutants are a common occurrence in Brassica oleracea L. and they have been documented in most crop varieties of the species including cabbage, kale, broccoli, and collard. Glossy phenotypes have been of particular interest to researchers due to observations that they influence insect behavior...

  4. Phenotypic mutant library: potential for gene discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid development of high throughput and affordable Next- Generation Sequencing (NGS) techniques has renewed interest in gene discovery using forward genetics. The conventional forward genetic approach starts with isolation of mutants with a phenotype of interest, mapping the mutation within a s...

  5. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

    PubMed Central

    Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

    2016-01-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  6. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks.

    PubMed

    Yang, Hee-Jeong; Bogomolnaya, Lydia M; Elfenbein, Johanna R; Endicott-Yazdani, Tiana; Reynolds, M Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin; McClelland, Michael; Andrews-Polymenis, Helene

    2016-04-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

  7. GAMPMS: Genetic algorithm managed peptide mutant screening.

    PubMed

    Long, Thomas; McDougal, Owen M; Andersen, Tim

    2015-06-30

    The prominence of endogenous peptide ligands targeted to receptors makes peptides with the desired binding activity good molecular scaffolds for drug development. Minor modifications to a peptide's primary sequence can significantly alter its binding properties with a receptor, and screening collections of peptide mutants is a useful technique for probing the receptor-ligand binding domain. Unfortunately, the combinatorial growth of such collections can limit the number of mutations which can be explored using structure-based molecular docking techniques. Genetic algorithm managed peptide mutant screening (GAMPMS) uses a genetic algorithm to conduct a heuristic search of the peptide's mutation space for peptides with optimal binding activity, significantly reducing the computational requirements of the virtual screening. The GAMPMS procedure was implemented and used to explore the binding domain of the nicotinic acetylcholine receptor (nAChR) α3β2-isoform with a library of 64,000 α-conotoxin (α-CTx) MII peptide mutants. To assess GAMPMS's performance, it was compared with a virtual screening procedure that used AutoDock to predict the binding affinity of each of the α-CTx MII peptide mutants with the α3β2-nAChR. The GAMPMS implementation performed AutoDock simulations for as few as 1140 of the 64,000 α-CTx MII peptide mutants and could consistently identify a set of 10 peptides with an aggregated binding energy that was at least 98% of the aggregated binding energy of the 10 top peptides from the exhaustive AutoDock screening.

  8. Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (I) mutant generation and characterization

    PubMed Central

    2014-01-01

    Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under

  9. Analysis of Sporulation Mutants II. Mutants Blocked in the Citric Acid Cycle

    PubMed Central

    Fortnagel, Peter; Freese, Ernst

    1968-01-01

    Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from 14C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in α-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation. Images PMID:4967197

  10. Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

    PubMed

    Fedorenko, Inna V; Fang, Bin; Koomen, John M; Gibney, Geoffrey T; Smalley, Keiran S M

    2014-10-01

    Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

  11. Intact Interval Timing in Circadian CLOCK Mutants

    PubMed Central

    Cordes, Sara; Gallistel, C. R.

    2008-01-01

    While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/− and −/− mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing. PMID:18602902

  12. Acriflavine-Resistant Mutant of Streptococcus cremoris†

    PubMed Central

    Sinha, R.P.

    1977-01-01

    Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40°C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures. PMID:907329

  13. Characterization of Helicobacter pylori urease mutants.

    PubMed Central

    Segal, E D; Shon, J; Tompkins, L S

    1992-01-01

    The association between Helicobacter pylori, gastritis, and peptic ulcer is well established, and the association of infection with gastric cancer has been noted in several developing countries. However, the pathogenic mechanism(s) leading to disease states has not been elucidated. The H. pylori urease is thought to be a determinant of pathogenicity, since the enzyme is produced by all H. pylori clinical isolates. Evidence indicates that some H. pylori strains are more cytotoxic than others, with a correlation between the activity of the urease and the presence of a vacuolating cytotoxin having been made. However, the number of cytotoxins remains unknown at this time. The relationship between the urease and cytotoxicity has previously been examined with chemical inhibitors. To examine the role of the urease and its relationship to cytotoxicity, urease-deficient mutants were produced following ethyl methanesulfonate mutagenesis of H. pylori 87A300. Two mutants (the ure1 and ure5 mutants) which were entirely deficient in urease activity (Ure-) were selected. Characterization of the isolates at the protein level showed that the urease subunits lacked the ability to complex and form the active urease enzyme. The ure1 mutant was shown to be sensitive to the effects of low pH in vitro and exhibited no cytotoxicity to eucaryotic cells, whereas the parental strain (Ure+) produced a cytotoxic effect in the presence of urea. Interaction between the H. pylori Ure+ and Ure- strains and Caco-2 cells appeared to be similar in that both bacterial types elicited pedestal formation and actin condensation. These results indicate that the H. pylori urease may have many functions, among them (i) protecting H. pylori against the acidic environment of the stomach, (ii) acting as a cytotoxin, with human gastric cells especially susceptible to its activity, and (iii) disrupting cell tight junctions in such a manner that the cells remain viable but an ionic flow between the cells occurs

  14. Arabidopsis MET1 cytosine methyltransferase mutants.

    PubMed Central

    Kankel, Mark W; Ramsey, Douglas E; Stokes, Trevor L; Flowers, Susan K; Haag, Jeremy R; Jeddeloh, Jeffrey A; Riddle, Nicole C; Verbsky, Michelle L; Richards, Eric J

    2003-01-01

    We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

  15. Isolation of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Chidambaram, M; Sharma, B; Petras, S F; Reese, C P; Froshauer, S; Weinstock, G M

    1995-01-01

    Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica. PMID:7868223

  16. Mutant Sodium Channel for Tumor Therapy

    PubMed Central

    Tannous, Bakhos A; Christensen, Adam P; Pike, Lisa; Wurdinger, Thomas; Perry, Katherine F; Saydam, Okay; Jacobs, Andreas H; García-Añoveros, Jaime; Weissleder, Ralph; Sena-Esteves, Miguel; Corey, David P; Breakefield, Xandra O

    2009-01-01

    Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by the delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein–Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, noninfected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells become susceptible and death is rapid with no time for the tumor cells to become resistant. PMID:19259066

  17. Mutant p53: One, No One, and One Hundred Thousand.

    PubMed

    Walerych, Dawid; Lisek, Kamil; Del Sal, Giannino

    2015-01-01

    Encoded by the mutated variants of the TP53 tumor suppressor gene, mutant p53 proteins are getting an increased experimental support as active oncoproteins promoting tumor growth and metastasis. p53 missense mutant proteins are losing their wild-type tumor suppressor activity and acquire oncogenic potential, possessing diverse transforming abilities in cell and mouse models. Whether various mutant p53s differ in their oncogenic potential has been a matter of debate. Recent discoveries are starting to uncover the existence of mutant p53 downstream programs that are common to different mutant p53 variants. In this review, we discuss a number of studies on mutant p53, underlining the advantages and disadvantages of alternative experimental approaches that have been used to describe the numerous mutant p53 gain-of-function activities. Therapeutic possibilities are also discussed, taking into account targeting either individual or multiple mutant p53 proteins in human cancer.

  18. Isolation and Preliminary Characterization of Developmental Mutants from Microsporum gypseum

    PubMed Central

    Leighton, T. J.; Stock, J. J.

    1970-01-01

    Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed. PMID:4992372

  19. Targeting Oncogenic Mutant p53 for Cancer Therapy.

    PubMed

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

    Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels.

  20. An annotated database of Arabidopsis mutants of acyl lipid metabolism

    SciTech Connect

    McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; Kim, Ryeo Jin; Yang, Weili; Shorrosh, Basil; Suh, Mi Chung; Ohlrogge, John

    2014-12-10

    Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.

  1. An annotated database of Arabidopsis mutants of acyl lipid metabolism

    DOE PAGES

    McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

    2014-12-10

    Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes ofmore » mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

  2. Registration of two allelic erect leaf mutants of sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two allelic sorghum [Sorghum bicolor (L.) Moench] erect leaf (erl) mutants were isolated from an Annotated Individually-pedigreed Mutagenized Sorghum (AIMS) mutant library developed at the Plant Stress and Germplasm Development Unit, at Lubbock, Texas. The two mutants, erl1-1 and erl1-2, were isol...

  3. Mutant models of prolonged life span.

    PubMed

    Mahler, J F

    2001-01-01

    Aging is an important biological process that affects all creatures. For humans, age-related diseases and the question of why we age and die also have tremendous social and philosophical impact. We can therefore expect that models to study mechanisms of the aging process will always attract much interest. Until recently, the mutant model approach to study molecular mechanisms of aging has been limited to lower animals such as yeast, worms, and flies. However, given the current power of genetic technology in mammals, we can expect that phenotypes of prolonged life span will increasingly be seen in mice and subject to evaluation by pathologists. A brief review of current models is presented.

  4. Pleiotropic aspartate taxis and serine taxis mutants of Escherichia coli.

    PubMed

    Reader, R W; Tso, W W; Springer, M S; Goy, M F; Adler, J

    1979-04-01

    Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.

  5. Auxin physiology of the tomato mutant diageotropical

    SciTech Connect

    Daniel, S.G.; Rayle, D.L. ); Cleland, R.E. )

    1989-11-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  6. Zebrafish Genomic Instability Mutants and Cancer Susceptibility

    PubMed Central

    Moore, Jessica L.; Rush, Lindsay M.; Breneman, Carol; Mohideen, Manzoor-Ali P. K.; Cheng, Keith C.

    2006-01-01

    Somatic loss of tumor suppressor gene function comprising the second hit of Knudson's two-hit hypothesis is important in human cancer. A genetic screen was performed in zebrafish (Danio rerio) to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic-eye assay that models this second hit. The assay, based on a visible test for loss of wild-type gene function at a single locus, golden, is representative of genomewide events. Twelve ENU-induced genomic instability (gin) mutations were isolated. Most mutations showed weak dominance in heterozygotes and all showed a stronger phenotype in homozygotes. Trans-heterozygosity for 7 of these mutations showed greatly enhanced instability. A variety of spontaneous tumors were found in heterozygous adults from all gin lines, consistent with the expectation that genomic instability (mutator) mutations can accelerate carcinogenesis. The incidence of spontaneous cancer at 30–34 months was increased 9.6-fold in heterozygotes for the mutant with the strongest phenotype, gin-10. Tumors were seen in skin, colon, kidney, liver, pancreas, ovary, testis, and neuronal tissues, with multiple tumors in some fish. The study of these mutants will add to our understanding of the mechanisms of somatic loss of gene function and how those mechanisms contribute to cancer susceptibility. PMID:16888336

  7. Too Many Mutants with Multiple Mutations

    PubMed Central

    Drake, John W.

    2007-01-01

    It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These “multiples” appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts. PMID:17687667

  8. The LORE1 insertion mutant resource.

    PubMed

    Małolepszy, Anna; Mun, Terry; Sandal, Niels; Gupta, Vikas; Dubin, Manu; Urbański, Dorian; Shah, Niraj; Bachmann, Asger; Fukai, Eigo; Hirakawa, Hideki; Tabata, Satoshi; Nadzieja, Marcin; Markmann, Katharina; Su, Junyi; Umehara, Yosuke; Soyano, Takashi; Miyahara, Akira; Sato, Shusei; Hayashi, Makoto; Stougaard, Jens; Andersen, Stig U

    2016-10-01

    Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

  9. Mutants of Arabidopsis thaliana with altered phototropism

    NASA Technical Reports Server (NTRS)

    Khurana, J. P.; Poff, K. L.

    1989-01-01

    Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and "second positive" phototropism. However, while the amplitude for "first positive" phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in "first positive" phototropism are shifted to higher fluence by a factor of 20-30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 micromole m-2 to 2700 micromoles m-2, but shows normal gravitropism. Strain JK345 shows no "first positive" phototropism, and reduced gravitropism and "second positive" phototropism. Strain JK229 shows no measurable "first positive" phototropism, but normal gravitropism and "second positive" phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. "first positive" and "second positive" phototropism contain at least one common element; and 3. "first positive" phototropism can be substantially altered without any apparent alteration of "second positive" phototropism.

  10. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, James P.

    1997-01-01

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

  11. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, J.P.

    1997-07-29

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

  12. Auxin physiology of the tomato mutant diageotropica

    NASA Technical Reports Server (NTRS)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  13. Thiostrepton-resistant mutants of Thermus thermophilus

    PubMed Central

    Cameron, Dale M.; Thompson, Jill; Gregory, Steven T.; March, Paul E.; Dahlberg, Albert E.

    2004-01-01

    Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic. PMID:15199170

  14. Effects of mutant rat dynamin on endocytosis

    PubMed Central

    1993-01-01

    Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non- coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2- terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over- expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis. PMID:8335685

  15. Too many mutants with multiple mutations.

    PubMed

    Drake, John W

    2007-01-01

    It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These "multiples" appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts.

  16. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity.

    PubMed

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A

    2017-03-07

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1(R132H) mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  17. Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants.

    PubMed

    McCammon, M T

    1996-09-01

    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn- ("ACetate Nonutilizing") mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism.

  18. Mutants of Saccharomyces Cerevisiae with Defects in Acetate Metabolism: Isolation and Characterization of Acn(-) Mutants

    PubMed Central

    McCammon, M. T.

    1996-01-01

    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn(-) (``ACetate Nonutilizing'') mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism. PMID:8878673

  19. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    SciTech Connect

    Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G; Chesler, Elissa J; Johnson, Dabney K; Goldowitz, Daniel

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  20. Forward genetic screen for auxin-deficient mutants by cytokinin.

    PubMed

    Wu, Lei; Luo, Pan; Di, Dong-Wei; Wang, Li; Wang, Ming; Lu, Cheng-Kai; Wei, Shao-Dong; Zhang, Li; Zhang, Tian-Zi; Amakorová, Petra; Strnad, Miroslav; Novák, Ondřej; Guo, Guang-Qin

    2015-07-06

    Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

  1. Hoxc13 mutant mice lack external hair.

    PubMed

    Godwin, A R; Capecchi, M R

    1998-01-01

    Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. Consistent with the expected pattern for a paralogous group 13 member, early embryonic Hoxc13 expression is found in the nails and tail. Hoxc13 is also expressed in vibrissae, in the filiform papillae of the tongue, and in hair follicles throughout the body; a pattern that apparently violates spatial colinearity. Mice carrying mutant alleles of Hoxc13 have been generated by gene targeting. Homozygotes have defects in every region in which gene expression is seen. The most striking defect is brittle hair resulting in alopecia (hairless mice). One explanation for this novel role is that Hoxc13 has been recruited for a function common to hair, nail, and filiform papilla development.

  2. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer

    PubMed Central

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed. PMID:26618142

  3. Characterization of Halobacterium halobium mutants defective in taxis.

    PubMed

    Sundberg, S A; Alam, M; Lebert, M; Spudich, J L; Oesterhelt, D; Hazelbauer, G L

    1990-05-01

    Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

  4. Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

    SciTech Connect

    Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E. )

    1991-10-01

    The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

  5. Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity.

    PubMed

    Upchurch, R G; Walker, D C; Rollins, J A; Ehrenshaft, M; Daub, M E

    1991-10-01

    We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

  6. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer.

    PubMed

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed.

  7. Isolation and characterization of Klebsiella pneumoniae unencapsulated mutants

    SciTech Connect

    Benedi, V.J.; Ciurana, B.; Tomas, J.M.

    1989-01-01

    Klebsiella pneumoniae mutants were obtained after UV irradiation and negative selection with anticapsular serum. Unencapsulation, rather than expression of a structurally altered capsule, was found in the mutants. The mutant strains showed no alterations in their outer membrane proteins and lipopolysaccharide, and a great similarity with the wild type in the properties tested (serum resistance, antimicrobial sensitivity, and lipopolysaccharide-specific bacteriophage sensitivity), with the exception of a higher cell surface hydrophobicity and resistance to bacteriophage FC3-9.

  8. Mutant Proteins--Enzymes to Hydrolyze Toxic Organophosphates.

    DTIC Science & Technology

    1987-06-15

    strains of infectious bacteria and the serine protease’ lytic protease. We also employ novel chemical modifications of ; mutant proteins to achieve...mutant RTEM -1 8-lactamase. We have previously generated and characterized mutants of RTEM -1 .- lactamase with all possible amino acid substitutions (site...denaturation than wild-type -lactamase. Uniquely among class A B- lactamases, the RTEM -1 (and RTEM -2) enzymes contain a single disulfide bond between Cys

  9. Division pattern of a round mutant of Escherichia coli.

    PubMed Central

    Cooper, S

    1997-01-01

    A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads. This implies that successive division planes of the round mutant are parallel rather than perpendicular. These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W. D. Donachie, S. Addinall, and K. Begg, Bioessays 17:569-576, 1995). PMID:9287016

  10. [Eremothecium ashbyii mutants resistant to 2,6-diaminopurine].

    PubMed

    Stepanov, A I; Beburov, M Iu; Zhdanov, V G

    1975-01-01

    3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

  11. Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

    PubMed

    Goodrich-Tanrikulu, M; Stafford, A E; Lin, J T; Makapugay, M I; Fuller, G; McKeon, T A

    1994-10-01

    New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

  12. Optimized cell transplantation using adult rag2 mutant zebrafish

    PubMed Central

    Tang, Qin; Abdelfattah, Nouran S.; Blackburn, Jessica S.; Moore, John C.; Martinez, Sarah A.; Moore, Finola E.; Lobbardi, Riadh; Tenente, Inês M.; Ignatius, Myron S.; Berman, Jason N.; Liwski, Robert S.; Houvras, Yariv; Langenau, David M.

    2014-01-01

    Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer. PMID:25042784

  13. Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

    DTIC Science & Technology

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina Politi...CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER 6...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Phenotypic changes have been observed in EGFR mutant lung cancers that become resistant to targeted

  14. Defining New Treatment Approaches for KRAS-Mutant Lung Cancer

    DTIC Science & Technology

    2014-10-01

    AWARD NUMBER: W81XWH-13-1-0225 TITLE: Defining New Treatment Approaches for KRAS- Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Eric Collisson...TITLE AND SUBTITLE Defining New Treatment Approaches for KRAS- Mutant Lung Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-13-1-0225 5c...one RAS-dependent. This Aim is underway and has verified that KRAS is indeed essential in the KRAS mutant mouse cell lines. Specific Aim 2. To

  15. Growth and development of maize that contains mutant tubulin genes

    SciTech Connect

    Susan M. Wick

    2004-07-23

    Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

  16. Identification of mutant monoclonal antibodies with increased antigen binding.

    PubMed

    Pollock, R R; French, D L; Gefter, M L; Scharff, M D

    1988-04-01

    Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.

  17. [Pigment composition and photosynthetic activity of pea chlorophyll mutants].

    PubMed

    Ladygin, V G

    2003-01-01

    Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthine; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO2 assimilation and oxygen production in the mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29-30% lower than in the control plants; in their hybrids, however, it is 1.5-2 higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that the mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus.

  18. Sulphate metabolism of selenate-resistant Schizosaccharomyces pombe mutants.

    PubMed

    Bánszky, Luca; Simonics, Tibor; Maráz, Anna

    2003-10-01

    Selenate-resistant mutants were obtained from several strains of Schizosaccharomyces pombe. The obtained mutants all belonged to the same genetic complementation group. They were low in sulphate uptake activity and in ATP sulphurylase activity. They grew on medium containing sulphite, thiosulphate, cysteine or glutathione but not methionine as the sole source of sulphur. From these results, the mutants were concluded to carry mutations in the ATP sulphurylase gene. Inability of the mutants to utilize methionine as a sulphur source is rationalized by the absence of the reverse transsulphurylation pathway in this organism; wild type strains must utilize methionine as a sulphur source after it is degraded to give rise to sulphate.

  19. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    MedlinePlus

    ... in liver cells. The urea cycle processes excess nitrogen, generated when protein is broken down by the ... the urea cycle, a reaction in which excess nitrogen compounds are incorporated into the cycle to be ...

  20. Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency

    PubMed Central

    Ehinger, Johannes K.; Piel, Sarah; Ford, Rhonan; Karlsson, Michael; Sjövall, Fredrik; Frostner, Eleonor Åsander; Morota, Saori; Taylor, Robert W.; Turnbull, Doug M.; Cornell, Clive; Moss, Steven J.; Metzsch, Carsten; Hansson, Magnus J.; Fliri, Hans; Elmér, Eskil

    2016-01-01

    Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [13C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction. PMID:27502960

  1. Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

    PubMed

    Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

    2016-01-01

    Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

  2. Photoperiod Affects the Phenotype of Mitochondrial Complex I Mutants.

    PubMed

    Pétriacq, Pierre; de Bont, Linda; Genestout, Lucie; Hao, Jingfang; Laureau, Constance; Florez-Sarasa, Igor; Rzigui, Touhami; Queval, Guillaume; Gilard, Françoise; Mauve, Caroline; Guérard, Florence; Lamothe-Sibold, Marlène; Marion, Jessica; Fresneau, Chantal; Brown, Spencer; Danon, Antoine; Krieger-Liszkay, Anja; Berthomé, Richard; Ribas-Carbo, Miquel; Tcherkez, Guillaume; Cornic, Gabriel; Pineau, Bernard; Gakière, Bertrand; De Paepe, Rosine

    2017-01-01

    Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8 Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis.

  3. Plasmidless, photosynthetically incompetent mutants of Rhodospirillum rubrum.

    PubMed Central

    Kuhl, S A; Wimer, L T; Yoch, D C

    1984-01-01

    Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation. Images PMID:6434514

  4. Mutant Huntingtin Disrupts the Nuclear Pore Complex.

    PubMed

    Grima, Jonathan C; Daigle, J Gavin; Arbez, Nicolas; Cunningham, Kathleen C; Zhang, Ke; Ochaba, Joseph; Geater, Charlene; Morozko, Eva; Stocksdale, Jennifer; Glatzer, Jenna C; Pham, Jacqueline T; Ahmed, Ishrat; Peng, Qi; Wadhwa, Harsh; Pletnikova, Olga; Troncoso, Juan C; Duan, Wenzhen; Snyder, Solomon H; Ranum, Laura P W; Thompson, Leslie M; Lloyd, Thomas E; Ross, Christopher A; Rothstein, Jeffrey D

    2017-04-05

    Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets.

  5. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant

    PubMed Central

    Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape. PMID:27610363

  6. New types of Escherichia coli recombination-deficient mutants.

    PubMed

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  7. New types of Escherichia coli recombination-deficient mutants.

    PubMed Central

    Freifelder, D

    1976-01-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency. PMID:789362

  8. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, O.E.; Pan, D.

    1994-07-19

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating. 2 figs.

  9. Structurally altered capsular polysaccharides produced by mutant bacteria

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1995-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  10. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant.

    PubMed

    Guo, Da-Long; Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape.

  11. Mutant strain of C. acetobutylicum and process for making butanol

    DOEpatents

    Jain, Mahendra K.; Beacom, Daniel; Datta, Rathin

    1993-01-01

    A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

  12. Gravitropism in roots of intermediate-starch mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Wright, J. B.; Caspar, T.

    1996-01-01

    Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

  13. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, Oliver E.; Pan, David

    1994-01-01

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating.

  14. A Mutant Hunt Using the C-Fern (Ceratopteris Richardii)

    ERIC Educational Resources Information Center

    Calie, Patrick J.

    2005-01-01

    A modification of the popular C-Fern system, the tropical fern Ceratopteris richardii is developed in which students plate out a genetically mixed set of fern spores and then select for specific mutants. This exercise can provide students with an experience in plant mutant selection and can be used as a platform to expose students to a diverse…

  15. Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

    PubMed Central

    Hurst, Sheldon; Rowedder, Holli; Michaels, Brandye; Bullock, Hannah; Jackobeck, Ryan; Abebe-Akele, Feseha; Durakovic, Umjia; Gately, Jon; Janicki, Erik

    2015-01-01

    ABSTRACT The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont. PMID

  16. Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin

    PubMed Central

    Tan, Z; Dai, W; van Erp, T G M; Overman, J; Demuro, A; Digman, M A; Hatami, A; Albay, R; Sontag, E M; Potkin, K T; Ling, S; Macciardi, F; Bunney, W E; Long, J D; Paulsen, J S; Ringman, J M; Parker, I; Glabe, C; Thompson, L M; Chiu, W; Potkin, S G

    2015-01-01

    Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT. PMID:26100538

  17. Misfolded opsin mutants display elevated β-sheet structure.

    PubMed

    Miller, Lisa M; Gragg, Megan; Kim, Tae Gyun; Park, Paul S-H

    2015-10-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.

  18. A short flagella mutant of Dunaliella sallina (Chlorophyta, Cholorophyceae).

    PubMed

    Vismara, Rosa; Verni, Franco; Barsanti, Laura; Evangelista, Valtere; Gualtieri, Paolo

    2004-01-01

    Dunaliella salina (Chlorophyta, Chlorophyceae) is a unicellular wall-less biflagellate alga. In this paper we describe a spontaneous mutant of D. salina, isolated from wild type cultures, which is characterized by very short flagella. The ultrastructure showed the basic 9 + 2 organization of wild-type flagella. Immunofluorescence localization of tubulin in this mutant confirmed the normal construction of the axoneme. Although, the mutant does not swim, still it is able to move and perform photobehavior. As shown by track reconstruction, and rotation movements, observed by means of reflection microscopy, this mutant can move, probably gliding by means of its stumpy flagella. A possible model to explain the mutant motion pattern is discussed.

  19. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    NASA Astrophysics Data System (ADS)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  20. Misfolded opsin mutants display elevated β -sheet structure

    DOE PAGES

    Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; ...

    2015-09-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate themore » aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.« less

  1. Architectural phenotypes in the transparent testa mutants of Arabidopsis thaliana

    PubMed Central

    Buer, Charles S.; Djordjevic, Michael A.

    2009-01-01

    Flavonoids are low molecular weight secondary plant metabolites with a myriad of functions. As flavonoids affect auxin transport (an important growth-controlling hormone) and are biologically active in eukaryotes, flavonoid mutants were expected to have undescribed architectural phenotypes. The Arabidopsis thaliana transparent testa (tt) mutants are compromised in the enzymatic steps or transcriptional regulators affecting flavonoid synthesis. tt mutant seedlings were grown on hard-slanted agar (a stress condition), under varying light conditions, and in soil to examine the resulting growth patterns. These tt mutants revealed a wide variety of architectural phenotypes in root and aerial tissues. Mutants with increased inflorescences, siliques, and lateral root density or reduced stature are traits that could affect plant yield or performance under certain environmental conditions. The regulatory genes affected in architectural traits may provide useful molecular targets for examination in other plants. PMID:19129166

  2. Extreme Vulnerability of IDH1 Mutant Cancers to NAD+ Depletion.

    PubMed

    Tateishi, Kensuke; Wakimoto, Hiroaki; Iafrate, A John; Tanaka, Shota; Loebel, Franziska; Lelic, Nina; Wiederschain, Dmitri; Bedel, Olivier; Deng, Gejing; Zhang, Bailin; He, Timothy; Shi, Xu; Gerszten, Robert E; Zhang, Yiyun; Yeh, Jing-Ruey J; Curry, William T; Zhao, Dan; Sundaram, Sudhandra; Nigim, Fares; Koerner, Mara V A; Ho, Quan; Fisher, David E; Roider, Elisabeth M; Kemeny, Lajos V; Samuels, Yardena; Flaherty, Keith T; Batchelor, Tracy T; Chi, Andrew S; Cahill, Daniel P

    2015-12-14

    Heterozygous mutation of IDH1 in cancers modifies IDH1 enzymatic activity, reprogramming metabolite flux and markedly elevating 2-hydroxyglutarate (2-HG). Here, we found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types. To identify other metabolic therapeutic targets, we systematically profiled metabolites in endogenous IDH1 mutant cancer cells after mutant IDH1 inhibition and discovered a profound vulnerability to depletion of the coenzyme NAD+. Mutant IDH1 lowered NAD+ levels by downregulating the NAD+ salvage pathway enzyme nicotinate phosphoribosyltransferase (Naprt1), sensitizing to NAD+ depletion via concomitant nicotinamide phosphoribosyltransferase (NAMPT) inhibition. NAD+ depletion activated the intracellular energy sensor AMPK, triggered autophagy, and resulted in cytotoxicity. Thus, we identify NAD+ depletion as a metabolic susceptibility of IDH1 mutant cancers.

  3. Human Liver Cell Trafficking Mutants: Characterization and Whole Exome Sequencing

    PubMed Central

    Yuan, Fei; Snapp, Erik L.; Novikoff, Phyllis M.; Suadicani, Sylvia O.; Spray, David C.; Potvin, Barry; Wolkoff, Allan W.; Stanley, Pamela

    2014-01-01

    The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α’’. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype. PMID:24466322

  4. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

    PubMed

    García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

    2013-12-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga.

  5. Human liver cell trafficking mutants: characterization and whole exome sequencing.

    PubMed

    Yuan, Fei; Snapp, Erik L; Novikoff, Phyllis M; Suadicani, Sylvia O; Spray, David C; Potvin, Barry; Wolkoff, Allan W; Stanley, Pamela

    2014-01-01

    The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

  6. Comparable clinical outcomes in patients with HER2-mutant and EGFR-mutant lung adenocarcinomas.

    PubMed

    Gow, Chien-Hung; Chang, Hou-Tai; Lim, Chor-Kuan; Liu, Chao-Yu; Chen, Jin-Shing; Shih, Jin-Yuan

    2017-05-01

    HER2 is a major proliferative driver in lung cancer. HER2 gene aberrations impact the prognosis of lung adenocarcinoma (ADC). A one-step reverse transcription-polymerase chain reaction was performed using RNA samples from 888 Asian lung cancer patients to detect HER2, EGFR, KRAS, ALK, and ROS1 mutations. The demographic data and treatment outcomes of HER2 mutation-positive lung ADC patients were analyzed and compared to those with HER2 mutation-negative tumors. HER2 mutation was identified in 40 (4.5%) lung ADC patients. HER2 mutations tended to occur in male patients with advanced-stage disease and never-smokers. A775_G776insYVMA (n = 22, 55%) was the most prevalent HER2 mutation, followed by P780_Y781insGSP (n = 4, 10%). For patients diagnosed with stage-IIIB/IV disease, HER2-mutant patients showed clinical outcomes comparable to EGFR-mutant patients (P = 0.721, log-rank test) and a better overall survival (OS) compared to patients lacking driver mutations in the investigated genes (P = 0.033, Breslow test). Specifically, lung ADC patients with stage-IV HER2-mutant tumors treated with chemotherapy or targeted agents, even without afatinib or anti-HER2 targeted therapy, showed similar clinical outcomes to lung ADC patients harboring EGFR exon 19 deletion or L858R mutations (P = 0.870). In addition, multivariate analysis indicated that HER2 mutation status was not a major risk factor for diminished OS in stage-IV lung cancer. In conclusion, lung ADC harboring HER2 mutations showed distinct characteristics from other driver mutations, including increased chemosensitivity with in advanced stage disease.

  7. Isolation of Mutants of Euglena gracilis With Impaired Photosynthesis 1

    PubMed Central

    Russell, George K.; Lyman, Harvard

    1968-01-01

    Four mutant strains of Euglena gracilis have been isolated after treatment of wild type cells with ultraviolet light or the chemical mutagen nitrosoguanidine. None of the mutants is capable of autotrophic growth or photosynthetic carbon dioxide fixation. The mutant strains contain normal amounts of the enzymes of the reductive pentose phosphate cycle and are qualitatively similar to the wild type in pigment composition, but are unable to carry out the Hill reaction (light induced reduction of 2,6-dichlorophenol indophenol). Isolated mutant plastids cannot photoreduce NADP with water as the electron donor but can carry out this reaction when the electron donating system is ascorbate and 2,6-dichlorophenol indophenol. Whole cells of the mutants show the light induced oxidation of cytochrome f by light reaction I but are unable to bring about cytochrome f reduction by light reaction II. The mutants appear to be blocked at or near light reaction II in the photosynthetic electron transport chain. The mutants may represent alterations of the chloroplast genome since the mutation isolation was carried out under conditions where chloroplast viability was severely impaired, but cell viability was unaffected. PMID:5700022

  8. Mouse infection and pathogenesis by Trypanosoma brucei motility mutants.

    PubMed

    Kisalu, Neville K; Langousis, Gerasimos; Bentolila, Laurent A; Ralston, Katherine S; Hill, Kent L

    2014-06-01

    The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.

  9. Temperature-sensitive rubisco mutant of Chlamydomonas. [Chlamydomonas reinhardtii

    SciTech Connect

    Chen, Z.; Spreitzer, R.J.; Chastain, C.J.

    1987-04-01

    The Chlamydomonas reinhardtii mutant 68-4PP is a temperature-sensitive mutant that lacks photosynthetic ability at 35/sup 0/C, but is able to grow photosynthetically at 25/sup 0/C. Genetic analysis indicated that 68-4PP is a chloroplast mutant that is allelic with known Rubisco large-subunit structural-gene mutants, implying that 68-4PP also resulted from a mutation in the large-subunit gene. The 68-4PP mutant has about 35% of the wild-type level of Rubisco holoenzyme and carboxylase activity when grown at 25/sup 0/C, but it has less than 10% of normal holoenzyme and carboxylase activity when grown at 35/sup 0/C. However, (/sup 35/S)-sulfate pulse labeling showed that Rubisco subunits were synthesized at normal rates at both temperatures. More significantly, the ratio of carboxylase activity in the absence and presence of oxygen at a limiting CO/sub 2/ concentration (6.6 ..mu..M) was about 2.2 for the mutant enzyme, as compared to about 3.0 for the wild-type enzyme. The decreased ratio of the mutant enzyme is maternally inherited, indicating that this reduced oxygen sensitivity results from a mutation in chloroplast DNA. The authors have recently cloned the 68-4PP Rubisco large-subunit gene, and DNA sequencing is in progress.

  10. Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

    PubMed Central

    Noel, K D; Stacey, G; Tandon, S R; Silver, L E; Brill, W J

    1982-01-01

    Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads. Images PMID:6956566

  11. Methods of producing protoporphyrin IX and bacterial mutants therefor

    SciTech Connect

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  12. A computational study of λ-lac mutants

    NASA Astrophysics Data System (ADS)

    Werner, Maria; Aurell, Erik

    2009-12-01

    We present a comprehensive, computational study of the properties of bacteriophage λ mutants designed by Atsumi and Little (2006 Proc. Natl. Acad. Sci. 103 4558-63). These phages underwent a genetic reconstruction where Cro was replaced by a dimeric form of the Lac repressor. To clarify the theoretical characteristics of these mutants, we built a detailed thermodynamic model. The mutants all have a different genetic wiring than the wild-type λ. One group lacks regulation of PRM by the lytic protein. These mutants only exhibit the lysogenic equilibrium, with no transiently active PR. The other group lacks the negative feedback from CI. In this group, we identify a handful of bi-stable mutants, although the majority only exhibit the lysogenic equilibrium. The experimental identification of functional phages differs from our predictions. From a theoretical perspective, there is no reason why only 4 out of 900 mutants should be functional. The differences between theory and experiment can be explained in two ways. Either, the view of the λ phage as a bi-stable system needs to be revised, or the mutants have in fact not undergone a modular replacement, as intended by Atsumi and Little, but constitute instead a wider systemic change.

  13. Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

    PubMed Central

    Noel, K D; Sanchez, A; Fernandez, L; Leemans, J; Cevallos, M A

    1984-01-01

    Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained. Images PMID:6325385

  14. Mutants of Myxococcus xanthus dsp defective in fibril binding.

    PubMed Central

    Chang, B Y; Dworkin, M

    1996-01-01

    The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils

  15. Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

    PubMed Central

    Gaillardin, C; Fournier, P; Sylvestre, G; Heslot, H

    1976-01-01

    Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests. PMID:1245461

  16. A dinoflagellate mutant with higher frequency of multiple fission.

    PubMed

    Lam, C M; Chong, C; Wong, J T

    2001-01-01

    The dinoflagellate Crypthecodinium cohnii Biecheler propagates by both binary and multiple fission. By a newly developed mutagenesis protocol based on using ethyl methanesulfonate and a cell size screening method, a cell cycle mutant, mf2, was isolated with giant cells which predominantly divide by multiple fission. The average cell size of the mutant mf2 is larger than the control C. cohnii. Cell cycle synchronization experiments suggest that mutant mf2, when compared with the control strain, has a prolonged G1 phase with a corresponding delay of the G2 + M phase.

  17. Third-chromosome mutagen-sensitive mutants of Drosophila melanogaster

    SciTech Connect

    Boyd, J.B.; Golino, M.D.; Shaw, K.E.S.; Osgood, C.J.; Green, M.M.

    1981-03-01

    A total of 34 third chromosomes of Drosophila melanogaster that render homozygous larvae hypersensitive to killing by chemical mutagens have been isolated. Genetic analyses have placed responsible mutations in more than eleven complementation groups. Mutants in three complementation groups are strongly sensitive to methyl methanesulfonate, those in one are sensitive to nitrogen mustard, and mutants in six groups are hypersensitive to both mutagens. Eight of the ten loci mapped fall within 15% of the genetic map that encompasses the centromere of chromosome 3. Mutants from four of the complementation groups are associated with moderate to strong meiotic effects in females. Preliminary biochemical analyses have implicated seven of these loci in DNA metabolism.

  18. Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

    PubMed

    Lalonde, R; Strazielle, C

    2011-04-15

    Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage.

  19. Brain dopamine and amino acid concentrations in Lurcher mutant mice.

    PubMed

    Reader, T A; Strazielle, C; Botez, M I; Lalonde, R

    1998-03-15

    Lurcher mutant mice are characterized by massive degeneration of the cerebellum, including Purkinje cells and granule cells, as well as for the loss of neurons from the inferior olive. Concentrations of dopamine and two of its metabolites and of several amino acid neurotransmitters were determined in the cerebellum and in other brain regions of these mutants. By comparison to wild-type mice of the same background strain, glutamate and taurine concentrations were reduced in the Lurcher cerebellum. No decrease was found for aspartate, gamma-aminobutyric acid (GABA), glycine, as well as dopamine and its metabolites. Moreover, no neurochemical alterations occurred in the brain stem, thalamus, or neostriatum of Lurcher mutants. A selective reduction of glutamate concentration was found in the hippocampus, while all amino acids measured were decreased in the entorhinal-piriform areas. These results indicate region-selective reductions of neurotransmitter concentrations in a mouse mutant with a defined cerebellar cortical pathology.

  20. A novel mutant mouse, joggle, with inherited ataxia.

    PubMed

    Chen, Ziyan; Hayasaka, Shizu; Takagishi, Yoshiko; Murata, Yoshiharu; Oda, Sen-ichi

    2006-07-01

    While establishing a new mouse strain, we discovered a novel mutant mouse that exhibited ataxia. Mating experiments showed that the mutant phenotype was due to a single autosomal recessive gene, which we have termed joggle (gene symbol: jog). The ataxia becomes apparent around postnatal day 12, when the mice first attempt to walk, and worsens thereafter. The life span of the mutant mouse is comparable to that of the wild-type mouse. After 21 days of age, the cerebellum weights of the jog/jog mice are significantly lower than those of the wild-type mice. These observations indicate that jog/jog mutant mice could be useful models for biomedical research.

  1. Endospore degradation in an oligosporogenic, crystalliferous mutant of Bacillus thuringiensis.

    PubMed

    Sierra-Martínez, Pável; Ibarra, Jorge E; de la Torre, Mayra; Olmedo, Gabriela

    2004-02-01

    We isolated a new oligosporogenic mutant from Bacillus thuringiensis var. kurstaki HD73 that retains the ability to produce insecticidal crystal inclusions. Sporulation in this mutant initiates in a manner similar to the wild-type strain, and under the electron microscope endospores are seen, but these do not reach maturity (except for 0.2% of them). At a late stage, the coat surrounding the forespore seems to lack shape and to be empty. Most mutant cells exhibit a well-formed bipyramidal crystal but are completely devoid of the forespore. The mutant has a functional SigK holoenzyme, which is required for the expression of genes involved in the formation of spore coat and cortex and for cry1A transcription from the BtII promoter. Defective maturation of spores could be due to an inadequate forespore coat or cortex structure resulting in the arrest of sporulation at late stage III or early stage IV.

  2. Overproduction of threonine by Saccharomyces cerevisiae mutants resistant to hydroxynorvaline.

    PubMed Central

    Ramos, C; Calderon, I L

    1992-01-01

    In this work, we isolated and characterized mutants that overproduce threonine from Saccharomyces cerevisiae. The mutants were selected for resistance to the threonine analog alpha-amino-beta-hydroxynorvalerate (hydroxynorvaline), and, of these, the ones able to excrete threonine to the medium were chosen. The mutant strains produce between 15 and 30 times more threonine than the wild type does, and, to a lesser degree, they also accumulate isoleucine. Genetic and biochemical studies have revealed that the threonine overproduction is, in all cases studied, associated with the presence in the strain of a HOM3 allele coding for a mutant aspartate kinase that is totally or partially insensitive to feedback inhibition by threonine. This enzyme seems, therefore, to be crucial in the regulation of threonine biosynthesis in S. cerevisiae. The results obtained suggest that this strategy could be efficiently applied to the isolation of threonine-overproducing strains of yeasts other than S. cerevisiae, even those used industrially. PMID:1622238

  3. Analysis of the aspartic acid metabolic pathway using mutant genes.

    PubMed

    Azevedo, R A

    2002-01-01

    Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants.

  4. Genetic analysis of sex chromosomal meiotic mutants in Drosophilia melanogaster.

    PubMed

    Baker, B S; Carpenter, A T

    1972-06-01

    A total of 209 ethyl methanesulfonate-treated X chromosomes were screened for meiotic mutants that either (1) increased sex or fourth chromosome nondisjunction at either meiotic division in males; (2) allowed recombination in such males; (3) increased nondisjunction of the X chromosome at either meiotic division in females; or (4) caused such females, when mated to males heterozygous for Segregation-Distorter (SD) and a sensitive homolog to alter the strength of meiotic drive in males.-Twenty male-specific meiotic mutants were found. Though the rates of nondisjunction differed, all twenty mutants were qualitatively similar in that (1) they alter the disjunction of the X chromosome from the Y chromosome; (2) among the recovered sex-chromosome exceptional progeny, there is a large excess of those derived from nullo-XY as compared to XY gametes; (3) there is a negative correlation between the frequency of sex-chromosome exceptional progeny and the frequency of males among the regular progeny. In their effects on meiosis these mutants are similar to In(1)sc(4L)sc(8R), which is deleted for the basal heterochromatin. These mutants, however, have normal phenotypes and viabilities when examined as X/0 males, and furthermore, a mapping of two of the mutants places them in the euchromatin of the X chromosome. It is suggested that these mutants are in genes whose products are involved in insuring the proper functioning of the basal pairing sites which are deleted in In(1)sc(4L)sc(8R), and in addition that there is a close connection, perhaps causal, between the disruption of normal X-Y pairing (and, therefore, disjunction) and the occurrence of meiotic drive in the male.-Eleven mutants were found which increased nondisjunction in females. These mutants were characterized as to (1) the division at which they acted; (2) their effect on recombination; (3) their dominance; (4) their effects on disjunction of all four chromosome pairs. Five female mutants caused a nonuniform

  5. Resistant mechanism study of benzalkonium chloride selected Salmonella Typhimurium mutants.

    PubMed

    Guo, Wei; Cui, Shenghui; Xu, Xiao; Wang, Haoyan

    2014-02-01

    Benzalkonium chloride is one of the invaluable biocides that is extensively used in healthcare settings as well as in the food processing industry. After exposing wild-type Salmonella Typhimurium 14028s or its AcrAB inactivation mutant to gradually increasing levels of benzalkonium chloride, resistance mutants S-41, S-150, S-AB-23, S-AB-38, and S-AB-73 were selected and these mutants also showed a 2-64-fold stable minimum inhibitory concentration (MIC) increase to chloramphenicol, ciprofloxacin, nalidixic acid, and tetracycline. In S-41 and S-150, the expression of acrB was increased 2.7- and 7.6-fold, and ΔtolC or ΔacrAB mutants of S-41 and S-150 showed the same MICs to all tested antimicrobials as the equivalent Salmonella Typhimurium 14028s mutants. However, in S-AB-23, S-AB-38, and S-AB-73, the expression of acrF was increased 96-, 230-, and 267-fold, respectively, and ΔtolC or ΔacrEF mutants of S-AB-23, S-AB-38, and S-AB-73 showed the similar MICs to all tested antimicrobials as the ΔtolC mutant of Salmonella Typhimurium 14028s. Our data showed that constitutively over-expressed AcrAB working through TolC was the main resistance mechanism in ST14028s benzalkonium chloride resistance mutants. However, after AcrAB had been inactivated, benzalkonium chloride-resistant mutants could still be selected and constitutively over-expressed, AcrEF became the dominant efflux pump working through TolC and being responsible for the increasing antimicrobial resistance. These data indicated that different mechanisms existed for acrB and acrF constitutive over-expression. Since exposure to benzalkonium chloride may lead to Salmonella mutants with a decreased susceptibility to quinolones, which is currently one of the drugs of choice for the treatment of life-threatening salmonelosis, research into the pathogenesis and epidemiology of the benzalkonium chloride resistance mutants will be of increasing importance.

  6. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

    Kerr, Emma M; Gaude, Edoardo; Turrell, Frances K; Frezza, Christian; Martins, Carla P

    2016-03-03

    The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.

  7. Thymidine kinase mutants obtained by random sequence selection.

    PubMed

    Munir, K M; French, D C; Loeb, L A

    1993-05-01

    Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

  8. Mutant huntingtin impairs immune cell migration in Huntington disease

    PubMed Central

    Kwan, Wanda; Träger, Ulrike; Davalos, Dimitrios; Chou, Austin; Bouchard, Jill; Andre, Ralph; Miller, Aaron; Weiss, Andreas; Giorgini, Flaviano; Cheah, Christine; Möller, Thomas; Stella, Nephi; Akassoglou, Katerina; Tabrizi, Sarah J.; Muchowski, Paul J.

    2012-01-01

    In Huntington disease (HD), immune cells are activated before symptoms arise; however, it is unclear how the expression of mutant huntingtin (htt) compromises the normal functions of immune cells. Here we report that primary microglia from early postnatal HD mice were profoundly impaired in their migration to chemotactic stimuli, and expression of a mutant htt fragment in microglial cell lines was sufficient to reproduce these deficits. Microglia expressing mutant htt had a retarded response to a laser-induced brain injury in vivo. Leukocyte recruitment was defective upon induction of peritonitis in HD mice at early disease stages and was normalized upon genetic deletion of mutant htt in immune cells. Migration was also strongly impaired in peripheral immune cells from pre-manifest human HD patients. Defective actin remodeling in immune cells expressing mutant htt likely contributed to their migration deficit. Our results suggest that these functional changes may contribute to immune dysfunction and neurodegeneration in HD, and may have implications for other polyglutamine expansion diseases in which mutant proteins are ubiquitously expressed. PMID:23160193

  9. Antigenic and virulence properties of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Petras, S F; Chidambaram, M; Illyes, E F; Froshauer, S; Weinstock, G M; Reese, C P

    1995-01-01

    Antigenic properties of two mutants of Pasteurella haemolytica, strains 59B0071 and 59B0072, that do not produce detectable leukotoxin were investigated. Western blot (immunoblot) analysis with a number of polyclonal sera from animals recovering from pasteurellosis revealed that both mutants secreted a variety of antigens that were also present in cultures of several wild-type strains. These antigens ranged from about 100 to 15 kDa. Mutant strain 59B0071 was found to be totally deficient in leukotoxin, as judged not only by Western blotting but also by cytotoxicity assays with bovine lymphoma (BL-3) cells or bovine polymorphonuclear cells as targets. The mutant strain 59B0071 had normal levels of a secreted sialylglycoprotease, however. When strains were tested for virulence in goat and cattle challenge experiments, a reduction in mortality and lung lesions was observed with the mutant 59B0071 in comparison with results obtained with wild-type strains. These results are consistent with an important role for leukotoxin in P. haemolytica virulence and suggest that leukotoxin-negative mutants may be useful tools in the investigation of other virulence properties involved in P. haemolytica infections. PMID:7868224

  10. Isolation of New Gravitropic Mutants under Hypergravity Conditions

    PubMed Central

    Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T.

    2016-01-01

    Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis. PMID:27746791

  11. RECOMBINATIONS OF MUTANT PHAGES OF BACILLUS MEGATHERIUM 899A

    PubMed Central

    Murphy, James S.

    1953-01-01

    A group of mutant phages stemming from the virus of B. megatherium 899a (lysogenic), growing on a sensitive B. megatherium strain (KM), have been studied with respect to their recombination reactions. All these mutants and many of their recombinations can be recognized by a characteristic plaque morphology. A similar group of phages have been isolated directly from a culture of B. megatherium 899a in this laboratory. Previous work has shown that when two different plaque mutant phages both infect essentially all the bacteria in a culture, a characteristic per cent of recombinants is produced. This percentage depends on the two recombinants used, each pair having its own value. Hershey and coworkers (2–5) have demonstrated with coli-phage T2, that the percentages of recombination found can be handled mathematically and that they demonstrate the existence of a relationship between the mutations entirely comparable to crossover percentages as used in gene locus maps in genetics. This has been found to hold true for the phages studied in the present work. Only one "linkage group" has been detected and all the mutants studied showed low percentages of recombination (0.8 to 7.6). B. megatherium 899a phage and some of its mutants have been examined with an electron microscope and no differences have been detected between the different mutant strains. PMID:13109115

  12. Biofilm formation-defective mutants in Pseudomonas putida.

    PubMed

    López-Sánchez, Aroa; Leal-Morales, Antonio; Jiménez-Díaz, Lorena; Platero, Ana I; Bardallo-Pérez, Juan; Díaz-Romero, Alberto; Acemel, Rafael D; Illán, Juan M; Jiménez-López, Julia; Govantes, Fernando

    2016-07-01

    Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida.

  13. Quantitative analysis of triple-mutant genetic interactions.

    PubMed

    Braberg, Hannes; Alexander, Richard; Shales, Michael; Xu, Jiewei; Franks-Skiba, Kathleen E; Wu, Qiuqin; Haber, James E; Krogan, Nevan J

    2014-08-01

    The quantitative analysis of genetic interactions between pairs of gene mutations has proven to be effective for characterizing cellular functions, but it can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed triple-mutant analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, which is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principal actors are deleted. TMA has also uncovered double-mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete and measures interactions for up to 30 double mutants against a library of 1,536 single mutants.

  14. Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities.

    PubMed

    Koduru, P R; Rao, M K

    1980-05-01

    Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these - 'white', 'white tipped green', 'patchy white', 'white virescent', 'white striping 1', 'white striping 2', 'white striping 4', 'fine striping', 'chlorina' and 'yellow virescent' showed monogenic recessive inheritance and the remaining three - 'yellow striping', 'yellow green' and 'light green' seedling phenotypes showed digenic recessive inheritance. The genes for (i) 'white tipped green' (wr) and 'yellow virescent' (yv) and (ii) 'patchy white' (pw) and 'white striping 1' (wst 1) showed independent assortment. Further, the genes for 'white' (w), 'white tipped green' (wr) and 'yellow virescent' (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants - 'white tipped green', 'patchy white', 'white striping 1', 'white striping 2', 'fine striping', 'chlorina', 'yellow virescent', 'yellow striping', 'yellow green' and 'light green' phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in 'white virescent' there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F1's (mutant x control green). In F1's of six of the mutants - 'white tip', 'patchy white', 'chlorina', 'yellow virescent', 'fine striping' and 'yellow striping' the quantity of chlorophyll was almost equal to the wild type. In the F1's of three of the mutants - 'white striping 1', 'white striping 2' and 'light green' an intermediate value between the mutant and wild types was observed. In 'yellow virescent' retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants - 'white tipped green, 'white virescent', 'fine striping', 'chlorina', 'yellow striping', 'yellow green' and 'light green' defective

  15. Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant

    PubMed Central

    Chen, Junmei; Thammina, Chandra; Li, Wei; Yu, Hao; Yer, Huseyin; El-Tanbouly, Rania; Marron, Manon; Katin-Grazzini, Lorenzo; Chen, Yongqin; Inguagiato, John; McAvoy, Richard J.; Guillard, Karl; Zhang, Xian; Li, Yi

    2016-01-01

    Prostrate turf varieties are desirable because of their increased low mowing tolerance, heat resistance, traffic resistance and ground coverage compared with upright varieties. Mutation breeding may provide a powerful tool to create prostrate varieties, but there are no simple, straightforward methods to screen for such mutants. Elucidation of the molecular basis of the major ‘green revolution’ traits, dwarfism and semi-dwarfism, guided us to design a simple strategy for isolating dwarf mutants of perennial ryegrass (Lolium perenne L.). We have shown that gamma-ray-mediated dominant dwarf mutants can be easily screened for at the three-leaf stage. About 10% of dwarf mutant lines also displayed a prostrate phenotype at mature stages (>10 tillers). One prostrate line, Lowboy I, has been characterized in detail. Lowboy I had significantly shorter canopy, leaf blade and internode lengths compared with wild type. Lowboy I also exhibited greater tolerance to low mowing stress than wild type. Exogenous gibberellic acid (GA) restored Lowboy I to a wild-type phenotype, indicating that the dwarf and prostrate phenotypes were both due to GA deficiency. We further showed that phenotypes of Lowboy I were dominant and stably inherited through sexual reproduction. Prostrate turfgrass mutants are difficult to screen for because the phenotype is not observed at young seedling stages, therefore our method represents a simple strategy for easily isolating prostrate mutants. Furthermore, Lowboy I may provide an outstanding germplasm for breeding novel prostrate perennial ryegrass cultivars. PMID:26955481

  16. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    PubMed Central

    Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

    2009-01-01

    Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho) mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch-once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch-once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs. PMID:20161699

  17. Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

    PubMed Central

    Gomes, Cláudia; Martínez-Puchol, Sandra; Ruiz-Roldán, Lidia; Pons, Maria J.; del Valle Mendoza, Juana; Ruiz, Joaquim

    2016-01-01

    The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments. PMID:27667026

  18. Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants.

    PubMed

    Gomes, Cláudia; Martínez-Puchol, Sandra; Ruiz-Roldán, Lidia; Pons, Maria J; Del Valle Mendoza, Juana; Ruiz, Joaquim

    2016-09-26

    The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62-65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments.

  19. Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity

    PubMed Central

    Pan, Yanchun; Daito, Takuji; Sasaki, Yo; Chung, Yong Hee; Xing, Xiaoyun; Pondugula, Santhi; Swamidass, S. Joshua; Wang, Ting; Kim, Albert H.; Yano, Hiroko

    2016-01-01

    Although epigenetic abnormalities have been described in Huntington’s disease (HD), the causal epigenetic mechanisms driving neurodegeneration in HD cortex and striatum remain undefined. Using an epigenetic pathway-targeted drug screen, we report that inhibitors of DNA methyltransferases (DNMTs), decitabine and FdCyd, block mutant huntingtin (Htt)-induced toxicity in primary cortical and striatal neurons. In addition, knockdown of DNMT3A or DNMT1 protected neurons against mutant Htt-induced toxicity, together demonstrating a requirement for DNMTs in mutant Htt-triggered neuronal death and suggesting a neurodegenerative mechanism based on DNA methylation-mediated transcriptional repression. Inhibition of DNMTs in HD model primary cortical or striatal neurons restored the expression of several key genes, including Bdnf, an important neurotrophic factor implicated in HD. Accordingly, the Bdnf promoter exhibited aberrant cytosine methylation in mutant Htt-expressing cortical neurons. In vivo, pharmacological inhibition of DNMTs in HD mouse brains restored the mRNA levels of key striatal genes known to be downregulated in HD. Thus, disturbances in DNA methylation play a critical role in mutant Htt-induced neuronal dysfunction and death, raising the possibility that epigenetic strategies targeting abnormal DNA methylation may have therapeutic utility in HD. PMID:27516062

  20. Mutant laboratory mice with abnormalities in pigmentation: annotated tables.

    PubMed

    Nakamura, Motonobu; Tobin, Desmond J; Richards-Smith, Beverly; Sundberg, John P; Paus, Ralf

    2002-01-01

    Mammalian pigment cell research has recently entered a phase of significantly increased activity due largely to the exploitation of the many mutant mouse stocks that are coming on stream. Numerous transgenic, targeted mutagenesis (so-called 'knockouts'), conditional (so-called 'gene switch') and spontaneous mutant mice develop abnormal coat color phenotypes. The number of mice that exhibit such abnormalities is increasing exponentially as genetic engineering methods become routine. Since defined abnormalities in such mutant mice provide important clues to the as yet often poorly understood functional roles of many gene products, this overview includes a corresponding, annotated table of mutant mice with pigmentation alterations. These range from early developmental defects via a large array of coat color abnormalities to a melanoma metastasis model. This overview should provide helpful pointers to investigators who are looking for mouse models to explore or to compare functional activities of genes of interest and for comparing coat color phenotypes of spontaneous or genetically engineered mouse mutants with novel ones. Secondly, this review includes a table of mouse models of specific human diseases with genetically defined pigmentation abnormalities. In summary, this annotated table should serve as a useful reference for anyone interested in the molecular controls of pigmentation.

  1. AraC regulatory protein mutants with altered effector specificity.

    PubMed

    Tang, Shuang-Yan; Fazelinia, Hossein; Cirino, Patrick C

    2008-04-16

    The AraC regulatory protein of the Escherichia coli ara operon has been engineered to activate transcription in response to D-arabinose and not in response to its native effector L-arabinose. Two different AraC mutant libraries, each with four randomized binding pocket residues, were subjected to FACS-mediated dual screening using a GFP reporter. Both libraries yielded mutants with the desired switch in effector specificity, and one mutant we describe maintains tight repression in the absence of effector. The presence of 100 mM L-arabinose does not influence the response of the reported mutants to D-arabinose, and the mutants are not induced by other sugars tested (D-xylose, D-fucose, D-lyxose). Co-expression of the FucP transporter in E. coli enabled induction by D-arabinose in the 0.1 mM range. Our results demonstrate the power of dual screening for altering AraC inducer specificity and represent steps toward the design of customized in vivo molecular reporters and gene switches for metabolic engineering.

  2. Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants.

    PubMed

    D'Argenio, David A; Calfee, M Worth; Rainey, Paul B; Pesci, Everett C

    2002-12-01

    Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few.

  3. The Tennessee Mouse Genome Consortium: Identification of ocular mutants

    SciTech Connect

    Jablonski, Monica M.; Wang, Xiaofei; Lu, Lu; Miller, Darla R; Rinchik, Eugene M; Williams, Robert; Goldowitz, Daniel

    2005-06-01

    The Tennessee Mouse Genome Consortium (TMGC) is in its fifth year of a ethylnitrosourea (ENU)-based mutagenesis screen to detect recessive mutations that affect the eye and brain. Each pedigree is tested by various phenotyping domains including the eye, neurohistology, behavior, aging, ethanol, drug, social behavior, auditory, and epilepsy domains. The utilization of a highly efficient breeding protocol and coordination of various universities across Tennessee makes it possible for mice with ENU-induced mutations to be evaluated by nine distinct phenotyping domains within this large-scale project known as the TMGC. Our goal is to create mutant lines that model human diseases and disease syndromes and to make the mutant mice available to the scientific research community. Within the eye domain, mice are screened for anterior and posterior segment abnormalities using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, eye weight, histology, and immunohistochemistry. As of January 2005, we have screened 958 pedigrees and 4800 mice, excluding those used in mapping studies. We have thus far identified seven pedigrees with primary ocular abnormalities. Six of the mutant pedigrees have retinal or subretinal aberrations, while the remaining pedigree presents with an abnormal eye size. Continued characterization of these mutant mice should in most cases lead to the identification of the mutated gene, as well as provide insight into the function of each gene. Mice from each of these pedigrees of mutant mice are available for distribution to researchers for independent study.

  4. A combinatorial strategy for treating KRAS-mutant lung cancer.

    PubMed

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T; Gainor, Justin F; Corcoran, Ryan B; Engelman, Jeffrey A; Rudin, Charles M; Rosen, Neal; Lowe, Scott W

    2016-06-30

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

  5. Mutant number distribution in an exponentially growing population

    NASA Astrophysics Data System (ADS)

    Keller, Peter; Antal, Tibor

    2015-01-01

    We present an explicit solution to a classic model of cell-population growth introduced by Luria and Delbrück (1943 Genetics 28 491-511) 70 years ago to study the emergence of mutations in bacterial populations. In this model a wild-type population is assumed to grow exponentially in a deterministic fashion. Proportional to the wild-type population size, mutants arrive randomly and initiate new sub-populations of mutants that grow stochastically according to a supercritical birth and death process. We give an exact expression for the generating function of the total number of mutants at a given wild-type population size. We present a simple expression for the probability of finding no mutants, and a recursion formula for the probability of finding a given number of mutants. In the ‘large population-small mutation’ limit we recover recent results of Kessler and Levine (2014 J. Stat. Phys. doi:10.1007/s10955-014-1143-3) for a fully stochastic version of the process.

  6. Assessment of development and chemotaxis in Dictyostelium discoideum mutants.

    PubMed

    Artemenko, Yulia; Swaney, Kristen F; Devreotes, Peter N

    2011-01-01

    Studies using the social amoeba Dictyostelium discoideum have greatly contributed to the current understanding of the signaling network that underlies chemotaxis. Since directed migration is essential for normal D. discoideum multicellular development, mutants with chemotactic impairments are likely to have abnormal developmental morphologies. We have used multicellular development as a readout in a screen of mutants to identify new potential regulators of chemotaxis. In this chapter, we describe how mutants generated by restriction enzyme-mediated integration (REMI) are analyzed, from assessment of development to detailed characterization of 3',5'-cyclic adenosine monophosphate (cAMP)-induced responses. Two complementary approaches, plating cells either clonally on a bacterial lawn or as a population on non-nutrient agar, are used to evaluate multicellular development. Once mutants with aberrant developmental phenotypes are identified, their chemotaxis toward cAMP is assessed by both small population and micropipette assays. Furthermore, mutants are tested for defects in both general and specific signaling pathways by examining the recruitment of actin-binding LimE(Δcoil) or PIP3-binding PH domains to the plasma membrane in response to cAMP stimulation.

  7. Nanoformulated cell-penetrating survivin mutant and its dual actions

    PubMed Central

    Sriramoju, Bhasker; Kanwar, Rupinder K; Kanwar, Jagat R

    2014-01-01

    In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A) against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH) cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells), and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid) nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. PMID:25045261

  8. Emergence of HA mutants during influenza virus pneumonia.

    PubMed

    Manríquez, Maria Eugenia Vázquez; Makino, Akiko; Tanaka, Motoko; Abe, Yasuhisa; Yoshida, Hiroyuki; Morioka, Ichiro; Arakawa, Soichi; Takeshima, Yasuhiro; Iwata, Kentaro; Takasaki, Jin; Manabe, Toshie; Nakaya, Takaaki; Nakamura, Shota; Iglesias, Anjarath Lorena Higuera; Rossales, Rosa Maria Rivera; Mirabal, Erika Pena; Ito, Tateki; Kitazawa, Toshio; Oka, Teruaki; Yamashita, Makoto; Kudo, Koichiro; Shinya, Kyoko

    2012-01-01

    During the influenza pandemic of 2009, the number of viral pneumonia cases showed a marked increase in comparison with seasonal influenza viruses. Mutations at amino acid 222 (D222G mutations) in the virus hemagglutinin (HA) molecule, known to alter the receptor-recognition properties of the virus, were detected in a number of the more severely-affected patients in the early phases of the pandemic. To understand the background for the emergence of the mutant amino acid D222G in human lungs, we conducted histological examinations on lung specimens of patients from Mexico who had succumbed in the pandemic. Prominent regenerative and hyperplastic changes in the alveolar type II pneumocytes, which express avian-type sialoglycan receptors in the respiratory tract of severely affected individuals, were observed in the Mexican patients. An infection model utilizing guinea pigs, which was chosen in order to best simulate the sialic acid distribution of severe pneumonia in human patients, demonstrated an increase of D222G mutants and a delay in the diminution of mutants in the lower respiratory tract in comparison to the upper respiratory tract. Our data suggests that the predominance of avian-type sialoglycan receptors in the pneumonic lungs may contribute to the emergence of viral HA mutants. This data comprehensively illustrates the mechanisms for the emergence of mutants in the clinical samples.

  9. Genetics and characterization of an open flower mutant in chickpea.

    PubMed

    Srinivasan, Samineni; Gaur, Pooran M

    2012-01-01

    The chickpea (Cicer arietinum L.) is a self-pollinated grain legume with cleistogamous flowers. A spontaneous open-flower mutant, designated OFM-3, was identified in which reproductive organs were not enclosed by the keel petals and thus remained exposed. All 10 stamens in this mutant were free, whereas these are in diadelphous (9 fused + 1 free) condition in normal chickpea flowers. A large number of pods (73%) remained unfilled (empty) in OFM-3, though its pollen fertility was as high as the standard cultivars. The open-flower trait was found to be recessive and controlled by a single gene. OFM-3 was crossed with earlier reported open-flower mutants, ICC 16341 and ICC 16129, to establish trait relationships of genes controlling open flower traits in these mutants. It was found that each of these mutants has a unique gene for open flower trait. The genes controlling open flower trait in ICC 16341, ICC 16129, and OFM-3 were designated ofl-1, ofl-2, and ofl-3, respectively. Breeding lines with open flower trait and higher percentage of filled pods have been developed from the progenies of the crosses of OFM-3 with normal-flowered lines. The open flower trait offers opportunity for exploring hybrid technology in the chickpea.

  10. Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuroi)

    SciTech Connect

    Klittich, C.J.R.; Leslie, J.F.

    1988-03-01

    Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactors that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. The authors concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.

  11. 6-Aminonicotinamide-resistant mutants of Salmonella typhimurium.

    PubMed Central

    Hughes, K T; Cookson, B T; Ladika, D; Olivera, B M; Roth, J R

    1983-01-01

    Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD. PMID:6222034

  12. A combinatorial strategy for treating KRAS mutant lung cancer

    PubMed Central

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P.; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T.; Gainor, Justin F.; Corcoran, Ryan B.; Engelman, Jeffrey A.; Rudin, Charles M.; Rosen, Neal; Lowe, Scott W.

    2016-01-01

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proven difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, an FDA-approved MEK inhibitor that acts downstream of KRAS to suppress signaling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA (shRNA) screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signaling rebound and adaptive drug resistance. As a consequence, genetic or pharmacologic inhibition of FGFR1 in combination with trametinib enhances tumor cell death in vitro and in vivo. This compensatory response shows distinct specificities – it is dominated by FGFR1 in KRAS mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patient tumors treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

  13. Mutants of Arabidopsis with altered regulation of starch degradation

    SciTech Connect

    Caspar, T.; Lin, Tsanpiao; Kakefuda, G.; Benbow, L.; Preiss, J.; Somerville, C. )

    1991-04-01

    Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes {alpha}-amylase, {beta}-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutant at a locus designated sex 1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.

  14. A yeast sir2 mutant temperature sensitive for silencing.

    PubMed

    Wang, Chia-Lin; Landry, Joseph; Sternglanz, Rolf

    2008-12-01

    A screen for Saccharomyces cerevisiae temperature-sensitive silencing mutants identified a strain with a point mutation in the SIR2 gene. The mutation changed Ser276 to Cys. This amino acid is in the highly conserved NAD(+) binding pocket of the Sir2 family of proteins. Haploid strains of either mating type carrying the mutation were severely defective at mating at 37 degrees but normal at 25 degrees . Measurements of RNA from the HMR locus demonstrated that silencing was lost rapidly upon shifting the mutant from the low to the high temperature, but it took >8 hours to reestablish silencing after a shift back to 25 degrees . Silencing at the rDNA locus was also temperature sensitive. On the other hand, telomeric silencing was totally defective at both temperatures. Enzymatic activity of the recombinant wild-type and mutant Sir2 protein was compared by three different assays. The mutant exhibited less deacetylase activity than the wild-type protein at both 37 degrees and 25 degrees . Interestingly, the mutant had much more NAD(+)-nicotinamide exchange activity than wild type, as did a mutation in the same region of the protein in the Sir2 homolog, Hst2. Thus, mutations in this region of the NAD(+) binding pocket of the protein are able to carry out cleavage of NAD(+) to nicotinamide but are defective at the subsequent deacetylation step of the reaction.

  15. Purkinje cell compartmentation in the cerebellum of the lysosomal Acid phosphatase 2 mutant mouse (nax - naked-ataxia mutant mouse).

    PubMed

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.

  16. Comparison of Non-Mutant and Mutant Waxy Genes in Rice and Maize

    PubMed Central

    Okagaki, R. J.; Wessler, S. R.

    1988-01-01

    The waxy gene, which is responsible for the synthesis of amylose in endosperm and pollen, is genetically well characterized in many grasses including maize and rice. Homology between the previously cloned maize waxy gene and the rice gene has facilitated our cloning of a 15-kb HindIII fragment that contains the entire rice gene. A comparison of the restriction maps of the maize and rice genes indicates that many restriction sites within translated exons are conserved. In addition, the rice gene encodes a 2.4-kb transcript that programs the in vitro synthesis of a 64-kD pre-protein which is efficiently precipitated with maize waxy antisera. We demonstrate that these gene products are altered in rice strains containing mutant waxy genes. Southern blot analysis of 16 rice strains, ten containing waxy mutations, reveals that the waxy gene and flanking restriction fragments are virtually identical. These results contrast dramatically with the high level of insertions and deletions associated with restriction fragment length polymorphism and spontaneous mutations among the waxy alleles of maize. PMID:2906308

  17. Changes in temperature preferences and energy homeostasis in dystroglycan mutants.

    PubMed

    Takeuchi, Ken-Ichi; Nakano, Yoshiro; Kato, Utako; Kaneda, Mizuho; Aizu, Masako; Awano, Wakae; Yonemura, Shigenobu; Kiyonaka, Shigeki; Mori, Yasuo; Yamamoto, Daisuke; Umeda, Masato

    2009-03-27

    Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

  18. Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

    PubMed

    Liu, Wan-Chun; Kohn, Jessica; Szwed, Sarah K; Pariser, Eben; Sepe, Sharon; Haripal, Bhagwattie; Oshimori, Naoki; Marsala, Martin; Miyanohara, Atsushi; Lee, Ramee

    2015-11-01

    Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

  19. Effects of population growth on the success of invading mutants.

    PubMed

    Ashcroft, Peter; Smith, Cassandra E R; Garrod, Matthew; Galla, Tobias

    2017-03-18

    Understanding if and how mutants reach fixation in populations is an important question in evolutionary biology. We study the impact of population growth has on the success of mutants. To systematically understand the effects of growth we decouple competition from reproduction; competition follows a birth-death process and is governed by an evolutionary game, while growth is determined by an externally controlled branching rate. In stochastic simulations we find non-monotonic behaviour of the fixation probability of mutants as the speed of growth is varied; the right amount of growth can lead to a higher success rate. These results are observed in both coordination and coexistence game scenarios, and we find that the 'one-third law' for coordination games can break down in the presence of growth. We also propose a simplified description in terms of stochastic differential equations to approximate the individual-based model.

  20. How Life History Can Sway the Fixation Probability of Mutants.

    PubMed

    Li, Xiang-Yi; Kurokawa, Shun; Giaimo, Stefano; Traulsen, Arne

    2016-07-01

    In this work, we study the effects of demographic structure on evolutionary dynamics when selection acts on reproduction, survival, or both. In contrast to the previously discovered pattern that the fixation probability of a neutral mutant decreases while the population becomes younger, we show that a mutant with a constant selective advantage may have a maximum or a minimum of the fixation probability in populations with an intermediate fraction of young individuals. This highlights the importance of life history and demographic structure in studying evolutionary dynamics. We also illustrate the fundamental differences between selection on reproduction and selection on survival when age structure is present. In addition, we evaluate the relative importance of size and structure of the population in determining the fixation probability of the mutant. Our work lays the foundation for also studying density- and frequency-dependent effects in populations when demographic structures cannot be neglected.

  1. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  2. Isolation and characterization of yeast monomorphic mutants of Candida albicans.

    PubMed Central

    Elorza, M V; Sentandreu, R; Ruiz-Herrera, J

    1994-01-01

    A method was devised for the isolation of yeast monomorphic (LEV) mutants of Candida albicans. By this procedure, about 20 stable yeast-like mutants were isolated after mutagenesis with ethyl methane sulfonate. The growth rate of the mutants in different carbon sources, both fermentable and not, was indistinguishable from that of the parental strain, but they were unable to grow as mycelial forms after application of any of the common effective inducers, i.e., heat shock, pH alterations, proline addition, or use of GlcNAc as the carbon source. Studies performed with one selected strain demonstrated that it had severe alterations in the chemical composition of the cell wall, mainly in the levels of chitin and glucans, and in specific mannoproteins, some of them recognizable by specific polyclonal and monoclonal antibodies. It is suggested that these structural alterations hinder the construction of a normal hyphal wall. Images PMID:8157600

  3. Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot

    PubMed Central

    Crick, Alexandra P; Babbs, Christian; Brown, Jennifer M; Morriss-Kay, Gillian M

    2003-01-01

    The pre-axial polydactylous mouse mutant Doublefoot has 6–9 digits per limb but lacks anteroposterior polarity (there is no biphalangeal digit 1). It differs from other polydactylous mutants in showing normal Shh expression, but polarizing activity (shown by mouse-chick grafting experiments) and hedgehog signalling activity (shown by expression of Ptc1) are present throughout the distal mesenchyme. The Dbf mutation has not yet been identified. Here we review current understanding of this mutant, and briefly report new results indicating (1) that limb bud expansion is concomitant with ectopic Ihh expression and with extension of the posterior high cell proliferation rate into the anterior region, and (2) that the Dbf mutation is epistatic to Shh in the limb. PMID:12587916

  4. Ferritin Mutants of Escherichia coli Are Iron Deficient and Growth Impaired, and fur Mutants are Iron Deficient

    PubMed Central

    Abdul-Tehrani, Hossein; Hudson, Aaron J.; Chang, Yung-Sheng; Timms, Andrew R.; Hawkins, Chris; Williams, John M.; Harrison, Pauline M.; Guest, John R.; Andrews, Simon C.

    1999-01-01

    Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of “reactive ferrous iron.” It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling

  5. Genetic interactions among homologous recombination mutants in Candida albicans.

    PubMed

    Bellido, Alberto; Andaluz, Encarnación; Gómez-Raja, Jonathan; Álvarez-Barrientos, Alberto; Larriba, Germán

    2015-01-01

    rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes.

  6. Metabolic flexibility of a butyrate pathway mutant of Clostridium acetobutylicum.

    PubMed

    Yoo, Minyeong; Croux, Christian; Meynial-Salles, Isabelle; Soucaille, Philippe

    2017-01-31

    Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg(-1)) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.

  7. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

    PubMed Central

    Vuimo, Tatiana A.; Surov, Stepan S.; Ovsepyan, Ruzanna A.; Korneeva, Vera A.; Vorobiev, Ivan I.; Orlova, Nadezhda A.; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V.; Ataullakhanov, Fazoil I.; Panteleev, Mikhail A.

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  8. Differential thermosensitivity in mixed syndrome cardiac sodium channel mutants.

    PubMed

    Abdelsayed, Mena; Peters, Colin H; Ruben, Peter C

    2015-09-15

    Cardiac arrhythmias are often associated with mutations in SCN5A the gene that encodes the cardiac paralogue of the voltage-gated sodium channel, NaV 1.5. The NaV 1.5 mutants R1193Q and E1784K give rise to both long QT and Brugada syndromes. Various environmental factors, including temperature, may unmask arrhythmia. We sought to determine whether temperature might be an arrhythmogenic trigger in these two mixed syndrome mutants. Whole-cell patch clamp was used to measure the biophysical properties of NaV 1.5 WT, E1784K and R1193Q mutants. Recordings were performed using Chinese hamster ovary (CHOk1) cells transiently transfected with the NaV 1.5 α subunit (WT, E1784K, or R1193Q), β1 subunit, and eGFP. The channels' voltage-dependent and kinetic properties were measured at three different temperatures: 10ºC, 22ºC, and 34ºC. The E1784K mutant is more thermosensitive than either WT or R1193Q channels. When temperature is elevated from 22°C to 34°C, there is a greater increase in late INa and use-dependent inactivation in E1784K than in WT or R1193Q. However, when temperature is lowered to 10°C, the two mutants show a decrease in channel availability. Action potential modelling using Q10 fit values, extrapolated to physiological and febrile temperatures, show a larger transmural voltage gradient in E1784K compared to R1193Q and WT with hyperthermia. The E1784K mutant is more thermosensitive than WT or R1193Q channels. This enhanced thermosensitivity may be a mechanism for arrhythmogenesis in patients with E1784K sodium channels.

  9. Purification of recombinant C-reactive protein mutants

    PubMed Central

    Thirumalai, Avinash; Singh, Sanjay K.; Hammond, David J.; Gang, Toh B.; Ngwa, Donald N.; Pathak, Asmita; Agrawal, Alok

    2017-01-01

    C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule. The mechanisms through which CRP functions in inflammatory diseases are not fully defined; however, the ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role. One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to employ site-directed mutagenesis to create mutants for experimentation. For example, CRP mutants incapable of binding to phosphocholine are generated to investigate the importance of the phosphocholine-binding property of CRP in mediating host defense. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. While the methods to purify wild-type CRP are well established, different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either calcium or phosphocholine. In this article, we report the methods used to purify pentameric recombinant wild-type and mutant CRP expressed in and secreted by mammalian cells. PMID:1460031

  10. Callus cultures of tomato mutants: I. Nutritional requirements.

    PubMed

    Ulrich, J M; Mackinney, G

    1969-01-01

    Callus from hypocotyl, stem, and fruit tissue of tomato mutants was grown on a complex pea extract medium. The genotypes responded differently to the levels of nutrients and stimulators or inhibitors in the medium. Hypocotyl callus of yellow (r) tomato required K(2) SO(4) for quick establishment and continued steady growth for several months; callus of this mutant could also grow with 0.5 % dimethyl sulfoxide in the medium, although growth was less than the control. The red ghost (r(+) gh) mutant is sensitive to a toxic component in the pea extract, and makes its best growth with the standard minerals and vitamins, but in 1/2 concentration pea extract plus 5 % coconut water. Tangerine (t), red lutescent stem (r(+) l(2) ), and r(+) gh are mutants which respond differently to thiourea: t grows about the same at all concentrations, r(+) gh grows best at low thiourea, and r(+) l(2) grows best at the specific level of 20 mg/l thiourea. The recent active t or r(+) l(1) and r(+) l(2) isolates require supplementary auxin to which the older, slow-growing isolates do not respond. However, there is variation in growth response of different isolates of the same mutant. The several red (r(+) ) cultures are similar in their slow growth, but somewhat different in responses to specific nutrients. The recent (+) isolate is one of the most active cultures, in comparison to the slow growth of t callus isolated in 1964. It is therefore concluded that growth is affected both by the specific requirements of the mutant and by the age and vigor of isolates.

  11. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  12. Isolation of a Defective Prion Mutant from Natural Scrapie

    PubMed Central

    Migliore, Sergio; Cosseddu, Gian Mario; Pirisinu, Laura; Riccardi, Geraldina; Nonno, Romolo

    2016-01-01

    It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of

  13. Repair effects of laser on mutants of filamentous fungi

    NASA Astrophysics Data System (ADS)

    Zhao, Yansheng; Xiao, Canpeng; Qian, Hailun; Su, Baoliang; Hu, Yujun; Deng, Jianhui

    1999-09-01

    The paper reports that penicillin-producing strains and lovastatin-producing strains were irradiated by UV and subsequently by laser (632.8 nm), and the reparation rate reached 297% and 264%. High-yield mutant was selected with improved potency of 24.5% and 30%, respectively; Gibberellin producing strains were treated with chemical agent LiCl, and then irradiated with 632.8 nm laser. One mutant with 189.6% increased potency was obtained. The experimental results indicated that using laser irradiation after UV or chemical agent mutation was a new useful method in breeding high-yield strains.

  14. Mutants of Arabidopsis as tools for physiology and molecular biology

    SciTech Connect

    Somerville, C.R.; Artus, N.; Browse, J.; Caspar, T.; Estelle, M.; Haughn, G.; Kunst, L.; Martinez, J.; McCourt, P.; Moffatt, B.

    1986-04-01

    The authors discuss the importance of developing a facile system for genetic analysis in higher plants which can be used to approach problems specific to plant biology in much the same way that molecular genetic approaches have been used in other classes of organisms such as yeast and Drosophila. Toward this end, they have developed methods for the isolation and analysis of mutants of Arabidopsis with specific alterations in photosynthesis, photorespiration, starch metabolism, lipid metabolism, purine metabolism, amino acid metabolism and phytohormone responses. The utility of this collection of mutants for studying problems in physiology and biochemistry is illustrated with selected examples.

  15. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  16. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    DOEpatents

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  17. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, M.; Millard, C.S.; Stols, L.

    1998-06-23

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.

  18. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2002-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  19. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  20. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    SciTech Connect

    Gibson, Susan I.

    2009-06-08

    Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affect sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation

  1. HBV genotypes prevalence, precore and basal core mutants in Morocco.

    PubMed

    Baha, Warda; Ennaji, My Mustapha; Lazar, Fatiha; Melloul, Marouane; El Fahime, Elmostafa; El Malki, Abdelouahad; Bennani, Abdelouaheb

    2012-08-01

    The study of hepatitis B virus (HBV) genomic heterogeneity has become a major issue in investigations aimed at understanding the relationship between HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection. The objective of the current study was to find out the pattern of HBV genotypes circulating in Morocco and to investigate the precore (PC) and basal core promoter (BCP) mutants' status in Moroccan chronic hepatitis B patients. Viral genotypes were determined in 221 chronic carriers using INNO-LiPA HBV assay and hemi-nested PCR. Phylogenetic analysis was performed in 70 samples, and multiplex PCR method was used to confirm some genotyping results. PC and CP mutants were determined using Inno-Lipa. All isolates were successfully genotyped. The genotype distribution was D in 90.45% of cases, A (5.9%), E (1 case), and mixed genotypes (5 A/D and 2 D/F) in 3.17% patients. HBV carried in the HBV/D samples could be assigned to D7 (63.3%), D1 (32.7%) and 2% of strains to each D4 and D5, all HBV/A belonged to A2 subgenotype and HBV/E strain could not be sub-genotyped. In 70 studied strains, HBV mutants were detected in 88.6% of cases; PC mutants were detected in (40%) of patients and 21.5% present a mixture of wild type and G1896A mutation. BCP mutants were observed in 65.7% of cases, 22.9% were found to have the T1762/1764A double mutation, 18.6% had A1762/1764T mutation and 22.9% of patients showed the A1762T/G1764A double mutation with either A1762T/G1764T mutation. Co-infection by PC and BCP mutants was detected in 52.9% of cases. Movement from place to place most likely shapes the observed genotype distribution and consequent prevalence of genotypes other than A2 or D7 in this population. High circulation of PC and BCP mutants is common in chronic hepatitis B infection in Morocco.

  2. Analysis of l-glycerol-3-phosphate dehydrogenase mutants in Drosophila melanogaster: complementation for intracellular degradation of the mutant polypeptide.

    PubMed

    Bewley, G C; DeZurik, J M; Pagelson, G

    1980-01-01

    Null and low activity alleles at the genetic locus coding for L-Glycerol-3-phosphate dehydrogenase (alpha-GPDH, NAD+ oxidoreductase, E.C. 1.1.1.8) in Drosophila melanogaster have been analyzed by a combination of rocket immunoelectrophoresis, interallelic complementation, and two-dimensional gel electrophoresis. In addition to proving information on the molecular weight, charged state, and steady state level of CRM in each of these mutants, it is suggested that each mutation has resulted in a genetic lesion within the structural element, Gpdh+. CRM levels appear to be the result of differential sensitivity to the normal intracellular degradative process and the CRM- mutants represent "hypersensitive" alleles, such that the mutant polypeptide does not accumulate in the intracellular environment.

  3. Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis.

    PubMed

    Dalbadie-McFarland, G; Neitzel, J J; Richards, J H

    1986-01-28

    We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.

  4. Rest Mutant zebrafish swim erratically and display atypical spatial preferences

    PubMed Central

    Moravec, Cara E.; Li, Edward; Maaswinkel, Hans; Kritzer, Mary F.; Weng, Wei; Sirotkin, Howard I.

    2015-01-01

    The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest. PMID:25712696

  5. Wheat ABA-insensitive mutants result in reduced grain dormancy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes the isolation of wheat mutants in the hard red spring Scarlet resulting in reduced sensitivity to the plant hormone abscisic acid (ABA) during seed germination. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature seeds. Wheat sensitivity t...

  6. 'Green revolution' genes encode mutant gibberellin response modulators.

    PubMed

    Peng, J; Richards, D E; Hartley, N M; Murphy, G P; Devos, K M; Flintham, J E; Beales, J; Fish, L J; Worland, A J; Pelica, F; Sudhakar, D; Christou, P; Snape, J W; Gale, M D; Harberd, N P

    1999-07-15

    World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

  7. Rest mutant zebrafish swim erratically and display atypical spatial preferences.

    PubMed

    Moravec, Cara E; Li, Edward; Maaswinkel, Hans; Kritzer, Mary F; Weng, Wei; Sirotkin, Howard I

    2015-05-01

    The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest.

  8. Computer modelling of dynamics of Ser92X deoxymyoglobin mutants.

    PubMed

    Nowak, W

    1998-01-01

    The hydrogen bond between His93 and Ser92, recently discovered in crystal structures of myoglobins (Mbs), may contribute to the oxygen storage capacity of the heme proteins through a stabilization of the proximal ligand. The possible influence of this H-bond on the geometry of the heme proximal side and ligand binding properties of Mb were computationally studied using model proteins with point mutations affecting this bond. The results of the computer modelling of Ser92X (X = Ala, Ile, Thr, Val) mutants of human (H) and sperm whale (SW) Mbs are presented. The OPLS-AMBER-CHARMM forcefield was used in the calculations. Several 10-50 ps molecular dynamics simulations (300 K, in vacuo) were performed. Our results show that the Ser92X mutants are stable molecules. In the wild types and Ser92Thr mutants, the H-bond studied is observed only for a relatively short period of time. It is expected that in both HMb and SW Mb molecules the impact of the proximal histidine interaction with the Ser92(F7) residue on the iron reactivity is rather low. However, the limited torsional flexibility of the proximal histidine imidazole ring was found in hydrogen bonding mutants. This effect may be attributed to the specific long range electrostatic interactions.

  9. Modeling dynamics of mutants in heterogeneous stem cell niche

    NASA Astrophysics Data System (ADS)

    Shahriyari, L.; Mahdipour-Shirayeh, A.

    2017-02-01

    Studying the stem cell (SC) niche architecture is a crucial step for investigating the process of oncogenesis and obtaining an effective stem cell therapy for various cancers. Recently, it has been observed that there are two groups of SCs in the SC niche collaborating with each other to maintain tissue homeostasis: border stem cells (BSCs), which are responsible in controlling the number of non-stem cells as well as stem cells, and central stem cells (CeSCs), which regulate the SC niche. Here, we develop a bi-compartmental stochastic model for the SC niche to study the spread of mutants within the niche. The analytic calculations and numeric simulations, which are in perfect agreement, reveal that in order to delay the spread of mutants in the SC niche, a small but non-zero number of SC proliferations must occur in the CeSC compartment. Moreover, the migration of BSCs to CeSCs delays the spread of mutants. Furthermore, the fixation probability of mutants in the SC niche is independent of types of SC division as long as all SCs do not divide fully asymmetrically. Additionally, the progeny of CeSCs have a much higher chance than the progeny of BSCs to take over the entire niche.

  10. Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma.

    PubMed

    Gilbert-Ross, Melissa; Konen, Jessica; Koo, Junghui; Shupe, John; Robinson, Brian S; Wiles, Walter Guy; Huang, Chunzi; Martin, W David; Behera, Madhusmita; Smith, Geoffrey H; Hill, Charles E; Rossi, Michael R; Sica, Gabriel L; Rupji, Manali; Chen, Zhengjia; Kowalski, Jeanne; Kasinski, Andrea L; Ramalingam, Suresh S; Fu, Haian; Khuri, Fadlo R; Zhou, Wei; Marcus, Adam I

    2017-03-09

    Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre-induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

  11. Modelling the Evolution and Spread of HIV Immune Escape Mutants

    PubMed Central

    Fryer, Helen R.; Frater, John; Duda, Anna; Roberts, Mick G.; Phillips, Rodney E.; McLean, Angela R.

    2010-01-01

    During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level. PMID:21124991

  12. Characterization of xylitol-utilizing mutants of Erwinia uredovora.

    PubMed Central

    Doten, R C; Mortlock, R P

    1985-01-01

    Of the four pentitols ribitol, xylitol, D-arabitol, and L-arabitol, Erwinia uredovora was able to utilize only D-arabitol as a carbon and energy source. Although attempts to isolate ribitol- or L-arabitol-utilizing mutants were unsuccessful, mutants able to grow on xylitol were isolated at a frequency of 9 X 10(-8). Xylitol-positive mutants constitutively synthesized both a novel NAD-dependent xylitol-4-dehydrogenase, which oxidized xylitol to L-xylulose, and an L-xylulokinase. The xylitol dehydrogenase had a Km for xylitol of 48 mM and showed best activity with xylitol and D-threitol as substrates. However, D-threitol was not a growth substrate for E. uredovora, and its presence did not induce either dehydrogenase or kinase activity. Attempts to determine the origin of the xylitol catabolic enzymes were unsuccessful; neither enzyme was induced on any growth substrate or in the presence of any polyol tested. Analysis of xylitol-negative mutants isolated after Tn5 mutagenesis suggested that the xylitol dehydrogenase and the L-xylulokinase structural genes were components of two separate operons but were under common regulatory control. Images PMID:2981816

  13. Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

    PubMed Central

    Konen, Jessica; Koo, Junghui; Robinson, Brian S.; Wiles, Walter Guy; Huang, Chunzi; Martin, W. David; Behera, Madhusmita; Smith, Geoffrey H.; Hill, Charles E.; Rossi, Michael R.; Sica, Gabriel L.; Rupji, Manali; Chen, Zhengjia; Kowalski, Jeanne; Kasinski, Andrea L.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Marcus, Adam I.

    2017-01-01

    Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors. PMID:28289710

  14. Insulator dysfunction and oncogene activation in IDH mutant gliomas.

    PubMed

    Flavahan, William A; Drier, Yotam; Liau, Brian B; Gillespie, Shawn M; Venteicher, Andrew S; Stemmer-Rachamimov, Anat O; Suvà, Mario L; Bernstein, Bradley E

    2016-01-07

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

  15. IGFBP2 expression predicts IDH-mutant glioma patient survival.

    PubMed

    Huang, Lin Eric; Cohen, Adam L; Colman, Howard; Jensen, Randy L; Fults, Daniel W; Couldwell, William T

    2017-01-03

    Mutations of the isocitrate dehydrogenase (IDH) 1 and 2 genes occur in ~80% of lower-grade (WHO grade II and grade III) gliomas. Mutant IDH produces (R)-2-hydroxyglutarate, which induces DNA hypermethylation and presumably drives tumorigenesis. Interestingly, IDH mutations are associated with improved survival in glioma patients, but the underlying mechanism for the difference in survival remains unclear. Through comparative analyses of 286 cases of IDH-wildtype and IDH-mutant lower-grade glioma from a TCGA data set, we report that IDH-mutant gliomas have increased expression of tumor-suppressor genes (NF1, PTEN, and PIK3R1) and decreased expression of oncogenes(AKT2, ARAF, ERBB2, FGFR3, and PDGFRB) and glioma progression genes (FOXM1, IGFBP2, and WWTR1) compared with IDH-wildtype gliomas. Furthermore, each of these genes is prognostic in overall gliomas; however, within the IDH-mutant group, none remains prognostic except IGFBP2 (encodinginsulin-like growth factor binding protein 2). Through validation in an independent cohort, we show that patients with low IGFBP2 expressiondisplay a clear advantage in overall and disease-free survival, whereas those with high IGFBP2 expressionhave worse median survival than IDH-wildtype patients. These observations hold true across different histological and molecular subtypes of lower-grade glioma. We propose therefore that an unexpected biological consequence of IDH mutations in glioma is to ameliorate patient survival by promoting tumor-suppressor signaling while inhibiting that of oncogenes, particularly IGFBP2.

  16. Searching and Mining Visually Observed Phenotypes of Maize Mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are thousands of maize mutants, which are invaluable resources for plant research. Geneticists use them to study underlying mechanisms of biochemistry, cell biology, cell development, and cell physiology. To streamline the understanding of such complex processes, researchers need the most curr...

  17. Immunochemical Characterization of Plaque Mutants of Simian Virus 40

    PubMed Central

    Ozer, H. L.; Takemoto, K. K.; Kirschstein, R. L.; Axelrod, D.

    1969-01-01

    Analysis of large and small plaque mutants of simian virus 40 using antisera prepared against each has revealed quantitative and possibly qualitative antigenic differences for each plaque type. A sensitive micro radioisotope precipitation test permitted evaluation of immunochemical similarities and differences of capsid antigens by inhibition of precipitation. PMID:4306300

  18. Genetic characterization of glossy-leafed mutant broccoli lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glossy mutants of Brassica oleracea L. have reduced or altered epicuticular wax on the surface of their leaves as compared to wild-type plants, conveying a shiny green appearance. Mutations conferring glossiness are common and have been found in most B. oleracea crop varieties, including cauliflower...

  19. Mutations and Misconceptions: The Isolation and Study of Mutant Bacteria.

    ERIC Educational Resources Information Center

    Corner, Thomas R.

    1992-01-01

    Describes simple, inexpensive activities for teaching students about mutants and mutations in bacteria. Explains how to isolate bacteria from soil and leaves and how to grow bacteria on agar or in broth. Describes how to construct a gradient plate for finding the minimum inhibitory concentration of a substance and how to use this set up to find…

  20. Characterization of Caenorhabditis Elegans Lectin-Binding Mutants

    PubMed Central

    Link, C. D.; Silverman, M. A.; Breen, M.; Watt, K. E.; Dames, S. A.

    1992-01-01

    We have identified 45 mutants of Caenorhabditis elegans that show ectopic surface binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA). These mutations are all recessive and define six genes: srf-2, srf-3, srf-4, srf-5, srf-8 and srf-9. Mutations in these genes fall into two phenotypic classes: srf-2, -3, -5 mutants are grossly wild-type, except for their lectin-binding phenotype; srf-4, -8, -9 mutants have a suite of defects, including uncoordinated movement, abnormal egg laying, and defective copulatory bursae morphogenesis. Characterization of these pleiotropic mutants at the cellular level reveals defects in the migration of the gonadal distal tip cell and in axon morphology. Unexpectedly, the pleiotropic mutations also interact with mutations in the lin-12 gene, which encodes a putative cell surface receptor involved in the control of cell fate. We propose that the underlying defect in the pleiotropic mutations may be in the general processing or secretion of extracellular proteins. PMID:1516818

  1. Some Experiments with Respiratory Deficient Mutants of Yeast (Saccharomyces cerevisiae)

    ERIC Educational Resources Information Center

    Freeland, P. W.

    1978-01-01

    Methods are described for the induction and identification of respiratory deficient mutants in yeast. Practical schemes are given to enable students to obtain dose-response information for physical and chemical mutagens such as heat, ultraviolet light, or acriflavine. A simple test for environmental mutagens is described. (Author/MA)

  2. alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti.

    PubMed Central

    Duncan, M J; Fraenkel, D G

    1979-01-01

    A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth. PMID:762018

  3. Cuticle surface proteins of wild type and mutant Caenorhabditis elegans.

    PubMed

    Blaxter, M L

    1993-03-25

    The molecular components of the surface of the free-living nematode Caenorhabditis elegans have been identified by surface-specific radioiodination. Four compartments were defined by fractionation of labeled wild type (N2 strain) adult hermaphrodites. Organic solvents extracted cuticular lipids. Homogenization in detergents released a single, non-collagenous, hydrophobic protein. This is not glycosylated and is a heterodimer of 6.5- and 12-kDa subunits. The third compartment, proteins solubilized by reducing agents, included both the cuticular collagens and the heterodimer. Residual material corresponds to the cuticlin fraction. Larval stages showed a similar pattern, except that the dauer larva had an additional 37-kDa detergent-soluble protein. Other species of rhabditid nematodes displayed similar profiles, and comparison with parasitic species suggests that this simple pattern may be primitive in the Nematoda. A C. elegans strain mutant in cuticular collagen (rol-6) had a pattern identical to that of wild type, but another morphological mutant (dpy-3) [corrected] and several mutants that differ in surface reactivity to antibody and lectins (srf mutants) also had striking differences in surface labeling patterns.

  4. Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases.

    PubMed

    Soya, Naoto; Fang, Ying; Palcic, Monica M; Klassen, John S

    2011-05-01

    The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing α-(1 → 3)-N-acetylgalactosaminyltransferase (GTA) and α-(1 → 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.

  5. Molecular mapping and characterization of the silkworm apodal mutant

    PubMed Central

    Chen, Peng; Tong, Xiao-Ling; Fu, Ming-Yue; Hu, Hai; Song, Jiang-Bo; He, Song-Zhen; Gai, Ting-Ting; Dai, Fang-Yin; Lu, Cheng

    2016-01-01

    The morphological diversity of insects is important for their survival; in essence, it results from the differential expression of genes during development of the insect body. The silkworm apodal (ap) mutant has degraded thoracic legs making crawling and eating difficult and the female is sterile, which is an ideal subject for studying the molecular mechanisms of morphogenesis. Here, we confirmed that the infertility of ap female moths is a result of the degradation of the bursa copulatrix. Positional cloning of ap locus and expression analyses reveal that the Bombyx mori sister of odd and bowl (Bmsob) gene is a strong candidate for the ap mutant. The expression of Bmsob is down-regulated, while the corresponding Hox genes are up-regulated in the ap mutant compared to the wild type. Analyses with the dual luciferase assay present a declined activity of the Bmsob promoter in the ap mutant. Furthermore, we demonstrate that Bmsob can inhibit Hox gene expression directly and by suppressing the expression of other genes, including the BmDsp gene. The results of this study are an important contribution to our understanding of the diversification of insect body plan. PMID:26738847

  6. Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

    PubMed

    Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

    2012-07-15

    Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia.

  7. Two-Pore Channels: Lessons from Mutant Mouse Models

    PubMed Central

    Ruas, Margarida; Galione, Antony; Parrington, John

    2016-01-01

    Recent interest in two-pore channels (TPCs) has resulted in a variety of studies dealing with the functional role and mechanism of action of these endo-lysosomal proteins in diverse physiological processes. With the availability of mouse lines harbouring mutant alleles for Tpcnl and/or Tpcn2 genes, several studies have made use of them to validate, consolidate and discover new roles for these channels not only at the cellular level but, importantly, also at the level of the whole organism. The different mutant mouse lines that have been used were derived from distinct genetic manipulation strategies, with the aim of knocking out expression of TPC proteins. However, the expression of different residual TPC sequences predicted to occur in these mutant mouse lines, together with the varied degree to which the effects on Tpcn expression have been studied, makes it important to assess the true knockout status of some of the lines. In this review we summarize these Tpcn mutant mouse lines with regard to their predicted effect on Tpcn expression and the extent to which they have been characterized. Additionally, we discuss how results derived from studies using these Tpcn mutant mouse lines have consolidated previously proposed roles for TPCs, such as mediators of NAADP signalling, endo-lysosomal functions, and pancreatic β cell physiology. We will also review how they have been instrumental in the assignment of new physiological roles for these cation channels in processes such as membrane electrical excitability, neoangiogenesis, viral infection and brown adipose tissue and heart function, revealing, in some cases, a specific contribution of a particular TPC isoform. PMID:27330869

  8. Identification and Characterization of Aspergillus Nidulans Mutants Defective in Cytokinesis

    PubMed Central

    Harris, S. D.; Morrell, J. L.; Hamer, J. E.

    1994-01-01

    Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokinesis mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis. PMID:8150280

  9. Selection and phenotypic characterization of nonhemagglutinating mutants of Porphyromonas gingivalis.

    PubMed Central

    Chandad, F; Mayrand, D; Grenier, D; Hinode, D; Mouton, C

    1996-01-01

    To further investigate the relationship between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis, three spontaneous mutants of the type strain ATCC 33277 were selected by a hemadsorption procedure. They were characterized for hemagglutination, trypsin-like and lectin-binding activities, and hydrophobicity and for the presence of fimbriae. The presence of the 42-kDa (the fimbrilin subunit) and the 43- and 49-kDa (the HA-Ag2 components) polypeptides was investigated by immunoblotting using polyclonal and monoclonal antibodies directed to fimbriae and to the hemagglutinating adhesin HA-Ag2. Cells from two of the three mutants (M1 and M2) exhibited no or little hemagglutination activity and very low trypsin-like activity and did not show the 43- and 49-kDa polypeptides. Abnormal fimbriation in M1 was deduced from the following observations of cells grown for 18 h: absence of the 42-kDa polypeptide and of a 14-kDa polypeptide and no fimbriae visible on electron micrographs. While the cells of mutant M2, irrespective of the age of the culture, were found to lack the 43- and 49-kDa polypeptides and hemagglutination activity, the supernatants of cultures grown for 72 h had high hemagglutination and trypsin-like activities and revealed the presence of the 42-, 43-, and 49-kDa polypeptides. This suggests that M2 may be missing some molecules which anchor the components to the cell surface. Mutant M3 showed levels of activities similar to those of the parental strain but lacked the 43-kDa polypeptide. Other pleiotropic effects observed for the mutants included loss of dark pigmentation and lower hydrophobicity. The data from this study fuel an emerging consensus whereby fimbriation, hemagglutination, and proteolytic activities, as well as other functions in P. gingivalis, are intricate. PMID:8641806

  10. Impaired stretch modulation in potentially lethal cardiac sodium channel mutants.

    PubMed

    Banderali, Umberto; Juranka, Peter F; Clark, Robert B; Giles, Wayne R; Morris, Catherine E

    2010-01-01

    The presence of two slowly inactivating mutants of the cardiac sodium channel (hNa(V)1.5), R1623Q and R1626P, associate with sporadic Long-QT3 (LQT3) syndrome, and may contribute to ventricular tachyarrhythmias and/or lethal ventricular disturbances. Cardiac mechanoelectric feedback is considered a factor in such sporadic arrhythmias. Since stretch and shear forces modulate hNa(V)1.5 gating, detailed electrophysiological study of LQT-Na(V)1.5 mutant channel alpha subunit(s) might provide insights. We compared recombinant R1623Q and WT currents in control vs. stretched membrane of cell-attached patches of Xenopus oocytes. Macroscopic current was monitored before, during, and after stretch induced by pipette suction. In either mutant Na(+) channel, peak current at small depolarizations could be more than doubled by stretch. As in WT, R1623Q showed reversible and stretch intensity dependent acceleration of current onset and decay at all voltages, with kinetic coupling between these two processes retained during stretch. These two Na(V)1.5 channel alpha subunits differed in the absolute extent of kinetic acceleration for a given stretch intensity; over a range of intensities, R1623Q inactivation speed increased significantly less than did WT. The LQT3 mutant R1626P also retained its kinetic coupling during stretch. Whereas WT stretch-difference currents (I(Na)(V,t) without stretch minus I(Na)(V,t) with stretch) were mostly inhibitory (equivalent to outward current), they were substantially (R1623Q) or entirely (R1626P) excitatory for the LQT3 mutants. If stretch-modulated Na(V)1.5 current (i.e., brief excitation followed by accelerated current decay) routinely contributes to cardiac mechanoelectric feedback, then during hemodynamic load variations, the abnormal stretch-modulated components of R1623Q and R1626P current could be pro-arrhythmic.

  11. Towards an informative mutant phenotype for every bacterial gene

    DOE PAGES

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; ...

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

  12. Towards an informative mutant phenotype for every bacterial gene

    SciTech Connect

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjen; Leon, Dacia; Arkin, Adam P.; Skerker, Jeffrey M.

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.

  13. Arabidopsis Mutant bik1 Exhibits Strong Resistance to Plasmodiophora brassicae

    PubMed Central

    Chen, Tao; Bi, Kai; He, Zhangchao; Gao, Zhixiao; Zhao, Ying; Fu, Yanping; Cheng, Jiasen; Xie, Jiatao; Jiang, Daohong

    2016-01-01

    Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1, and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2, and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40–50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 [non-expresser of PR genes that regulate systemic acquired resistance (SAR)] mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms. PMID:27679580

  14. Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport.

    PubMed Central

    Bryan, L E; Kwan, S

    1981-01-01

    Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells. PMID:6791588

  15. Comparative stability of dihydrofolate reductase mutants in vitro and in vivo.

    PubMed

    Leontiev, V V; Uversky, V N; Gudkov, A T

    1993-01-01

    Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.

  16. Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Prakash, Louise; Prakash, Satya

    1977-01-01

    We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev ) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups. PMID:195865

  17. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

  18. Pharmacodynamic assessment based on mutant prevention concentrations of fluoroquinolones to prevent the emergence of resistant mutants of Streptococcus pneumoniae.

    PubMed

    Homma, Tomoyuki; Hori, Toshihiko; Sugimori, Giichi; Yamano, Yoshinori

    2007-11-01

    The objective of this study was to investigate the relationship between pharmacokinetic and pharmacodynamic parameters, on the basis of the mutant prevention concentration (MPC) concept, and the emergence of resistant mutants of Streptococcus pneumoniae to fluoroquinolone antibacterials. Some clinical isolates with various MIC and MPC values of moxifloxacin and levofloxacin were exposed under conditions simulating the time-concentration curves observed when moxifloxacin (400 or 80 mg, once a day) or levofloxacin (200 mg, twice a day) was orally administered by using an in vitro pharmacodynamic model. The decrease in susceptibility was evaluated by altering the population analysis profiles after moxifloxacin or levofloxacin treatment for 72 h. When the area under the concentration-time curve from 0 to 24 h (AUC(0-24))/MPC and peak concentration (C(max))/MPC were above 13.41 and 1.20, respectively, complete eradication occurred and no decrease in susceptibility was observed. On the other hand, when AUC(0-24)/MPC and C(max)/MPC were below 0.84 and 0.08, respectively, the susceptibility decreased. However, the time inside the mutant selective window and the time above the MPC did not show any correlation with the decrease in susceptibility. These results suggest that AUC(0-24)/MPC and C(max)/MPC are important parameters for predicting the emergence of resistant mutants and that higher values indicate greater effectiveness.

  19. Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

    PubMed

    Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph).

  20. Analysis of Mycobacterium avium subsp. paratuberculosis Mutant Libraries Reveals Loci-dependent Transposition Biases and Strategies to Novel Mutant Discovery.

    PubMed

    Rathnaiah, Govardhan; Bannantine, John P; Bayles, Darrell O; Zinniel, Denise K; Stabel, Judith R; Gröhn, Yrjö T; Barletta, Raúl G

    2016-02-16

    Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne's disease, is one of the most important bacterial pathogens in ruminants. A thorough understanding of MAP pathogenesis is needed to develop new vaccines and diagnostic tests. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis compared the insertion sites for the mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99% of all MAP genes. Analysis of the MAP K-10 genome indicated that 710 of all open reading frames do not possess IS1096 recognition sites, while only 37 do not have the recognition site for MycoMarT7. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096 derived transposons. Analysis of MycomarT7 and Tn5367 mutants showed that Tn5367 has a predilection to insert within intergenic regions, suggesting that MycoMarT7 is more adequate to generate a comprehensive library. However, we uncovered the novel finding that both transposons have loci-dependent biases with Tn5367 being the most skewed. These loci-dependent transposition biases lead to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Herein, we also demonstrated a useful platform for gene discovery and analysis by isolating three novel mutants for each transposon.

  1. Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase

    PubMed Central

    Franke, Kamila B.; Bukau, Bernd; Mogk, Axel

    2017-01-01

    The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings. PMID:28275610

  2. Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase.

    PubMed

    Franke, Kamila B; Bukau, Bernd; Mogk, Axel

    2017-01-01

    The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings.

  3. Increased Anxiety in Offspring Reared by Circadian Clock Mutant Mice

    PubMed Central

    Koizumi, Hiroko; Kurabayashi, Nobuhiro; Watanabe, Yuto; Sanada, Kamon

    2013-01-01

    The maternal care that offspring receive from their mothers early in life influences the offspring’s development of emotional behavior in adulthood. Here we found that offspring reared by circadian clock-impaired mice show elevated anxiety-related behavior. Clock mutant mice harboring a mutation in Clock, a key component of the molecular circadian clock, display altered daily patterns of nursing behavior that is fragmented during the light period, instead of long bouts of nursing behavior in wild-type mice. Adult wild-type offspring fostered by Clock mutant mice exhibit increased anxiety-related behavior. This is coupled with reduced levels of brain serotonin at postnatal day 14, whose homeostasis during the early postnatal period is critical for normal emotional behavior in adulthood. Together, disruption of the circadian clock in mothers has an adverse impact on establishing normal anxiety levels in offspring, which may increase their risk of developing anxiety disorders. PMID:23776596

  4. Selection screen for novel photorespiratory mutants of barley

    SciTech Connect

    Hall, N.P.; Kendall, A.C.; Turner, J.C.; Wallsgrove R.M.; Keys, A.J.

    1987-04-01

    Selfed seed from a catalase mutant of barley (RPr 79/4) was treated with the mutagen N-nitroso-N-methyl urea, which is known to induce mutations in both chloroplast and nuclear genomes. Treated seed was grown to maturity at 0.8% CO/sub 2/, until the second leaf emerged, then plants were transferred to air under high light intensity for 5 days. Those plants which did not show the characteristic phenotype of the catalase mutant, silvering of the leaves, were selected and maintained in high CO/sub 2/. These should include plants with mutations upstream catalase (i.e. non-producers of H/sub 2/O/sub 2/); for example, those affecting glycollate oxidase, phosphoglycollate phosphatase and RuBP oxygenase, in addition to catalase revertants. Preliminary experiments showed a high (7%) frequency of pigment mutations and one plant was selected for further study.

  5. Nucleophosmin leukemogenic mutant activates Wnt signaling during zebrafish development

    PubMed Central

    Barbieri, Elisa; Deflorian, Gianluca; Pezzimenti, Federica; Valli, Debora; Saia, Marco; Meani, Natalia

    2016-01-01

    Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein with both oncogenic and tumor suppressor functions. Mutations of the NPM1 gene are the most frequent genetic alterations in acute myeloid leukemia (AML) and result in the expression of a mutant protein with aberrant cytoplasmic localization, NPMc+. Although NPMc+ causes myeloproliferation and AML in animal models, its mechanism of action remains largely unknown. Here we report that NPMc+ activates canonical Wnt signaling during the early phases of zebrafish development and determines a Wnt-dependent increase in the number of progenitor cells during primitive hematopoiesis. Coherently, the canonical Wnt pathway is active in AML blasts bearing NPMc+ and depletion of the mutant protein in the patient derived OCI-AML3 cell line leads to a decrease in the levels of active β-catenin and of Wnt target genes. Our results reveal a novel function of NPMc+ and provide insight into the molecular pathogenesis of AML bearing NPM1 mutations. PMID:27486814

  6. Dedifferentiation of Neurons Precedes Tumor Formation in lola Mutants

    PubMed Central

    Southall, Tony D.; Davidson, Catherine M.; Miller, Claire; Carr, Adrian; Brand, Andrea H.

    2014-01-01

    Summary The ability to reprogram differentiated cells into a pluripotent state has revealed that the differentiated state is plastic and reversible. It is evident, therefore, that mechanisms must be in place to maintain cells in a differentiated state. Transcription factors that specify neuronal characteristics have been well studied, but less is known about the mechanisms that prevent neurons from dedifferentiating to a multipotent, stem cell-like state. Here, we identify Lola as a transcription factor that is required to maintain neurons in a differentiated state. We show that Lola represses neural stem cell genes and cell-cycle genes in postmitotic neurons. In lola mutants, neurons dedifferentiate, turn on neural stem cell genes, and begin to divide, forming tumors. Thus, neurons rather than stem cells or intermediate progenitors are the tumor-initiating cells in lola mutants. PMID:24631403

  7. Characterizing visible and invisible cell wall mutant phenotypes.

    PubMed

    Carpita, Nicholas C; McCann, Maureen C

    2015-07-01

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

  8. Improved solubility of replication factor C (RFC) Walker A mutants.

    PubMed

    Marzahn, Melissa R; Bloom, Linda B

    2012-06-01

    Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.

  9. GREEN FLUORESCENT PIGMENT ACCUMULATED BY A MUTANT OF CELLVIBRIO GILVUS.

    PubMed

    LOVE, S H; HULCHER, F H

    1964-01-01

    Love, Samuel H. (Bowman Gray School of Medicine, Winston-Salem, N.C.), and Frank H. Hulcher. Green fluorescent pigment accumulated by a mutant of Cellvibrio gilvus. J. Bacteriol. 87:39-45. 1964.-A mutant of Cellvibrio gilvus, designated strain 139A, liberated a green, fluorescent pigment into the surrounding culture medium. A study of the factors which affected the accumulation of this pigment led to the development of a chemically defined medium which supported maximal pigment accumulation in aerated, liquid cultures. d-Glucose, glycine or l-serine, l-phenylalanine, l-proline, and l-lysine comprised the organic components of this medium. The visible absorption spectrum of the pigment showed a maximal band at 400 mmu (pH 7.0). A difference spectrum between reduced and oxidized pigment showed loss of the band at 400 mmu upon oxidation. However, a methanol-extractable, flavinelike compound occurred in the wild strain but not in the mutant. Ferric ions added to the defined medium stimulated growth, with a concomitant reduction of pigment accumulation. Pigment was formed at a maximal rate during the stationary growth phase, and the highest yield was obtained by 18 hr. Organic solvents did not extract the pigment from water solutions. One and sometimes two, compounds absorbing at 400 mmu could be eluted by ion-exchange chromatography on Cellex-P (H(+)), which was used to separate the pigment from other components in the culture supernatants so that the radioactivity of the pigment could be measured. The mutant synthesized C(14)-labeled pigment from d-glucose-U-C(14) and from each of four amino acids (glycine-1-C(14), l-phenylalanine-U-C(14), l-proline-U-C(14), and l-lysine-U-C(14). Delta-Amino-levulenic acid-4-C(14) did not contribute C(14) to the pigment.

  10. Prion propagation in cells expressing PrP glycosylation mutants.

    PubMed

    Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-04-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

  11. Construction and characterization of avian Escherichia coli cya crp mutants.

    PubMed

    Peighambari, S M; Gyles, C L

    1998-01-01

    We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.

  12. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    DTIC Science & Technology

    2014-08-01

    to ~20% of melanoma with no effective treatment. Targeting palmitoyl acyltansferases (PATs) involved in N-RAS regulation could be a novel strategy to...treat N-RAS mutant melanoma . The objective of the project is to identify PATs responsible for NRAS activation in melanoma cells using chemical...have carried out PATs profiling in melanoma cells using chemical probes and mRNA profiling. We have identified candidate PATs highly expressed in NRAS

  13. Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes.

    PubMed

    Nwachukwu, Res; Shahbazi, A; Wang, L; Ibrahim, S; Worku, M; Schimmel, K

    2012-03-29

    The main objective of this research is to develop, by adaptive evolution, mutant strains of Enterobacter aerogenes ATCC 13048 that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The mutant will be used for high ethanol fermentation from glycerol feedstock. Ethanol production from pure (P-) and recovered (R-) glycerol using the stock was evaluated. A six-tube-subculture-generations method was used for developing the mutant. This involved subculturing the organism six consecutive times in tubes containing the same glycerol and ethanol concentrations at the same culture conditions. Then, the glycerol and/or ethanol concentration was increased and the six subculture generations were repeated. A strain capable of growing in 200 g/L glycerol and 30 g/L ethanol was obtained. The ability of this mutant, vis-à-vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant ethanol yield, was assessed. Tryptic soy broth without dextrose (TSB) was used as the fermentation medium. Fermentation products were analyzed using HPLC.In a 20 g/L glycerol TSB, E. aerogenes ATCC 13048 converted 18.5 g/L P-glycerol and 17.8 g/L R-glycerol into 12 and 12.8 g/L ethanol, respectively. In a 50 g/L P-glycerol TSB, it utilized only 15.6 g/L glycerol; but the new strain used up 39 g/L, yielding 20 g/L ethanol after 120 h, an equivalence of 1.02 mol ethanol/mol-glycerol. This is the highest ethanol yield reported from glycerol bioconversion. The result of this P-glycerol fermentation can be duplicated using the R-glycerol from biodiesel production.

  14. Generating amphioxus Hedgehog knockout mutants and phenotype analysis.

    PubMed

    Hui, Wang; Guang, Li; Yiquan, Wang

    2015-10-01

    The amphioxus is a promising animal model for evolutionary-developmental studies due to its key position on the animal phylogenetic tree. In the present study, we reported a genetically modified amphioxus strain on the Hedgehog (Hh) gene locus using the TALEN method. The result showed that our TALEN pair injection could bring about 34% mutations in the amphioxus Hh coding region. Further analysis on the F(0) gametic DNA revealed that the mutations had entered into gametes. So, we paired one F(0) male carrying an 8 bp deletion with a wild-type (WT) female, and carefully nursed the F(1) embryos up to adulthood. We then screened F(1) individually via analyzing their genomic DNA from a tiny tail tip, and obtained eight heterozygous mutants from the F(1) offspring. Moreover, our observation on the F(2) embryos generated by mating F(1) mutants also revealed that about 25% of early larvae developed aberrantly with head and tail curving ventrally, agenesis of the mesoblastic tissue under their anterior notochord, and no mouth opening. With the larva growth, deformities (such as twist of head and tail, mouth absent, ventrally localized endostyle and gill slits) became more severe, and eventually those malformed larvae died due to no food intake. Genetic analysis showed that all these deformed embryos were homozygous mutants and the ratio of Hh hetorozygotes vs WT agreed with Mondel's law. WT amphioxus larvae are asymmetric with the mouth on the left and gill slits on the right side. However, the homozygous mutant larvae became left-right symmetric with the gill slits on the ventral side, indicating a conserved role of Hedgehog signaling in establishing the left-right embryonic axis.

  15. Lactate dehydrogenase A silencing in IDH mutant gliomas

    PubMed Central

    Chesnelong, Charles; Chaumeil, Myriam M.; Blough, Michael D.; Al-Najjar, Mohammad; Stechishin, Owen D.; Chan, Jennifer A.; Pieper, Russell O.; Ronen, Sabrina M.; Weiss, Samuel; Luchman, H. Artee; Cairncross, J. Gregory

    2014-01-01

    Background Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. Methods We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant (mt) and IDH wild-type (wt) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. Results We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDHmt gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDHmt derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDHwt), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDHmt glioblastomas. Conclusion To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDHmt gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis. PMID:24366912

  16. Human ARF4 expression rescues sec7 mutant yeast cells.

    PubMed Central

    Deitz, S B; Wu, C; Silve, S; Howell, K E; Melançon, P; Kahn, R A; Franzusoff, A

    1996-01-01

    Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics. PMID:8668142

  17. Metabolite profiling of two low phytic acid (lpa) rice mutants.

    PubMed

    Frank, Thomas; Meuleye, Bertrand Seumo; Miller, Andreas; Shu, Qing-Yao; Engel, Karl-Heinz

    2007-12-26

    Two low phytic acid (lpa) rice mutant lines, Os-lpa-XS110-1 and Os-lpa-XS110-2, were grown together with their parent wild-type variety Xiushui 110 in four field trials. HPLC analysis of inositol phosphates in the seeds produced demonstrated that compared to the wild-type, the reduction in phytic acid content in Os-lpa-XS110-1 (-46%) was more pronounced than that in Os-lpa-XS110-2 (-23%). Lower inositol phosphates (InsP 3, InsP 4, InsP 5) were not detected in the mutants. The lpa mutants and the wild-type rice were subjected to comparative metabolite profiling by capillary gas chromatography. On average, 34% (Os-lpa-XS110-1) and 42% (Os-lpa-XS110-2) of the detected peaks were statistically significantly different between wild-type and mutants. However, only a few of these differences could be consistently observed for all field trials. Identification and quantification of the consistently different metabolites revealed that contents of myo-inositol and raffinose were increased in Os-lpa-XS110-1 but decreased in Os-lpa-XS110-2 compared to the wild-type. In addition, Os-lpa-XS110-1 exhibited increased levels of galactose and galactinol. Consideration of these metabolic changes in light of the routes involved in the biosynthesis of phytic acid indicated a disturbance in the early biosynthetic pathway of phytic acid in Os-lpa-XS110-2 (similar to the lpa-1 type mutation in maize) and a mutation event affecting phosphorylation of myo-inositol in Os-lpa-XS110-1 (similar to the lpa-3-type mutation).

  18. IGFBP2 expression predicts IDH-mutant glioma patient survival

    PubMed Central

    Huang, Lin Eric; Cohen, Adam L.; Colman, Howard; Jensen, Randy L.; Fults, Daniel W.; Couldwell, William T.

    2017-01-01

    Mutations of the isocitrate dehydrogenase (IDH) 1 and 2 genes occur in ~80% of lower-grade (WHO grade II and grade III) gliomas. Mutant IDH produces (R)-2-hydroxyglutarate, which induces DNA hypermethylation and presumably drives tumorigenesis. Interestingly, IDH mutations are associated with improved survival in glioma patients, but the underlying mechanism for the difference in survival remains unclear. Through comparative analyses of 286 cases of IDH-wildtype and IDH-mutant lower-grade glioma from a TCGA data set, we report that IDH-mutant gliomas have increased expression of tumor-suppressor genes (NF1, PTEN, and PIK3R1) and decreased expression of oncogenes(AKT2, ARAF, ERBB2, FGFR3, and PDGFRB) and glioma progression genes (FOXM1, IGFBP2, and WWTR1) compared with IDH-wildtype gliomas. Furthermore, each of these genes is prognostic in overall gliomas; however, within the IDH-mutant group, none remains prognostic except IGFBP2 (encodinginsulin-like growth factor binding protein 2). Through validation in an independent cohort, we show that patients with low IGFBP2 expressiondisplay a clear advantage in overall and disease-free survival, whereas those with high IGFBP2 expressionhave worse median survival than IDH-wildtype patients. These observations hold true across different histological and molecular subtypes of lower-grade glioma. We propose therefore that an unexpected biological consequence of IDH mutations in glioma is to ameliorate patient survival by promoting tumor-suppressor signaling while inhibiting that of oncogenes, particularly IGFBP2. PMID:27852048

  19. Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data

    PubMed Central

    Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C.; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J.; Aghera, Nilesh; Varadarajan, Raghavan

    2016-01-01

    Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. PMID:27563054

  20. Pollen embryogenesis to induce, detect, and analyze mutants

    SciTech Connect

    Constantin, M.J.

    1981-01-01

    The development of fully differentiated plants from individual pollen grains through a series of developmental phases that resemble embryogenesis beginning with the zygote was demonstrated during the mid-1960's. This technology opened the door to the use of haploid plants (sporophytes with the gametic number of chromosomes) for plant breeding and genetic studies, biochemical and metabolic studies, and the selection of mutations. Although pollen embryogenesis has been demonstrated successfully in numerous plant genera, the procedure cannot as yet be used routinely to generate large populations of plants for experiments. Practical results from use of the technology in genetic toxicology research to detect mutations have failed to fully realize the theoretical potential; further developments of the technology could overcome the limitations. Pollen embryogenesis could be used to develop plants from mutant pollen grains to verify that genetic changes are involved. Through either spontaneous or induced chromosome doubling, these plants can be made homozygous and used to analyze genetically the mutants involved. The success of this approach will depend on the mutant frequency relative to the fraction of pollen grains that undergo embryogenesis; these two factors will dictate population size needed for success. Research effort is needed to further develop pollen embryogenesis for use in the detection of genotoxins under both laboratory and in situ conditions.

  1. PRRT2 Mutant Leads to Dysfunction of Glutamate Signaling

    PubMed Central

    Li, Ming; Niu, Fenghe; Zhu, Xilin; Wu, Xiaopan; Shen, Ning; Peng, Xiaozhong; Liu, Ying

    2015-01-01

    Paroxysmal kinesigenic choreoathetosis (PKC) is an inherited disease of the nervous system. We previously identified PRRT2 as the causative gene of PKC. However, as little is known about the function of PRRT2, elucidating its function will benefit not only PKC studies, but also many other related disorders. Here, we reveal higher levels of glutamate in the plasma of PKC patients and the culture medium of neurons following knock-out Prrt2 expression. Using double immunostaining assays we confirm Prrt2 is located at the glutamatergic neurons in accordance with its function. Our co-immunoprecipitation assays reveal mutant PRRT2 interferes with SNAP25 and GRIA1 interactions, respectively. Furthermore, using live-labeling techniques, we confirmed co-transfection with mutant PRRT2 caused an increase in GRIA1 distribution on the cell surface. Therefore, our results suggest that mutant PRRT2, probably through its weakened interaction with SNAP25, affects glutamate signaling and glutamate receptor activity, resulting in the increase of glutamate release and subsequent neuronal hyperexcitability. PMID:25915028

  2. Disposal of iron by a mutant form of lipocalin 2

    PubMed Central

    Barasch, Jonathan; Hollmen, Maria; Deng, Rong; Hod, Eldad A.; Rupert, Peter B.; Abergel, Rebecca J.; Allred, Benjamin E.; Xu, Katherine; Darrah, Shaun F.; Tekabe, Yared; Perlstein, Alan; Wax, Rebecca; Bruck, Efrat; Stauber, Jacob; Corbin, Kaitlyn A.; Buchen, Charles; Slavkovich, Vesna; Graziano, Joseph; Spitalnik, Steven L.; Bao, Guanhu; Strong, Roland K.; Qiu, Andong

    2016-01-01

    Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe−/−) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway. PMID:27796299

  3. Novel mutants of NAB corepressors enhance activation by Egr transactivators.

    PubMed Central

    Svaren, J; Sevetson, B R; Golda, T; Stanton, J J; Swirnoff, A H; Milbrandt, J

    1998-01-01

    The NGFI-A binding corepressors NAB1 and NAB2 interact with a conserved domain (R1 domain) within the Egr1/NGFI-A and Egr2/Krox20 transactivators, and repress the transcription of Egr target promoters. Using a novel adaptation of the yeast two-hybrid screen, we have identified several point mutations in NAB corepressors that interfere with their ability to bind to the Egr1 R1 domain. Surprisingly, NAB proteins bearing some of these mutations increased Egr1 activity dramatically. The mechanism underlying the unexpected behavior of these mutants was elucidated by the discovery that NAB conserved domain 1 (NCD1) not only binds to Egr proteins but also mediates multimerization of NAB molecules. The activating mutants exert a dominant negative effect on NAB repression by multimerizing with native NAB proteins and preventing binding of endogenous NAB proteins with Egr transactivators. To examine NAB repression of a native Egr target gene, we show that NAB2 represses Egr2/Krox20-mediated activation of the bFGF/FGF-2 promoter, and that repression is reversed by coexpression of dominant negative NAB2. Because of their specific ability to alleviate NAB repression of Egr target genes, the dominant negative NAB mutants will be useful in elucidating the mechanism and function of NAB corepressors. PMID:9774344

  4. Temperature effect on a high stearic acid sunflower mutant.

    PubMed

    Fernández-Moya, Valle; Martínez-Force, Enrique; Garcés, Rafael

    2002-01-01

    Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids because of its neutral effect on serum cholesterol lipoproteins. Selection of a very high stearic acid sunflower (Helianthus annuus L.) line (CAS-14), with up to a 37.3% of stearic acid in the seed oil, and the relationship between the expression of this character and the growth temperature are presented. The mutant was selected from the M(2) progeny of 3000 mutagenized seeds (4 mM sodium azide mutagenesis treatment) by analysing the fatty acid composition of half-seed by gas liquid chromatography. In order to genetically fix the mutant character, plants were grown at high day/night temperatures during seed formation. We found that temperatures higher than 30/20 degrees C are required for good expression of the phenotype, the maximum stearic acid content being obtained at 39/24 degrees C. This behaviour is totally opposed to that observed in normal and previously isolated high-stearic acid sunflower lines that contain more stearic acid at low temperature. Thus, a new type of temperature regulation on the stearate desaturation must occur. This line is the sunflower mutant with the highest stearic acid content reported so far.

  5. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used to show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.

  6. Distinct Phyllosphere Bacterial Communities on Arabidopsis Wax Mutant Leaves

    PubMed Central

    Reisberg, Eva E.; Hildebrandt, Ulrich; Riederer, Markus; Hentschel, Ute

    2013-01-01

    The phyllosphere of plants is inhabited by diverse microorganisms, however, the factors shaping their community composition are not fully elucidated. The plant cuticle represents the initial contact surface between microorganisms and the plant. We thus aimed to investigate whether mutations in the cuticular wax biosynthesis would affect the diversity of the phyllosphere microbiota. A set of four Arabidopsis thaliana eceriferum mutants (cer1, cer6, cer9, cer16) and their respective wild type (Landsberg erecta) were subjected to an outdoor growth period and analysed towards this purpose. The chemical distinctness of the mutant wax phenotypes was confirmed by gas chromatographic measurements. Next generation amplicon pyrosequencing of the bacterial communities showed distinct community patterns. This observation was supported by denaturing gradient gel electrophoresis experiments. Microbial community analyses revealed bacterial phylotypes that were ubiquitously present on all plant lines (termed “core” community) while others were positively or negatively affected by the wax mutant phenotype (termed “plant line-specific“ community). We conclude from this study that plant cuticular wax composition can affect the community composition of phyllosphere bacteria. PMID:24223831

  7. Functional Analysis of Jasmonates in Rice through Mutant Approaches

    PubMed Central

    Dhakarey, Rohit; Kodackattumannil Peethambaran, Preshobha; Riemann, Michael

    2016-01-01

    Jasmonic acid, one of the major plant hormones, is, unlike other hormones, a lipid-derived compound that is synthesized from the fatty acid linolenic acid. It has been studied intensively in many plant species including Arabidopsis thaliana, in which most of the enzymes participating in its biosynthesis were characterized. In the past 15 years, mutants and transgenic plants affected in the jasmonate pathway became available in rice and facilitate studies on the functions of this hormone in an important crop. Those functions are partially conserved compared to other plant species, and include roles in fertility, response to mechanical wounding and defense against herbivores. However, new and surprising functions have also been uncovered by mutant approaches, such as a close link between light perception and the jasmonate pathway. This was not only useful to show a phenomenon that is unique to rice but also helped to establish this role in plant species where such links are less obvious. This review aims to provide an overview of currently available rice mutants and transgenic plants in the jasmonate pathway and highlights some selected roles of jasmonate in this species, such as photomorphogenesis, and abiotic and biotic stress. PMID:27135235

  8. Mouse mutants from chemically mutagenized embryonic stem cells.

    PubMed

    Munroe, R J; Bergstrom, R A; Zheng, Q Y; Libby, B; Smith, R; John, S W; Schimenti, K J; Browning, V L; Schimenti, J C

    2000-03-01

    The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

  9. Molecular Imaging of Metabolic Reprograming in Mutant IDH Cells.

    PubMed

    Viswanath, Pavithra; Chaumeil, Myriam M; Ronen, Sabrina M

    2016-01-01

    Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG). In turn, 2-HG, which has been termed an "oncometabolite," inhibits key α-KG-dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprograming that extends beyond 2-HG production, and this reprograming often differs from what has been previously reported in other cancer types. In this review, we will discuss in detail what is known to date about the metabolic reprograming of mutant IDH cells, and how this reprograming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells, and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  10. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    PubMed

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  11. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    PubMed

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

  12. Characterization of Saccharomyces cerevisiae ubiquinone-deficient mutants.

    PubMed

    Schultz, J R; Clarke, C F

    1999-01-01

    Ubiquinol (QH2) is a lipid-soluble molecule that participates in cellular redox reactions. Previous studies have shown that yeast mutants lacking QH2 are hypersensitive to treatment with polyunsaturated fatty acids (PUFAs) indicating that QH2 can function as an antioxidant in vivo. In this study the effect of 1 mM linolenic acid on levels of Q6 and Q6H2 is assessed in both wild-type and respiration-deficient (atp2 delta) strains. The response of Q-deficient mutants to other forms of oxidative stress is further characterized to define those conditions where QH2 acts as an antioxidant. Endogenous antioxidant defense systems were also assessed in wild-type, Q-deficient, and atp2 delta strains. Superoxide dismutase (SOD) activity decreased and catalase activity increased in both Q-deficient and atp2 delta mutants compared to wild-type cells, suggesting that such changes result from the loss of respiration rather than the lack of Q.

  13. Genes and Alcohol Consumption: Studies with Mutant Mice

    PubMed Central

    Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

    2017-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

  14. Temperature Sensitivity of Neural Tube Defects in Zoep Mutants.

    PubMed

    Ma, Phyo; Swartz, Morgan R; Kindt, Lexy M; Kangas, Ashley M; Liang, Jennifer Ostrom

    2015-12-01

    Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation.

  15. Yeast mutants affecting possible quality control of plasma membrane proteins.

    PubMed

    Li, Y; Kane, T; Tipper, C; Spatrick, P; Jenness, D D

    1999-05-01

    Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.

  16. Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

    PubMed Central

    Elmonem, Mohamed A.; Khalil, Ramzi; Khodaparast, Ladan; Khodaparast, Laleh; Arcolino, Fanny O.; Morgan, Joseph; Pastore, Anna; Tylzanowski, Przemko; Ny, Annelii; Lowe, Martin; de Witte, Peter A.; Baelde, Hans J.; van den Heuvel, Lambertus P.; Levtchenko, Elena

    2017-01-01

    The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis. PMID:28198397

  17. Temperature-sensitive yeast mutants defective in mitochondrial inheritance.

    PubMed

    McConnell, S J; Stewart, L C; Talin, A; Yaffe, M P

    1990-09-01

    The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

  18. Homeotic transformation of cervical vertebrae in Hoxa-4 mutant mice.

    PubMed Central

    Horan, G S; Wu, K; Wolgemuth, D J; Behringer, R R

    1994-01-01

    Hoxa-4 (previously known as Hox-1.4) is a mouse homeobox-containing gene that is expressed in the presumptive hindbrain and spinal cord, prevertebrae, and other tissues during embryogenesis. To understand the role of Hoxa-4 during development, we generated Hoxa-4 mutant mice. Homozygous mutants were viable and fertile. Analysis of neonatal skeletons revealed the development of ribs on the seventh cervical vertebra at variable penetrance and expressivity. A low frequency of alterations in sternal morphogenesis was also observed. In addition, we analyzed the skeletons of transgenic mice that overexpress Hoxa-4 and found that the formation of the small rib anlagen that often develop on the seventh cervical vertebra was suppressed. Analysis of adult homozygous mutant skeletons revealed that the dorsal process normally associated with the second cervical vertebra was also found on the third cervical vertebra. These results demonstrate that Hoxa-4 plays a role in conferring positional information along the anteroposterior axis to specify the identity of the third and the seventh cervical vertebrae. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7809093

  19. Temperature Sensitivity of Neural Tube Defects in Zoep Mutants

    PubMed Central

    Ma, Phyo; Swartz, Morgan R.; Kindt, Lexy M.; Kangas, Ashley M.

    2015-01-01

    Abstract Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation. PMID:26366681

  20. Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant.

    PubMed Central

    Regan, SM; Moffatt, BA

    1990-01-01

    Microsporogenesis has been examined in wild-type Arabidopsis thaliana and the nuclear male-sterile mutant BM3 by cytochemical staining. The mutant lacks adenine phosphoribosyltransferase, an enzyme of the purine salvage pathway that converts adenine to AMP. Pollen development in the mutant began to diverge from wild type just after meiosis, as the tetrads of microspores were released from their callose walls. The first indication of abnormal pollen development in the mutant was a darker staining of the microspore wall due to an incomplete synthesis of the intine. Vacuole formation was delayed and irregular in the mutant, and the majority of the mutant microspores failed to undergo mitotic divisions. Enzyme activities of alcohol dehydrogenase and esterases decreased in the mutant soon after meiosis and were undetectable in mature pollen grains of the mutant. RNA accumulation was also diminished. These results are discussed in relation to the possible role(s) of adenine salvage in pollen development. PMID:12354970

  1. [Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis].

    PubMed

    Kurbatova, E M; Dutova, T A; Serkova, N N; Rabinovich, Ia M; Trotsenko, Iu A

    2004-05-01

    After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation

  2. Determination of the Mutant Prevention Concentration and the Mutant Selection Window of Topical Antimicrobial Agents against Propionibacterium acnes.

    PubMed

    Nakase, Keisuke; Nakaminami, Hidemasa; Toda, Yuta; Noguchi, Norihisa

    2017-01-01

    Determination of the mutant prevention concentration (MPC) and the mutant selection window (MSW) of antimicrobial agents used to treat pathogenic bacteria is important in order to apply effective antimicrobial therapies. Here, we determined the MPCs of the major topical antimicrobial agents against Propionibacterium acnes and Staphylococcus aureus which cause skin infections and compared their MSWs. Among the MPCs of nadifloxacin and clindamycin, the clindamycin MPC was determined to be the lowest against P. acnes. In contrast, the nadifloxacin MPC was the lowest against S. aureus. Calculations based on the minimum inhibitory concentrations and MPCs showed that clindamycin has the lowest MSW against both P. acnes and S. aureus. Nadifloxacin MSWs were 4-fold higher against P. acnes than against S. aureus. It is more likely for P. acnes to acquire resistance to fluoroquinolones than S. aureus. Therefore, topical application of clindamycin contributes very little to the emergence of resistant P. acnes and S. aureus strains.

  3. Identification of GRO1 as a critical determinant for mutant p53 gain of function.

    PubMed

    Yan, Wensheng; Chen, Xinbin

    2009-05-01

    Mutant p53 gain of function contributes to cancer progression, increased invasion and metastasis potentials, and resistance to anticancer therapy. The ability of mutant p53 to acquire its gain of function is shown to correlate with increased expression of progrowth genes, such as c-MYC, MDR1, and NF-kappaB2. However, most of the published studies to identify mutant p53 target genes were performed in a cell system that artificially overexpresses mutant p53. Thus, it remains unclear whether such mutant p53 targets can be regulated by endogenous physiological levels of mutant p53. Here, we utilized SW480 and MIA-PaCa-2 cells, in which endogenous mutant p53 can be inducibly knocked down, to identify mutant p53 target genes that potentially mediate mutant p53 gain of function. We found that knockdown of mutant p53 inhibits GRO1 expression, whereas ectopic expression of mutant R175H in p53-null HCT116 cells increases GRO1 expression. In addition, we found that endogenous mutant p53 is capable of binding to and activating the GRO1 promoter. Interestingly, ectopic expression of GRO1 can rescue the proliferative defect in SW480 and MIA-PaCa-2 cells induced by knockdown of mutant p53. Conversely, knockdown of endogenous GRO1 inhibits cell proliferation and thus abrogates mutant p53 gain of function in SW480 cells. Taken together, our findings define a novel mechanism by which mutant p53 acquires its gain of function via transactivating the GRO1 gene in cancer cells. Thus, targeting GRO1 for cancer therapy would be applicable to a large portion of human tumors with mutant p53, but the exploration of GRO1 as a potential target should take the mutation status of p53 into consideration.

  4. Analysis of spontaneous suppressor mutants from the photomixotrophically grown pmgA-disrupted mutant in the cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Nishijima, Yoshiki; Kanesaki, Yu; Yoshikawa, Hirofumi; Ogawa, Takako; Sonoike, Kintake; Nishiyama, Yoshitaka; Hihara, Yukako

    2015-12-01

    The pmgA-disrupted (ΔpmgA) mutant in the cyanobacterium Synechocystis sp. PCC 6803 suffers severe growth inhibition under photomixotrophic conditions. In order to elucidate the key factors enabling the cells to grow under photomixotrophic conditions, we isolated spontaneous suppressor mutants from the ΔpmgA mutant derived from a single colony. When the ΔpmgA mutant was spread on a BG11 agar plate supplemented with glucose, colonies of suppressor mutants appeared after the bleaching of the background cells. We identified the mutation site of these suppressor mutants and found that 11 mutants out of 13 had a mutation in genes related to the type 1 NAD(P)H dehydrogenase (NDH-1) complex. Among them, eight mutants had mutations within the ndhF3 (sll1732) gene: R32stop, W62stop, V147I, G266V, G354W, G586C, and deletion of 7 bp within the coding region. One mutant had one base insertion in the putative -10 box of the ndhC (slr1279) gene, leading to the decrease in the transcripts of the ndhCKJ operon. Two mutants had one base insertion and deletion in the coding region of cupA (sll1734), which is co-transcribed with ndhF3 and ndhD3 and comprises together a form of NDH-1 complex (NDH-1MS complex) involved in inducible high-affinity CO2 uptake. The results indicate that the loss of the activity of this complex effectively rescues the ΔpmgA mutant under photomixotrophic condition with 1 % CO2. However, little difference among WT and mutants was observed in the activities ascribed to the NDH-1MS complex, i.e., CO2 uptake and cyclic electron transport. This may suggest that the NDH-1MS complex has the third, currently unknown function under photomixotrophic conditions.

  5. Expression of an anthranilate synthase from maize mutant bf-1 in maize line HiII

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize mutant bf-1 was one of a series of maize mutants generated by radiation from the Bikini Atoll atomic bomb test in 1946. It is characterized by blue fluorescence in seedlings and anthers under ultraviolet illumination and by mutant plants giving off a characteristic grape-like odor due to the ...

  6. Isolation, characterization, and expression analyses of tryptophan aminotransferase genes in a maize dek18 mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The dek18 mutant of maize has decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology in maize seed development. Seeds of the dek18 mutants are smaller compared to wild type seeds and the vegetative d...

  7. Construction and physiological analysis of a Xanthomonas oryzae pv. oryzae recA mutant.

    PubMed

    Mongkolsuk, S; Rabibhadana, S; Sukchavalit, R; Vaughn, G

    1998-12-15

    A Xoo recA insertion inactivation mutant was constructed. The mutant, lacking RecA, showed increased sensitivity towards mutagen killing. This phenotype could be complemented by a cloned, functional recA. Unlike other bacteria, both the recA mutant and the parental strain had similar level of resistance to H2O2 killing and peroxide-induced mutagenesis.

  8. An improved method for rapid generation and screening of Bacillus thuringiensis phage-resistant mutants.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-11-01

    A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.

  9. A new fuzzless seed locus in an upland cotton (Gossypium hirsutum L.) mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Various fiber mutants of cotton have been reported since 1920. Two of the best characterized mutants are the naked seed loci, N1N1 and n2n2. Recently, a naked-tufted mutant called 9023n4t was developed from the cultivar SC 9023 through chemical mutagenesis. The objective of this research was to dete...

  10. Saccharomyces cerevisiae mutants resistant to catabolite repression: use in cheese whey hydrolysate fermentation

    SciTech Connect

    Bailey, R.B.; Benitez, T.; Woodward, A.

    1982-09-01

    Mutants of an industrial-type strain of Saccharomyces cerevisiae which rapidly and completely fermented equimolar mixtures of glucose and galactose to ethanol were isolated. These mutants fell into two general phenotypic classes based upon their fermentation kinetics and enzyme induction patterns. One class apparently specifically effects the utilization of galactose and allows sequential utilization of first glucose and then galactose in an anaerobic fermentation. The second class of mutants was resistant to general catabolite repression and produced maltase, invertase, and galactokinase in the presence of repressive levels of glucose. These mutants were completely dominant and appear to represent an as yet undescribed class of mutant. (Refs. 23).

  11. Xanthine Dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity.

    PubMed

    Browder, L W; Tucker, L; Wilkes, J

    1982-02-01

    Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin and cin mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.

  12. Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny.

    PubMed Central

    Miller, R V; Ku, C M

    1978-01-01

    Mutants of Pseudomonas aeruginosa with impaired ability to establish a lysogenic relationship with temperate bacteriophage (Les-) have been isolated. These les mutations map to two areas of the P. aeruginosa chromosomal map as determined by conjugational and transductional analyses. Two phenotypic classes of Les- mutants were identified. One class of mutations has pleiotropic effects on DNA metabolism. These mutants are unable to recombine genetic material acquired as a result of either conjugation or transduction (Rec-). In addition, the ability of these Les- Rec- mutants to repair UV-induced damage to bacteriophage is reduced (host-cell reactivation deficient, Hcr-). Mutants of the second class are Les-, Rec+, and Hcr+. PMID:96103

  13. Identification of An Arsenic Tolerant Double Mutant With a Thiol-Mediated Component And Increased Arsenic Tolerance in PhyA Mutants

    SciTech Connect

    Sung, D.Y.; Lee, D.; Harris, H.; Raab, A.; Feldmann, J.; Meharg, A.; Kumabe, B.; Komives, E.A.; Schroeder, J.I.; /SLAC, SSRL /Sydney U. /Aberdeen U. /UC, San Diego

    2007-04-06

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

  14. Proton movement and photointermediate kinetics in rhodopsin mutants.

    PubMed

    Lewis, James W; Szundi, Istvan; Kazmi, Manija A; Sakmar, Thomas P; Kliger, David S

    2006-05-02

    The role of ionizable amino acid side chains in the bovine rhodopsin activation mechanism was studied in mutants E134Q, E134R/R135E, H211F, and E122Q. All mutants exhibited bathorhodopsin stability on the 30 ns to 1 micros time scale similar to that of the wild type. Lumirhodopsin decay was also similar to that of the wild type except for the H211F mutant where early decay (20 micros) to a second form of lumirhodopsin was seen, followed by formation of an extremely long-lived Meta I(480) product (34 ms), an intermediate which forms to a much reduced extent, if at all, in dodecyl maltoside suspensions of wild-type rhodopsin. A smaller amount of a similar long-lived Meta I(480) product was seen after photolysis of E122Q, but E134Q and E134R/R135Q displayed kinetics much more similar to those of the wild type under these conditions (i.e., no Meta I(480) product). These results support the idea that specific interaction of His211 and Glu122 plays a significant role in deprotonation of the retinylidene Schiff base and receptor activation. Proton uptake measurements using bromcresol purple showed that E122Q was qualitatively similar to wild-type rhodopsin, with at least one proton being released during lumirhodopsin decay per Meta I(380) intermediate formed, followed by uptake of at least two protons per rhodopsin bleached on a time scale of tens of milliseconds. Different results were obtained for H211F, E134Q, and E134R/R135E, which all released approximately two protons per rhodopsin bleached. These results show that several ionizable groups besides the Schiff base imine are affected by the structural changes involved in rhodopsin activation. At least two proton uptake groups and probably at least one proton release group in addition to the Schiff base are present in rhodopsin.

  15. Mutant prevention concentrations of daptomycin for Enterococcus faecium clinical isolates.

    PubMed

    Sinel, Clara; Jaussaud, Clara; Auzou, Michel; Giard, Jean-Christophe; Cattoir, Vincent

    2016-10-01

    Owing to the emergence of vancomycin-resistant Enterococcus faecium, treatment of enterococcal infections has become challenging. Although spontaneous in vitro resistance frequencies are low, the emergence of resistance is increasingly reported during daptomycin therapy. The mutant selection window (MSW), comprised between the minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC), corresponds to the concentration range within which resistant mutants may be selected. Since no data are available for enterococci, the aim of this study was to determine MPCs and MSWs for 12 representative E. faecium clinical isolates. MICs and MPCs were determined by broth microdilution and agar dilution methods, respectively. A basic MSW-derived pharmacodynamic analysis was also performed using mean maximum plasma concentration (Cmax) values obtained with dosages from 4 to 12 mg/kg. MICs and MPCs of daptomycin ranged from 0.5 to 4 mg/L and from 2 to 32 mg/L, respectively, with no correlation between them. The wideness of MSWs ranged from 2× to 32× MIC. Mean plasma Cmax values of daptomycin were calculated from 55 to 174.5 mg/L when using a dosage from 4 to 12 mg/kg. All Cmax values were above the MPCs whatever the dosage. Taking into account the protein binding of daptomycin (ca. 90%), the unbound fraction Cmax was just within the MSW in 67-92% of strains at recommended dosages (4-6 mg/kg) and was above the MPC for the majority of strains only with the highest dosage (12 mg/kg). This study shows that free daptomycin Cmax values usually fell into MSWs when using lower dosages (<10 mg/kg).

  16. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    SciTech Connect

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

  17. Enhanced radiosensitization of p53 mutant cells by oleamide

    SciTech Connect

    Lee, Yoon-Jin; Chung, Da Yeon; Lee, Su-Jae; Ja Jhon, Gil; Lee, Yun-Sil . E-mail: yslee@kcch.re.kr

    2006-04-01

    Purpose: Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. Methods and Materials: NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. Results: Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. Conclusions: Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.

  18. Generation of targeted mouse mutants by embryo microinjection of TALENs.

    PubMed

    Wefers, Benedikt; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

    2014-08-15

    Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.

  19. Applications of mutant yeast strains with low glycogen storage capability

    NASA Technical Reports Server (NTRS)

    Petersen, G. R.; Schubert, W. W.; Stokes, B. O.

    1981-01-01

    Several strains of Hansenula polymorpha were selected for possible low glycogen storage characteristics based on a selective I2 staining procedure. The levels of storage carbohydrates in the mutant strains were found to be 44-70% of the levels in the parent strain for cultures harvested in stationary phase. Similar differences generally were not found for cells harvested in exponential phase. Yeast strains deficient in glycogen storage capability are valuable in increasing the relative protein value of microbial biomass and also may provide significant cost savings in substrate utilization in fermentative processes.

  20. Rhes, a Striatal Specific Protein, Mediates Mutant-Huntingtin Cytotoxicity

    PubMed Central

    Subramaniam, Srinivasa; Sixt, Katherine M.; Barrow, Roxanne; Snyder, Solomon H.

    2009-01-01

    Huntington's disease (HD) is caused by a polyglutamine repeat in the protein huntingtin (Htt) with mutant Htt (mHtt) expressed throughout the body and similarly in all brain regions. Yet, HD neuropathology is largely restricted to the corpus striatum. We report that the small guanine nucleotide–binding protein Rhes, which is localized very selectively to the striatum, binds physiologically to mHtt. Using cultured cells, we found Rhes induces sumoylation of mHtt, which leads to cytotoxicity. Thus, Rhes-mHtt interactions can account for the localized neuropathology of HD. PMID:19498170

  1. Use of Electroporation To Generate a Thiobacillus neapolitanus Carboxysome Mutant

    PubMed Central

    English, R. S.; Jin, S.; Shively, J. M.

    1995-01-01

    Two cloning vectors designed for use in Escherichia coli and the thiobacilli were constructed by combining a Thiobacillus intermedius plasmid replicon with a multicloning site, lacZ(prm1), and either a kanamycin or a streptomycin resistance gene. Conditions necessary for the introduction of DNA into T. intermedius and T. neapolitanus via electroporation were examined and optimized. By using optimal electroporation conditions, the gene encoding a carboxysome shell protein, csoS1A, was insertionally inactivated in T. neapolitanus. The mutant showed a reduced number of carboxysomes and an increased level of CO(inf2) necessary for growth. PMID:16535117

  2. DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

    PubMed Central

    Aron, G M; Purifoy, D J; Schaffer, P A

    1975-01-01

    Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies. PMID:169388

  3. A genetic screen for zebrafish mutants with hepatic steatosis identifies a locus required for larval growth.

    PubMed

    Hugo, Sarah E; Schlegel, Amnon

    2017-03-01

    In a screen for zebrafish larval mutants with excessive liver lipid accumulation (hepatic steatosis), we identified harvest moon (hmn). Cytoplasmic lipid droplets, surrounded by multivesicular structures and mitochondria whose cristae appeared swollen, are seen in hmn mutant hepatocytes. Whole body triacylglycerol is increased in hmn mutant larvae. When we attempted to raise mutants, which were morphologically normal at the developmental stage that the screen was conducted, to adulthood, we observed that most hmn mutants do not survive to the juvenile period when raised. An arrest in growth occurs in the late larval period without obvious organ defects. Maternal zygotic mutants have no additional defects, suggesting that the mutation affects a late developmental process. The developmental window between embryogenesis and the metamorphosis remains under-studied, and hmn mutants might be useful for exploring the molecular and anatomic processes occurring during this transition period.

  4. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms.

    PubMed

    Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie; Wang, Ran; Chen, Jian; Logothetis, Diomedes E; Zhang, Zhe

    2016-01-05

    Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects.

  5. Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation.

    PubMed

    Dutilleul, Christelle; Garmier, Marie; Noctor, Graham; Mathieu, Chantal; Chétrit, Philippe; Foyer, Christine H; de Paepe, Rosine

    2003-05-01

    To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H(2)O(2) and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.

  6. Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

    PubMed Central

    Hamam, Ahmed

    2012-01-01

    We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog (MscS), a Ca2+/H+ exchange protein (cax), and Ca2+-ATPases (nca-1, nca-2, nca-3)—as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H+-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca2+ levels, indicative of lesions in Ca2+ homeostasis. However, the net Ca2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca2+-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca2+] was elevated. Thus, although Ca2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H+-ATPase activity. PMID:22408225

  7. The phenotype alterations showed by the res tomato mutant disappear when the plants are grown under semi-arid conditions: Is the res mutant tolerant to multiple stresses?

    PubMed

    Garcia-Abellan, José O; Albaladejo, Irene; Egea, Isabel; Flores, Francisco B; Capel, Carmen; Capel, Juan; Angosto, Trinidad; Lozano, Rafael; Bolarin, Maria C

    2016-02-23

    The res (restored cell structure by salinity) mutant, recently identified as the first tomato mutant accumulating jasmonate (JA) without stress, exhibited important morphological alterations when plants were grown under control conditions but these disappeared under salt stress. Since the defense responses against stresses are activated in the res mutant as a consequence of the increased expression of genes from the JA biosynthetic and signaling pathways, the mutant may display a tolerance response not only to salt stress but also to multiple stresses. Here, we show that when res mutant plants are grown under the summer natural conditions of the Mediterranean area, with high temperatures and low relative humidity, the characteristic leaf chlorosis exhibited by the mutant disappears and leaves become dark green over time, with a similar aspect to WT leaves. Moreover, the mutant plants are able to achieve chlorophyll and fluorescence levels similar to those of WT. These results hint that research on res tomato mutant may allow very significant advances in the knowledge of defense responses activated by JA against multiple stresses.

  8. Reduced chlorophyll biosynthesis in heterozygous barley magnesium chelatase mutants.

    PubMed

    Braumann, Ilka; Stein, Nils; Hansson, Mats

    2014-05-01

    Chlorophyll biosynthesis is initiated by magnesium chelatase, an enzyme composed of three proteins, which catalyzes the insertion of Mg2+ into protoporphyrin IX to produce Mg-protoporphyrin IX. In barley (Hordeum vulgare L.) the three proteins are encoded by Xantha-f, Xantha-g and Xantha-h. Two of the gene products, XanH and XanG, belong to the structurally conserved family of AAA+ proteins (ATPases associated with various cellular activities) and form a complex involving six subunits of each protein. The complex functions as an ATP-fueled motor of the magnesium chelatase that uses XanF as substrate, which is the catalytic subunit responsible for the insertion of Mg2+ into protoporphyrin IX. Previous studies have shown that semi-dominant Xantha-h mutations result in non-functional XanH subunits that participate in the formation of inactive AAA complexes. In the present study, we identify severe mutations in the barley mutants xantha-h.38, -h.56 and -h.57. A truncated form of the protein is seen in xantha-h.38, whereas no XanH is detected in xantha-h.56 and -h.57. Heterozygous mutants show a reduction in chlorophyll content by 14-18% suggesting a slight semi-dominance of xantha-h.38, -h.56 and -h.57, which otherwise have been regarded as recessive mutations.

  9. Pyrin gene and mutants thereof, which cause familial Mediterranean fever

    DOEpatents

    Kastner, Daniel L.; Aksentijevichh, Ivona; Centola, Michael; Deng, Zuoming; Sood, Ramen; Collins, Francis S.; Blake, Trevor; Liu, P. Paul; Fischel-Ghodsian, Nathan; Gumucio, Deborah L.; Richards, Robert I.; Ricke, Darrell O.; Doggett, Norman A.; Pras, Mordechai

    2003-09-30

    The invention provides the nucleic acid sequence encoding the protein associated with familial Mediterranean fever (FMF). The cDNA sequence is designated as MEFV. The invention is also directed towards fragments of the DNA sequence, as well as the corresponding sequence for the RNA transcript and fragments thereof. Another aspect of the invention provides the amino acid sequence for a protein (pyrin) associated with FMF. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. The invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain at the C-terminal of the protein. These mutants include M6801, M694V, K695R, and V726A. Additionally, the invention includes methods for diagnosing a patient at risk for having FMF and kits therefor.

  10. Efficient construction of Haemophilus parasuis mutants based on natural transformation.

    PubMed

    Li, Junxing; Yuan, Xiufang; Xu, Lihua; Kang, Lei; Jiang, Jun; Wang, Yicheng

    2016-10-01

    Studies on virulence factors and pathogenecity of Haemophilus parasuis have long been hindered by a lack of a consistent system for genetic manipulation. In this study, competence was induced by transferring H. parasuis from rich medium to starvation medium media-IV (M-IV) and iscR gene deficient mutants of H. parasuis were generated efficiently. Transformation frequency varied from 4.1 × 10(-5) to 1.1 × 10(-8) when using circular plasmid, and increased to about 2- to 31-fold when transformed using linearized plasmid. Allele replacement occurred efficiently in 6 strains, which are transformable using both circular and linearized pTRU, but not in another 2 strains which could only be transformed using linearized plasmid. The iscR mutants were stable for at least 20 passages in vitro. Haemophilus parasuis strains vary extensively in natural transformation efficiency and the method established here allows for transformation of a larger spectrum of strains with an easily accessed plasmid. This provides important tools for genetic manipulation of H. parasuis.

  11. Boolean Network Model Predicts Knockout Mutant Phenotypes of Fission Yeast

    PubMed Central

    Davidich, Maria I.; Bornholdt, Stefan

    2013-01-01

    Boolean networks (or: networks of switches) are extremely simple mathematical models of biochemical signaling networks. Under certain circumstances, Boolean networks, despite their simplicity, are capable of predicting dynamical activation patterns of gene regulatory networks in living cells. For example, the temporal sequence of cell cycle activation patterns in yeasts S. pombe and S. cerevisiae are faithfully reproduced by Boolean network models. An interesting question is whether this simple model class could also predict a more complex cellular phenomenology as, for example, the cell cycle dynamics under various knockout mutants instead of the wild type dynamics, only. Here we show that a Boolean network model for the cell cycle control network of yeast S. pombe correctly predicts viability of a large number of known mutants. So far this had been left to the more detailed differential equation models of the biochemical kinetics of the yeast cell cycle network and was commonly thought to be out of reach for models as simplistic as Boolean networks. The new results support our vision that Boolean networks may complement other mathematical models in systems biology to a larger extent than expected so far, and may fill a gap where simplicity of the model and a preference for an overall dynamical blueprint of cellular regulation, instead of biochemical details, are in the focus. PMID:24069138

  12. Microfilament Distribution in Maize Meiotic Mutants Correlates with Microtubule Organization.

    PubMed Central

    Staiger, CJ; Cande, WZ

    1991-01-01

    Microtubules and microfilaments often codistribute in plants; their presumed interaction can be tested with drugs although it is not always clear that these are without side effects. In this study, we exploited mutants defective in meiotic cell division to investigate in a noninvasive way the relationship between the two cytoskeletal elements. By staining unfixed, permeabilized cells with rhodamine-phalloidin, spatial and temporal changes in microfilament distribution during maize meiosis were examined. In wild-type microsporocytes, a microtubule array that radiates from the nucleus disappeared during spindle formation and returned at late telophase. This result differed from the complex cytoplasmic microfilament array that is present at all stages, including karyokinesis and cytokinesis. During division, a second class of microfilaments also was observed in the spindle and phragmoplast. To analyze this apparent association of microtubules and microfilaments, we examined several meiotic mutants known to have stage-specific disruptions in their microtubule arrays. Two mutations that altered the number or form of meiotic spindles also led to a dramatic reorganization of F-actin. In contrast, rearrangement of nonspindle, cytoplasmic microtubules did not lead to concomitant changes in F-actin distribution. These results suggested that microtubules and microfilaments interact in a cell cycle-specific and site-specific fashion during higher plant meiosis. PMID:12324607

  13. Nonequivalent Loci and the distribution of mutant effects.

    PubMed Central

    Welch, J J; Waxman, D

    2002-01-01

    It has been observed repeatedly that the distribution of new mutations of a quantitative trait has a kurtosis (a statistical measure of the distribution's shape) that is systematically larger than that of a normal distribution. Here we suggest that rather than being a property of individual loci that control the trait, the enhanced kurtosis is highly likely to be an emergent property that arises directly from the loci being mutationally nonequivalent. We present a method of incorporating nonequivalent loci into quantitative genetic modeling and give an approximate relation between the kurtosis of the mutant distribution and the degree of mutational nonequivalence of loci. We go on to ask whether incorporating the experimentally observed kurtosis through nonequivalent loci, rather than at locus level, affects any biologically important conclusions of quantitative genetic modeling. Concentrating on the maintenance of quantitative genetic variation by mutation-selection balance, we conclude that typically nonequivalent loci yield a genetic variance that is of order 10% smaller than that obtained from the previous approaches. For large populations, when the kurtosis is large, the genetic variance may be <50% of the result of equivalent loci, with Gaussian distributions of mutant effects. PMID:12072483

  14. Aspergillus nidulans mutants defective in stc gene cluster regulation.

    PubMed Central

    Butchko, R A; Adams, T H; Keller, N P

    1999-01-01

    The genes involved in the biosynthesis of sterigmatocystin (ST), a toxic secondary metabolite produced by Aspergillus nidulans and an aflatoxin (AF) precursor in other Aspergillus spp., are clustered on chromosome IV of A. nidulans. The sterigmatocystin gene cluster (stc gene cluster) is regulated by the pathway-specific transcription factor aflR. The function of aflR appears to be conserved between ST- and AF-producing aspergilli, as are most of the other genes in the cluster. We describe a novel screen for detecting mutants defective in stc gene cluster activity by use of a genetic block early in the ST biosynthetic pathway that results in the accumulation of the first stable intermediate, norsolorinic acid (NOR), an orange-colored compound visible with the unaided eye. We have mutagenized this NOR-accumulating strain and have isolated 176 Nor(-) mutants, 83 of which appear to be wild type in growth and development. Sixty of these 83 mutations are linked to the stc gene cluster and are likely defects in aflR or known stc biosynthetic genes. Of the 23 mutations not linked to the stc gene cluster, 3 prevent accumulation of NOR due to the loss of aflR expression. PMID:10511551

  15. Pediococcus cerevisiae mutant with altered transport of folates.

    PubMed

    Mandelbaum-Shavit, F; Grossowicz, N

    1975-08-01

    A Pediococcus cerevisiae mutant that actively accumulated folate (PteGlu), in contrast to the wild-type, was also found to exhibit changes in the pattern of uptake of 5-methyl-tetrahydrofolate (5-CH3-H4PteGlu) and amethopterin. Most of the 5-CH3-H4PteGlue accumulated through a glucose- and temperature-dependent process, and a concentrative uptake was also found in gluocse-starved cells and in cells incubated at OC. About 75% of the accumulated 5-CH3-H4PteGlu exchanged with amethopterin. In contrast to the wild type, the mutant accumulated both diastereoisomers of 5-CH3-H4PteGlue by glucose-dependent and glucose-independent processes. Amethopterin and PteGlue competitively inhibited the uptake in both processes, with an apparent lower affinity of the carrier for PteGlu than for the analogue. p-Chloromercuribenzoate strongly inhibited the uptake (75%). The p-chloromercuribenzoate-nonsusceptible and temperature-independent uptake was also competed by amethopterin. Metabolic poisons like sodium azide, potassium fluoride, iodoacetate, and 2,4-dimitrophenol inhibited the glucose-dependent process. Uptake, in the absence of glucose, was enhanced by sodium azide and potassium fluoride.

  16. A Mutant of Arabidopsis with Increased Levels of Stearic Acid.

    PubMed Central

    Lightner, J.; Wu, J.; Browse, J.

    1994-01-01

    A mutation at the fab2 locus of Arabidopsis caused increased levels of stearate in leaves. The increase in leaf stearate in fab2 varied developmentally, and the largest increase occurred in young leaves, where stearate accounted for almost 20% of total leaf fatty acids. The fatty acid composition of leaf lipids isolated from the fab2 mutant showed increased stearate in all the major glycerolipids of both the chloroplast and extrachloroplast membranes. Although the stearate content was increased, the fab2 mutant still contained abundant amounts of 18:1, 18:2, and 18:3 fatty acids. These results are consistent with the expectations for a mutation partially affecting the action of the stromal stearoyl-acyl carrier protein desaturase. Positional analysis indicated that the extra 18:0 is excluded with high specificity from the sn-2 position of both chloroplast and extrachloroplast glycerolipids. Although stearate content was increased in all the major leaf membrane lipids, the amount of increase varied considerably among the different lipids, from a high of 25% of fatty acids in phosphatidylcholine to a low of 2.9% of fatty acids in monogalactosyldiacylglycerol. PMID:12232421

  17. Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity

    PubMed Central

    Crum, Mary A.; Sewell, B. Trevor; Benedik, Michael J.

    2016-01-01

    Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme’s thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its kcat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein–protein interfaces leading to the protein quaternary structure. PMID:27570524

  18. Energy Homeostasis Control in Drosophila Adipokinetic Hormone Mutants

    PubMed Central

    Gáliková, Martina; Diesner, Max; Klepsatel, Peter; Hehlert, Philip; Xu, Yanjun; Bickmeyer, Iris; Predel, Reinhard; Kühnlein, Ronald P.

    2015-01-01

    Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based on AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone [termed “adipokinetic hormone precursor-related peptide” (APRP)] is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. Here we employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, we provide evidence for a negative autoregulatory loop in Akh gene regulation. PMID:26275422

  19. Computational and Experimental Study of Neuroglobin and Mutants

    NASA Astrophysics Data System (ADS)

    Nelson, Lauren; Cho, Samuel; Kim-Shaprio, Daniel

    Neuroglobin (Ngb) is a hexacoordinated heme protein that is closely related to hemoglobin and myoglobin and normally found in the brain and nervous systems. It is involved in cellular oxygen homeostasis and reversibly binds to oxygen with a higher binding affinity than hemoglobin. To protect the brain tissue from hypoxic or ischemic conditions, Ngb increases oxygen availability. We have previously shown that a mutant form of Ngb reduces nitrite to nitric oxide 50x faster than myoglobin and 500x faster than hemoglobin. It also tightly binds to carbon monoxide (CO) with an association rate that is 500x faster than hemoglobin. To analyze the structure of neuroglobin and the characteristics causing these phenomena, we performed 3 sets of 1 microsecond molecular dynamic (MD) simulations of wild-type oxidized and reduced human Ngb and their C46A, C55A, H64L, and H64Q mutants. We also directly compare our MD simulations with time-resolved absorption spectroscopy. These studies will help identify treatments for diseases involving low nitric oxide availability and carbon monoxide poisoning. This research was supported by an NIH NSRA predoctoral fellowship in the Structural and Computational Biophysics Program training Grant (T32GM095440-05).

  20. JC polyomavirus mutants escape antibody-mediated neutralization.

    PubMed

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  1. Improved production of spiramycin by mutant Streptomyces ambofaciens.

    PubMed

    Jin, Zhi-hua; Cen, Pei-lin

    2004-06-01

    Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S. ambofaciens XC 2-37 were studied. The potency of S. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation. The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m(3) fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

  2. Identification of Drosophila Mutants Affecting Defense to an Entomopathogenic Fungus

    PubMed Central

    Lu, Hsiao-Ling; Wang, Jonathan B.; Brown, Markus A.; Euerle, Christopher; St. Leger, Raymond J.

    2015-01-01

    Fungi cause the majority of insect disease. However, to date attempts to model host–fungal interactions with Drosophila have focused on opportunistic human pathogens. Here, we performed a screen of 2,613 mutant Drosophila lines to identify host genes affecting susceptibility to the natural insect pathogen Metarhizium anisopliae (Ma549). Overall, 241 (9.22%) mutant lines had altered resistance to Ma549. Life spans ranged from 3.0 to 6.2 days, with females being more susceptible than males in all lines. Speed of kill correlated with within-host growth and onset of sporulation, but total spore production is decoupled from host genotypes. Results showed that mutations affected the ability of Drosophila to restrain rather than tolerate infections and suggested trade-offs between antifungal and antibacterial genes affecting cuticle and gut structural barriers. Approximately, 13% of mutations where in genes previously associated with host pathogen interactions. These encoded fast-acting immune responses including coagulation, phagocytosis, encapsulation and melanization but not the slow-response induction of anti-fungal peptides. The non-immune genes impact a wide variety of biological functions, including behavioral traits. Many have human orthologs already implicated in human disorders; while others were mutations in protein and non-protein coding genes for which disease resistance was the first biological annotation. PMID:26202798

  3. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

    Block, T.; Kon, C.; Breckenridge, B.M.

    1984-10-01

    PCl2 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ..mu..m 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(BETA,..gamma..-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, N/sub s/. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PCl2 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PCl2 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

  4. Flavonoid accumulation patterns of transparent testa mutants of arabidopsis

    NASA Technical Reports Server (NTRS)

    Peer, W. A.; Brown, D. E.; Tague, B. W.; Muday, G. K.; Taiz, L.; Murphy, A. S.

    2001-01-01

    Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.

  5. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  6. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    PubMed

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

  7. Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase[C][W][OPEN

    PubMed Central

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-01-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants. PMID:24357602

  8. Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin-abundant mutants and a new hypomorphic allele of GDSL lipase.

    PubMed

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-02-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

  9. A novel mutant p53 binding partner BAG5 stabilizes mutant p53 and promotes mutant p53 GOFs in tumorigenesis

    PubMed Central

    Yue, Xuetian; Zhao, Yuhan; Huang, Grace; Li, Jun; Zhu, Junlan; Feng, Zhaohui; Hu, Wenwei

    2016-01-01

    Tumor suppressor p53 is the most frequently mutated gene in human tumors. Many tumor-associated mutant p53 (mutp53) proteins gain new tumor-promoting activities, including increased proliferation, metastasis and chemoresistance of tumor cells, which are defined as gain-of-functions (GOFs). Mutp53 proteins often accumulate at high levels in human tumors, which is important for mutp53 to exert their GOFs. The mechanism underlying mutp53 proteins accumulation in tumors is not fully understood. Here, we report that BAG5, a member of Bcl-2-associated athanogene (BAG) family proteins, promotes mutp53 accumulation in tumors, which in turn enhances mutp53 GOFs. Mechanistically, BAG5 interacts with mutp53 proteins to protect mutp53 from ubiquitination and degradation by E3 ubiquitin ligases MDM2 and CHIP, which in turn promotes mutp53 protein accumulation and therefore GOFs in promoting cell proliferation, tumor growth, cell migration and chemoresistance. BAG5 is frequently overexpressed in many human tumors and the overexpression of BAG5 is associated with poor prognosis of cancer patients. Altogether, this study revealed that inhibition of mutp53 degradation by BAG5 is a novel and critical mechanism underlying mutp53 protein accumulation and GOFs in cancer. Furthermore, our results also uncovered that promoting mutp53 accumulation and GOFs is a novel mechanism of BAG5 in tumorigenesis. PMID:27807478

  10. Genome-Wide Screen for Oxalate-Sensitive Mutants of Saccharomyces cerevisiae▿ †

    PubMed Central

    Cheng, V.; Stotz, H. U.; Hippchen, K.; Bakalinsky, A. T.

    2007-01-01

    Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a Saccharomyces cerevisiae deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the rgp1Δ, ric1Δ, snf7Δ, vps16Δ, vps20Δ, and vps51Δ mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2Δ, vps16Δ, vps51Δ, ric1Δ, and rib4Δ mutants, was lethal. With the exception of the rib4Δ mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4Δ mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5. PMID:17644632

  11. Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    PubMed Central

    Abdul-Hussein, Saba; Rahl, Karin; Moslemi, Ali-Reza; Tajsharghi, Homa

    2013-01-01

    Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of β-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-β-TMEGFP mutant showed perinuclear aggregates. The G53ins-β-TMEGFP mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-β-TMEGFP and E122K-β-TMEGFP mutants induced the formation of rod-like structures in human cells. The N202K-β-TMEGFP mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant β-TMEGFP in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related β-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein. PMID:24039757

  12. A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

    PubMed

    Su, Shih-Heng; Krysan, Patrick J

    2016-12-01

    Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.

  13. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression.

    PubMed

    Lima, Juliana Oliveira; Pereira, Jorge Fernando; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de

    2017-02-09

    Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

  14. Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

    PubMed

    Porwollik, Steffen; Santiviago, Carlos A; Cheng, Pui; Long, Fred; Desai, Prerak; Fredlund, Jennifer; Srikumar, Shabarinath; Silva, Cecilia A; Chu, Weiping; Chen, Xin; Canals, Rocío; Reynolds, M Megan; Bogomolnaya, Lydia; Shields, Christine; Cui, Ping; Guo, Jinbai; Zheng, Yi; Endicott-Yazdani, Tiana; Yang, Hee-Jeong; Maple, Aimee; Ragoza, Yury; Blondel, Carlos J; Valenzuela, Camila; Andrews-Polymenis, Helene; McClelland, Michael

    2014-01-01

    We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

  15. Characterization of epitopes on the rabies virus glycoprotein by selection and analysis of escape mutants.

    PubMed

    Fallahi, Firouzeh; Wandeler, Alexander I; Nadin-Davis, Susan A

    2016-07-15

    The glycoprotein (G) is the only surface protein of the lyssavirus particle and the only viral product known to be capable of eliciting the production of neutralizing antibodies. In this study, the isolation of escape mutants resistant to monoclonal antibody (Mab) neutralization was attempted by a selection strategy employing four distinct rabies virus strains: the extensively passaged Evelyn Rokitnicki Abelseth (ERA) strain and three field isolates representing two bat-associated variants and the Western Canada skunk variant (WSKV). No escape mutants were generated from either of the bat-associated viral variants but two neutralization mutants were derived from the WSKV isolate. Seven independent ERA mutants were recovered using Mabs directed against antigenic sites I (four mutants) and IIIa (three mutants) of the glycoprotein. The cross-neutralization patterns of these viral mutants were used to determine the precise location and nature of the G protein epitopes recognized by these Mabs. Nucleotide sequencing of the G gene indicated that those mutants derived using Mabs directed to antigenic site (AS) III all contained amino acid substitutions in this site. However, of the four mutants selected with AS I Mabs, two bore mutations within AS I as expected while the remaining two carried mutations in AS II. WSKV mutants exhibited mutations at the sites appropriate for the Mabs used in their selection. All ERA mutant preparations were more cytopathogenic than the parental virus when propagated in cell culture; when in vivo pathogenicity in mice was examined, three of these mutants exhibited reduced pathogenicity while the remaining four mutants exhibited comparable pathogenic properties to those of the parent virus.

  16. Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

    PubMed

    Arai, Sayuri; Hayashihara, Kayoko; Kanamoto, Yuki; Shimizu, Kazunori; Hirokawa, Yasutaka; Hanai, Taizo; Murakami, Akio; Honda, Hiroyuki

    2017-04-12

    Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported.n this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72-h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. This article is protected by copyright. All rights reserved.

  17. Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

    PubMed

    Lambright, D G; Balasubramanian, S; Boxer, S G

    1989-05-05

    Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively

  18. MUTANT GENES REGULATING THE INDUCIBILITY OF KYNURENINE SYNTHESIS.

    PubMed

    RIZKI, T M

    1964-05-01

    Alterations in the cellular synthesis of kynurenine in the larval fatbody of Drosophila melanogaster may be obtained by feeding the precursor tryptophan or by changing the genotype. In the wild type Ore-R strain, autofluorescent kynurenine globules normally occur in the cells in the anterior regions of the fatbody designated as regions 1, 2, and 3. When tryptophan is included in the larval diet, kynurenine will develop throughout the entire fatbody, thus extending to the cells in regions 4, 5, and 6. In the fatbodies of both the sepia mutant strain and the mutant combinations of the suppressible vermilion alleles with the suppressor gene (su(2)-s, v(1) and su(2)-s, v(2)), kynurenine is found in the cells from region 1 through region 4. This involvement of additional cells in the synthesis of kynurenine occurs under the usual culture conditions for Drosophila. When sepia larvae are fed tryptophan, kynurenine appears in all of the cells of the fatbody. However, dietary tryptophan does not induce kynurenine production in cells in regions 5 and 6 in the mutant combination su(2)-s, v(1) or su(2)-s, v(2). In the latter strains, an increase in the quantity of kynurenine in the fatbody is detected, but this increase remains limited to the same cells in which kynurenine production is found under normal feeding conditions. When the v(36f) allele is combined with the su(2)-s allele, an extremely faint autofluorescence characteristic of kynurenine is found in some of the anteriormost fat cells of regions 1 and 2. This autofluorescence becomes intensified when tryptophan is fed to su(2)-s, v(36f) larvae. The genetic control of kynurenine synthesis in the cells of the fatbody of Drosophila melanogaster has been previously demonstrated. The present observations establish genetic regulation of the ability to induce kynurenine production within a cell through the administration of the inducer tryptophan. Kynurenine production has been considered as a unit function of the cell as a

  19. Increased volatile anesthetic requirement in short-sleeping Drosophila mutants

    PubMed Central

    Weber, Bernd; Schaper, Christian; Bushey, Daniel; Rohlfs, Marko; Steinfath, Markus; Tononi, Giulio; Cirelli, Chiara; Scholz, Jens; Bein, Berthold

    2009-01-01

    Background Anesthesia and sleep share physiological and behavioral similarities. The anesthetic requirement of the recently identified Drosophila mutant minisleeper and other Drosophila mutants was investigated. Methods Sleep and wakefulness were determined by measuring activity of individual wild-type and mutant flies. Based on the response of the flies at different concentrations of the volatile anesthetics isoflurane and sevoflurane, concentration-response curves were generated and EC50 values were calculated. Results The average amount of daily sleep in wild-type Drosophila (n=64) was 965 ±15 minutes and 1022 ± 29 in na[har38] p>0.05; n=32) (mean ± SEM, all p compared to wild-type and other shaker alleles). Shmns flies slept 584 ±13 minutes (n=64, p<0.01), Sh102 412 ± 22 minutes (n=32, p<0.01) and Sh120 782 ± 25 minutes (n=32, p<0.01). The EC50 values for isoflurane were 0.706 (95% confidence interval 0.649 to 0.764, n=661) and for sevoflurane 1.298 (1.180 to 1.416, n=522) in wild-type Drosophila, 1.599 (1.527 to 1.671, n=308) and 2.329 (2.177 to 2.482, n=282) in Sh102, 1.306 (1.212 to 1.400, n=393) and 2.013 (1.868 to 2.158, n=550) in Shmns, 0.957 (0.860 to 1.054, n=297) and 1.619 (1.508 to 1.731, n=386) in Sh120, and 0.6154 (0.581 to 0.649, n=360; p<0.05) and 0.9339 (0.823 to 1.041, n= 274) in na[har38], respectively (all p<0.01). Conclusions A single-gene mutation in Drosophila that causes an extreme reduction in daily sleep is responsible for a significant increase in the requirement of volatile anesthetics. This suggests that a single gene mutation affects both sleep behavior and anesthesia and sedation. PMID:19164958

  20. Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2α Kinase Levels in NRAS(Q61) Mutant Cells.

    PubMed

    Posch, Christian; Sanlorenzo, Martina; Vujic, Igor; Oses-Prieto, Juan A; Cholewa, Brian D; Kim, Sarasa T; Ma, Jeffrey; Lai, Kevin; Zekhtser, Mitchell; Esteve-Puig, Rosaura; Green, Gary; Chand, Shreya; Burlingame, Alma L; Panzer-Grümayer, Renate; Rappersberger, Klemens; Ortiz-Urda, Susana

    2016-10-01

    In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.

  1. Characterization of Cytokinetic Mutants Using Small Fluorescent Probes.

    PubMed

    Smertenko, Andrei; Moschou, Panagiotis; Zhang, Laining; Fahy, Deirdre; Bozhkov, Peter

    2016-01-01

    Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

  2. Antiphase synchronization in a flagellar-dominance mutant of Chlamydomonas.

    PubMed

    Leptos, Kyriacos C; Wan, Kirsty Y; Polin, Marco; Tuval, Idan; Pesci, Adriana I; Goldstein, Raymond E

    2013-10-11

    Groups of beating flagella or cilia often synchronize so that neighboring filaments have identical frequencies and phases. A prime example is provided by the unicellular biflagellate Chlamydomonas reinhardtii, which typically displays synchronous in-phase beating in a low-Reynolds number version of breaststroke swimming. We report the discovery that ptx1, a flagellar-dominance mutant of C. reinhardtii, can exhibit synchronization in precise antiphase, as in the freestyle swimming stroke. High-speed imaging shows that ptx1 flagella switch stochastically between in-phase and antiphase states, and that the latter has a distinct waveform and significantly higher frequency, both of which are strikingly similar to those found during phase slips that stochastically interrupt in-phase beating of the wild-type. Possible mechanisms underlying these observations are discussed.

  3. The genetics of Fraser syndrome and the blebs mouse mutants.

    PubMed

    Smyth, Ian; Scambler, Peter

    2005-10-15

    Fraser syndrome is a recessive multisystem disorder characterized by embryonic epidermal blistering, cryptophthalmos, syndactyly, renal defects and a range of other developmental abnormalities. More than 17 years ago, the family of four mapped mouse blebs mutants was proposed as models of this disorder, given their striking phenotypic overlaps. In the last few years, these loci have been cloned, uncovering a family of three large extracellular matrix proteins and an intracellular adapter protein which are required for normal epidermal adhesion early in development. The proteins have also been shown to play a crucial role in the development and homeostasis of the kidney. We review the cloning and characterization of these genes and explore the consequences of their loss.

  4. Identification of mutant firefly luciferases that efficiently utilize aminoluciferins.

    PubMed

    Harwood, Katryn R; Mofford, David M; Reddy, Gadarla R; Miller, Stephen C

    2011-12-23

    Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.

  5. Use of Arabidopsis eceriferum mutants to explore plant cuticle biosynthesis.

    PubMed

    Samuels, Lacey; DeBono, Allan; Lam, Patricia; Wen, Miao; Jetter, Reinhard; Kunst, Ljerka

    2008-05-31

    The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of pathogenic microorganisms. The cuticle is made up of a tough crosslinked polymer called "cutin" and a protective wax layer that seals the plant surface. The waxy layer of the cuticle is obvious on many plants, appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf thanks to light scattering crystals present in the wax. Because the cuticle is an essential adaptation of plants to a terrestrial environment, understanding the genes involved in plant cuticle formation has applications in both agriculture and forestry. Today, we'll show the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.

  6. Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice

    PubMed Central

    Leushacke, Marc; Li, Ling; Wong, Julin S.; Chiam, Poh Cheang; Rahmat, Siti Aishah Binte; Mann, Michael B.; Mann, Karen M.; Barker, Nick; Lozano, Guillermina; Terzian, Tamara; Lane, David P.

    2015-01-01

    The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy. PMID:26255629

  7. Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice.

    PubMed

    Goh, Amanda M; Xue, Yuezhen; Leushacke, Marc; Li, Ling; Wong, Julin S; Chiam, Poh Cheang; Rahmat, Siti Aishah Binte; Mann, Michael B; Mann, Karen M; Barker, Nick; Lozano, Guillermina; Terzian, Tamara; Lane, David P

    2015-07-20

    The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy.

  8. Curvature Dynamics of α-Synuclein Familial Parkinson Disease Mutants

    PubMed Central

    Perlmutter, Jason D.; Braun, Anthony R.; Sachs, Jonathan N.

    2009-01-01

    α-Synuclein remains a protein of interest due to its propensity to form fibrillar aggregates in neurodegenerative disease and its putative function in synaptic vesicle regulation. Herein, we present a series of atomistic molecular dynamics simulations of wild-type α-synuclein and three Parkinson disease familial mutants (A30P, A53T, and E46K) in two distinct environments. First, in order to match recent NMR experiments, we have simulated each protein bound to an SDS detergent micelle. Second, in order to connect more closely to the true biological environment, we have simulated the proteins bound to a 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid bilayer. In the micelle-bound case, we find that the wild type and all of the variants of α-synuclein flatten the underlying micelle, decreasing its surface area. A30P is known to lessen α-synuclein/membrane affinity and, consistent with experiment, destabilizes the simulated secondary structure. In the case of A53T, our simulations reveal a range of stabilizing hydrogen bonds that form with the threonine. In both environments, the E46K mutation, which is known to increase bilayer affinity, leads to an additional hydrogen bond between the protein and either the detergent or lipid. Simulations indicate that αS and its variants are less dynamic in the bilayer than in the micelle. Furthermore, the simulations of the mutants suggest how changes in the structure and dynamics of α-synuclein may affect its biological role. PMID:19126542

  9. Mutant HSP70 reverses autoimmune depigmentation in vitiligo.

    PubMed

    Mosenson, Jeffrey A; Zloza, Andrew; Nieland, John D; Garrett-Mayer, Elizabeth; Eby, Jonathan M; Huelsmann, Erica J; Kumar, Previn; Denman, Cecele J; Lacek, Andrew T; Kohlhapp, Frederick J; Alamiri, Ahmad; Hughes, Tasha; Bines, Steven D; Kaufman, Howard L; Overbeck, Andreas; Mehrotra, Shikhar; Hernandez, Claudia; Nishimura, Michael I; Guevara-Patino, Jose A; Le Poole, I Caroline

    2013-02-27

    Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell-mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

  10. A pedigree analysis of two homozygous mutant Gitelman syndrome cases.

    PubMed

    Luo, Jiewei; Yang, Xiao; Liang, Jixing; Li, Weihua

    2015-01-01

    Gitelman syndrome (GS) is a salt-wasting tubulointerstitial disease of autosomal recessive inheritance (OMIM613395) caused by genic mutation of SLC12A3, which codes thiazide-sensitive Na-Cl cotransporter (NCCT) gene. The gene mutation of the majority of GS patients is compound heterozygous. This study analyzes two cases of GS gene mutation and the clinical phenotype. Twenty patients of two GS pedigrees underwent direct sequence alignment of 26 exons of SLC12A3 to spot and locate mutant site. Proband A of Pedigree I had three mutant sites: Arg928Cys, a homozygote, missense mutation, and two homozygous silent mutations, Ala122Ala and Thr465Thr, and 8 members of Pedigree I carried Arg928Cy heterozygous mutation. Proband B of Pedigree II had a homozygote, Ser710X, and a termination codon was spotted, which would inevitably be translated into abridged and defective protein, and 7 members had Ser710X heterozygous mutation. The heterozygous mutation carriers of the two pedigrees often have stimulus-controlled hypokalemia after strenuous exercise. The parents of Proband A are cousins, a case of intermarriage. Both probands show hypokalemia, hypochloraemia, hypocalcinuria, hyperreninemia, and hyperaldosteronemia; Proband A has normal serum magnesium and increased urinary sodium excretion, while Proband B has hypomagnesemia and increased urinary magnesium ion excretion. Both probands have normal or lower blood pressure, weakness and numbness of lower extremities, muscular soreness, and occasional palpitations and chest discomfort. Proband A wearies easily and Proband B has occasional joint numbness and pain. These two homozygous mutations are responsible for the morbidity of two GS families and they show heterogenicity of clinical phenotype.

  11. A mutant gene that increases gibberellin production in Brassica

    SciTech Connect

    Rood, S.B. ); Williams, P.H. ); Pearce, D.; Pharis, R.P. ); Murofushi, Noboru ); Mander, L.N. )

    1990-07-01

    A single gene mutant (elongated internode (ein/ein)) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A{sub 3} (GA{sub 3}) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA{sub 1} and GA{sub 3} were estimated by gas chromatography-selected ion monitoring using ({sup 2}H)GA{sub 1} as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA{sub 20} and GA{sub 1}, and the rate of GA{sub 19} metabolism were simultaneously analyzed. Levels of GA{sub 1} and GA{sub 20} were 4.6- and 12.9-fold higher, respectively, and conversions to GA{sub 20} and GA{sub 1} were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA{sub 1} biosynthesis in ein, the conversion of ({sup 3}H)GA{sub 20} to ({sup 3}H) GA{sub 1} was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA{sub 1} biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A{sub 1} and A{sub 3}.

  12. PHB biosynthesis in catabolite repression mutant of Burkholderia sacchari.

    PubMed

    Lopes, Mateus Schreiner Garcez; Gosset, Guillermo; Rocha, Rafael Costa Santos; Gomez, José Gregório Cabrera; Ferreira da Silva, Luiziana

    2011-10-01

    Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.

  13. Mutants of plant genes for developing cancer vaccines.

    PubMed

    Massa, Silvia; Paolini, Francesca; Spanò, Laura; Franconi, Rosella; Venuti, Aldo

    2011-01-01

    Preventive Human Papillomavirus (HPV) vaccination is an expensive practice and it may be an insufficient tool to tackle cervical cancer worldwide. Therapeutic intervention is seeking for safe/effective vaccines inducing the activation of CD8+ cytotoxic T lymphocytes (CTLs) that is required to clear the tumor. Linking a tumor-specific antigen (i.e. the E7 oncoprotein of the 'high risk' HPVs) to molecules able to increase its immune 'visibility' represents a strategy to force the immune system to fight cancer. We focused on plants as sources of innovative immunostimulatory sequences. We have already shown the anti-cancer activity obtained by fusing E7GGG (a mutagenized E7 gene from the high risk HPV type 16) to the coat protein of a plant virus, the Potato Virus X. We are now investigating plant-derived carriers, such as the 'Ribosome inactivating proteins' (RIPs), so far used to develop immunotoxins for targeted cancer therapy. Beside toxicity, RIPs have other features (i.e. immunogenicity, ability to modulate immune functions and apoptosis induction) that could be useful tools to use in tumor immunotherapy. A non toxic mutant of saporin (SAP-KQ) was used as a carrier for the E7GGG gene in the context of a DNA-based vaccine. We show here that fusion constructs of SAP-KQ with E7GGG can induce E7-specific Immunoglobulins (IgGs), CTLs and Delayed-Type Hypersensitivity (DTH) affecting the growth of E7-expressing tumors in mice. These data demonstrate that mutant plant genes hold promise to improve the poor immunogenicity of tumor-associated cancer antigens and could contribute to the evolution of new cancer immunotherapy.

  14. A Rice Mutant Defective in Si Uptake1

    PubMed Central

    Ma, Jian Feng; Tamai, Kazunori; Ichii, Masahiko; Wu, Guo Feng

    2002-01-01

    Rice (Oryza sativa) accumulates silicon (Si) in the tops to levels up to 10.0% of shoot dry weight, but the mechanism responsible for high Si uptake by rice roots is not understood. We isolated a rice mutant (GR1) that is defective in active Si uptake by screening M2 seeds (64,000) of rice cv Oochikara that were treated with 10−3 m sodium azide for 6 h at 25oC. There were no phenotypic differences between wild type (WT) and GR1 except that the leaf blade of GR1 remained droopy when Si was supplied. Uptake experiments showed that Si uptake by GR1 was significantly lower than that by WT at both low and high Si concentrations. However, there was no difference in the uptake of other nutrients such as phosphorus and potassium. Si concentration in the xylem sap of WT was 33-fold that of the external solution, but that of GR1 was 3-fold higher than the external solution at 0.15 mm Si. Si uptake by WT was inhibited by metabolic inhibitors including NaCN and 2,4-dinitrophenol and by low temperature, whereas Si uptake by GR1 was not inhibited by these agents. These results suggest that an active transport system for Si uptake is disrupted in GR1. Analysis of F2 populations between GR1 and WT showed that roots with high Si uptake and roots with low Si uptake segregated at a 3:1 ratio, suggesting that GR1 is a recessive mutant of Si uptake. PMID:12481095

  15. Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production.

    PubMed

    Mehtani, Juhi; Arora, Neha; Patel, Alok; Jain, Priyanka; Pruthi, Parul A; Poluri, Kirshna Mohan; Pruthi, Vikas

    2017-03-21

    The aim of this work was to generate high lipid accumulating mutants of Chlorella minutissima (CM) using ethyl methyl sulphonate (EMS) as a random chemical mutagen. Amid the 5% surviving cells after exposure to EMS (2M), three fast growing mutants (CM2, CM5, CM7) were selected and compared with wild type for lipid productivity and biochemical composition. Among these mutants, CM7 showed the maximum biomass (2.4g/L) and lipid content (42%) as compared to wild type (1.5g/L; 27%). Further, the mutant showed high photosynthetic pigments with low starch content signifying the re-allocation of carbon flux to lipid. The obtained mutant showed no visible morphological changes in comparison to its WT. The fatty acid profile showed increase in monounsaturated fatty acids while decreased saturated and polyunsaturated fatty acids signifying good quality biodiesel. The mutant strain thus obtained can be optimized further and applied for enhanced biodiesel production.

  16. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

    PubMed Central

    Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S

    1995-01-01

    An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants. PMID:7770517

  17. Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

    SciTech Connect

    Chandra, P. Manish; Brannigan, James A.; Prabhune, Asmita; Pundle, Archana; Turkenburg, Johan P.; Dodson, G. Guy; Suresh, C. G.

    2005-01-01

    The production, crystallization and characterization of three inactive mutants of penicillin V acylase from B. sphaericus in their respective precursor and processed forms are reported. The space groups are different for the native enzyme and the mutants. The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.

  18. Evaluation of genetic variability in in vitro sodium azide-induced Dendrobium 'Earsakul' mutants.

    PubMed

    Wannajindaporn, A; Poolsawat, O; Chaowiset, W; Tantasawat, P A

    2014-07-24

    In vitro mutagenesis of Dendrobium 'Earsakul' was carried out by incubating the protocorm-like bodies in 0-5 mM sodium azide for 1 h. Twenty-eight putative mutants were evaluated for genetic variability compared to untreated control plants using inter-simple sequence repeat (ISSR) analysis. Polymorphic fragments were produced by 9 of 12 ISSR primers. A total of 173 amplified ISSR fragments varying in size from 140 to 5000 bp were obtained, 39 of which were polymorphic (22.5%). Of the 28 putative mutants, 15 (53.6%) showed altered genetic profiles compared to control and were identified as mutants. These results suggest that sodium azide can be effectively utilized to generate mutants in Dendrobium 'Earsakul', and ISSR provides a powerful tool that allows efficient early detection of these mutants. The identified mutants are currently being multiplied for further evaluation of their horticultural characteristics.

  19. Spontaneous Mutation at the Mtr Locus of Neurospora: The Spectrum of Mutant Types

    PubMed Central

    Stadler, D.; Macleod, H.; Dillon, D.

    1991-01-01

    We have isolated 135 strains of Neurospora which have mutations at the mtr locus resulting from independent spontaneous events. mtr is the structural gene for the neutral amino acid permease. The mutants have been characterized by their reversion behavior (both spontaneous and induced) and by hybridization studies of restriction digests of their DNA. About half of the mutants (54%) appear to result from single base-pair substitutions. Thirty-four percent have deletions, including some which extend into neighboring genes. Most of the remaining mutants have insertions. Several of the insertions are tandem duplications of 400-1000 bp and these mutants are unstable, reverting to mtr(+) with a high frequency. When tandem-duplication mutants go through a cross, they are modified: the mutant progeny are fully stable. This modification is probably due to RIP (repeat-induced point mutation). This process has an important bearing on the comparison of germinal to somatic mutation. PMID:1718818

  20. [LIGHT-DEPENDENT SYNTHESIS OF CELL MEMBRANES IN THE Brc-1 MUTANT OF CHLAMYDOMONAS REINHARDTII].

    PubMed

    Semenova, G A; Chekunova, E M; Ladygin, V G

    2015-01-01

    The structural organization of cells of the Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the light and in the dark has been studied. The Brc-1 mutant contains the brc-1 mutation in the nucleus gene LTS3. In the light, all membrane structures in mutant cells form normally and are well developed. In the dark under heterotrophic conditions, the mutant cells grew and divided well, however, all its cell membranes: plasmalemma, tonoplast, mitochondrial membranes, membranes of the nucleus shell and chloroplast, thylakoids, and the membranes of dictiosomes of the Golgi apparatus were not detected. In the dark under heterotrophic conditions, mutant cells well grow and divide. It were shown that a short-term (1-10 min) exposure of Brc-1 mutant cells to light leads to the restoration of all above-mentioned membrane structures. Possible reasons for the alterations of membrane structures are discussed.

  1. Isolation and preliminary characterization of auxotrophic and morphological mutants of the yeastlike form of Paracoccidioides brasiliensis.

    PubMed Central

    San Blas, F; Centeno, S

    1977-01-01

    N-methyl-N'-nitro-N-nitrosoguanidine, which is known to be a very effective mutagen in many systems, was used to induce mutants in the yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9, an imperfect fungus. Forty-three auxotrophic and 27 prototrophic morphological mutants were isolated after treatment with 50 mug of nitrosoguanidine per ml in 0.1 M citrate buffer, pH 5.0. Auxotrophic mutants required primarily either amino acids, purines, or pyrimidines. Some auxotrophs were also morphological mutants. The main morphological difference from the parental strain was the texture or the color of the yeast-like colonies. Only one prototrophic morphological mutant differed in the size and form of the yeastlike cells when compared with the parental strain. Suxotrophic mutants were used in pairwise combination to attempt heterokaryon formation without success. Images PMID:830638

  2. C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

    PubMed

    Kanteti, Rajani; Dhanasingh, Immanuel; El-Hashani, Essam; Riehm, Jacob J; Stricker, Thomas; Nagy, Stanislav; Zaborin, Alexander; Zaborina, Olga; Biron, David; Alverdy, John C; Im, Hae Kyung; Siddiqui, Shahid; Padilla, Pamela A; Salgia, Ravi

    2016-01-01

    We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

  3. Regulatory Mutations of Inositol Biosynthesis in Yeast: Isolation of Inositol-Excreting Mutants

    PubMed Central

    Greenberg, Miriam L.; Reiner, Barry; Henry, Susan A.

    1982-01-01

    The enzyme inositol-1-phosphate synthase (I-1-P synthase), product of the INO1 locus, catalyzes the synthesis of inositol-1-phosphate from the substrate glucose-6-phosphate. The activity of this enzyme is dramatically repressed in the presence of inositol. By selecting for mutants which overproduce and excrete inositol, we have identified mutants constitutive for inositol-1-phosphate synthase as well as a mutation in phospholipid biosynthesis. Genetic analysis of the mutants indicates that at least three loci (designated OPI1, OPI2 and OPI4) direct inositol-mediated repression of I-1-P synthase. Mutants of these loci synthesize I-1-P synthase constitutively. Three loci are unlinked to each other and to INO1, the structural gene for the enzyme. A mutant of a fourth locus, OPI3, does not synthesize I-1-P synthase constitutively, despite its inositol excretion phenotype. This mutant is preliminarily identified as having a defect in phospholipid synthesis. PMID:7047296

  4. In vivo fitness and virulence of a drug-resistant herpes simplex virus 1 mutant.

    PubMed

    Pesola, Jean M; Coen, Donald M

    2007-05-01

    Two important issues regarding a virus mutant that is resistant to an antiviral drug are its ability to replicate in animal hosts (in vivo fitness) relative to other genetic variants, including wild type, and its ability to cause disease. These issues have been investigated for a herpes simplex virus 1 mutant that is resistant to thiourea compounds, which inhibit encapsidation of viral DNA. Following corneal inoculation of mice, the mutant virus replicated very similarly to its wild-type parent in the eye, trigeminal ganglion and brain. The mutant virus was as lethal to mice as its wild-type parent following this route of inoculation. Indeed, it exhibited increased virulence. Thus, unlike most drug-resistant virus mutants, this mutant retained in vivo fitness and virulence.

  5. Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

    SciTech Connect

    Gu Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Takeda, Jun; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke . E-mail: jinkan@tom.life.h.kyoto-u.ac.jp

    2006-08-04

    Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2 mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.

  6. Isolation and characterization of outermost layer deficient mutant spores of Bacillus megaterium.

    PubMed

    Takubo, Y; Atarashi, M; Nishihara, T; Kondo, M

    1988-01-01

    Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.

  7. Genetic mapping and characterization of Pseudomonas aeruginosa mutants that hyperproduce exoproteins.

    PubMed Central

    Björklind, A; Wretlind, B; Möllegård, I; Schad, P A; Iglewski, B H; Cox, C D

    1985-01-01

    We isolated two mutants of Pseudomonas aeruginosa PAO with defective iron uptake. In contrast to the wild-type strain, the mutants produced extracellular protease activity in media containing high concentrations of salts or iron and hyperproduced elastase, staphylolytic enzyme, and exotoxin A in ordinary media (Xch mutants). The mutations were located in the 55' region of the chromosome, between the markers met-9011 and pyrD. PMID:3922951

  8. Germination-defective mutant of Neurospora crassa that responds to siderophores

    NASA Technical Reports Server (NTRS)

    Charlang, G.; Williams, N. P.

    1977-01-01

    A conditionally germination-defective mutant of Neurospora crassa has been found to be partially curable by ferricrocin and other siderophores. The mutant conidia rapidly lose their membrane-bound siderophores when suspended in buffer or growth media. Germination is consequently delayed unless large numbers of conidia are present (positive population effect). This indicates that the mutant has a membrane defect involving the siderophore attachment site.

  9. Immobilization of Organophosphorus Acid Anhydrolase Mutant Y212F on Silica Nanospheres

    DTIC Science & Technology

    2016-09-01

    IMMOBILIZATION OF ORGANOPHOSPHORUS ACID ANHYDROLASE MUTANT Y212F ON SILICA NANOSPHERES Disclaimer The findings in this report are not to be...Dec 2014 4. TITLE AND SUBTITLE Immobilization of Organophosphorus Acid Anhydrolase Mutant Y212F on Silica Nanospheres 5a. CONTRACT NUMBER...STATEMENT Approved for public release; distribution is unlimited. 13. SUPPLEMENTARY NOTES 14. ABSTRACT-LIMIT 200 We have engineered mutants of the

  10. Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity

    SciTech Connect

    Rosenberg, M.

    1984-10-01

    Enrichment for nonhydrophobic mutants of Serratia marcescens yielded two types: (i) a nonpigmented mutant which exhibited partial hydrophobic characteristics compared with the wild type, as determined by adherence to hexadecane and polystyrene; and (ii) a pigmented, nonhydrophobic mutant whose colonies were translucent with respect to those of the wild type. The data suggest that the pronounced cell surface hydrophobicity of the wild type is mediated by a combination of several surface f

  11. Proton suicide: general method for direct selection of sugar transport- and fermentation-defective mutants

    SciTech Connect

    Winkelman, J.W.; Clark, D.P.

    1984-11-01

    A positive selection procedure was devised for bacterial mutants incapable of producing acid from sugars by fermentation. The method relied on the production of elemental bromine from a mixture of bromide and bromate under acidic conditions. When wild-type Escherichia coli cells were plated on media containing a fermentable sugar and an equimolar mixture of bromide and bromate, most of the cells were killed but a variety of mutants unable to produce acid from the sugar survived. Among these mutants were those defective in (i) sugar uptake, (ii) the glycolytic pathway, and (iii) the excretion. There were also novel mutants with some presumed regulatory defects affecting fermentation.

  12. Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

    PubMed Central

    Dierks-Ventling, Christa; Tonelli, Chiara

    1982-01-01

    In excised pro1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [14C]proline, [14C]glutamic acid, [14C]glutamine, and [14C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO2 production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro1-1 mutant proline catabolism prevails over proline synthesis. PMID:16662144

  13. Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

    SciTech Connect

    Zerler, B.R.; Wallace, S.S.

    1984-02-01

    The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detecttable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants.

  14. Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly.

    PubMed Central

    Wang, Y X; Zhao, H; Harding, T M; Gomes de Mesquita, D S; Woldringh, C L; Klionsky, D J; Munn, A L; Weisman, L S

    1996-01-01

    In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole. Images PMID:8885233

  15. Production of amino acids by analog-resistant mutants of the cyanobacterium Spirulina platensis.

    PubMed Central

    Riccardi, G; Sora, S; Ciferri, O

    1981-01-01

    Mutants of Spirulina platensis resistant to 5-fluorotryptophan, beta-3-thienyl-alanine, ethionine, p-fluorophenylalanine, or azetidine-2-carboxylic acid were isolated. Some of these mutants appeared to be resistant to more than one analog and to overproduce the corresponding amino acids. A second group was composed of mutants that were resistant to one analog only. Of the latter mutants, one resistant to azetidine-2-carboxylic acid was found to overproduce proline only, whereas one resistant to fluorotryptophan and one resistant to ethionine did not overproduce any of the tested amino acids. PMID:6792182

  16. Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents.

    PubMed

    Rogers, P; Palosaari, N

    1987-12-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid, and the Spo mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum.

  17. Membrane function in lipid mutants of Arabidopsis. First year progress report

    SciTech Connect

    Browse, J.A.

    1993-06-01

    Progress on the biochemical characterization of the fad3 mutants deficient in 18:3 fatty acid synthesis and the fab2 mutant that accumulates increased amounts of 18:0 is described. Studies of the cell biology and physiology of the fab2 and fad2 mutants have provided evidence for some of the critical roles played by unsaturated fatty acids as components of plant membranes. Finally, the fab2 mutant has allowed us to carry out the first isolation and characterization of intergenic suppressor mutations in a higher plant.

  18. Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation

    SciTech Connect

    Messing, K.; Ferraris, J.; Bradley, W.E.; Swartz, J.; Seifert, A.M. )

    1989-10-01

    The frequency of hypoxanthine phosphoribosyl transferase (HPRT) mutants among peripheral T-lymphocytes of radiotherapy technicians primarily exposed to 60Co was measured by the T-cell cloning method. Mutant frequencies of these technicians in 1984 and 1986 were significantly higher than those of physiotherapy technicians who worked in a neighboring service, and correlated significantly with thermoluminescence dosimeter readings recorded during the 6 mo preceding mutant frequency determination. Correlations decreased when related to dose recorded over longer time intervals. HPRT mutant frequency determination in peripheral lymphocytes is a good measure of recently received biologically effective radiation dose in an occupationally exposed population.

  19. Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato

    PubMed Central

    Tal, M.; Imber, D.; Itai, C.

    1970-01-01

    The wilty tomato mutant, flacca, and the normal variety, Rheinlands Ruhm, were compared for kinetin-like activity in ontogeny. The mutant wilts easily because its stomata resist closure. This stomatal resistance decreases with age. The occurrence of a root factor which induces stomatal opening was inferred from grafting experiments. It was hypothesized that the excessive stomatal openings in the mutant may result from excess of kinetin-like activity in the leaf of that plant. In addition, it was suggested that the closure of stomata in the aging mutant is due to a decrease of kinetin-like activity with age. Kinetin-like activity in the leaf was determined by incorporation of labeled leucine. The concentration of cytokinins in root exudate and leaf extract was determined by the soybean callus assay. Evidence was presented of higher kinetin-like activity in the leaves of the mutant and higher cytokinin concentration in its root exudate. Cytokinin concentration in the shoot was found to be only slightly higher in the mutant than in the normal plants. Kinetin-like activity in the leaf and cytokinin concentration of root exudate decreased with age in both mutant and normal plants. Kinetin-like activity in the leaves of mutant plants, which phenocopy the normal variety as a result of continuous application of abscisic acid, was lower than in control mutant plants. The significance of these findings per se and in connection with stomatal behavior is discussed. PMID:16657469

  20. Purification and in vitro complementation of mutant histidinol dehydrogenases. [Salmonella typhimurium

    SciTech Connect

    Lee, S.Y.; Grubmeyer, C.T.

    1987-09-01

    The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the his01242 (constitutive overproducer) attenuator mutations and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl/sub 2/, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound /sup 54/Mn/sup 2 +/ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound /sup 54/Mn/sup 2 +/ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 20-mercaptoethanol was prevented by low levels of MnCl/sub 2/.

  1. Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose.

    PubMed Central

    Nevalainen, K M; Palva, E T

    1978-01-01

    Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain. Images PMID:564161

  2. Altered desferrioxamine-mediated iron utilization is a common trait of bald mutants of Streptomyces coelicolor.

    PubMed

    Lambert, Stéphany; Traxler, Matthew F; Craig, Matthias; Maciejewska, Marta; Ongena, Marc; van Wezel, Gilles P; Kolter, Roberto; Rigali, Sébastien

    2014-08-01

    Streptomyces coelicolor is an important model organism for developmental studies of filamentous GC-rich actinobacteria. The genetic characterization of mutants of S. coelicolor blocked at the vegetative mycelium stage, the so-called bald (bld) mutants that are unable to erect spore-forming aerial hyphae, has opened the way to discovering the molecular basis of development in actinomycetes. Desferrioxamine (DFO) production and import of ferrioxamines (FO; iron-complexed DFO) are key to triggering morphogenesis of S. coelicolor and we show here that growth of S. coelicolor on the reference medium for Streptomyces developmental studies is fully dependent on DFO biosynthesis. UPLC-ESI-MS analysis revealed that all bld mutants tested are affected in DFO biosynthesis, with bldA, bldJ, and ptsH mutants severely impaired in DFO production, while bldF, bldK, crr and ptsI mutants overproduce DFO. Morphogenesis of bldK and bldJ mutants was recovered by supplying exogenous iron. Transcript analysis showed that the bldJ mutant is impaired in expression of genes involved in the uptake of FO, whereas transcription of genes involved in both DFO biosynthesis and FO uptake is increased in bldK mutants. Our study allows proposing altered DFO production and/or FO uptake as a novel phenotypic marker of many S. coelicolor bld mutants, and strengthens the role of siderophores and iron acquisition in morphological development of actinomycetes.

  3. Expression of A/B zeins in single and double maize endosperm mutants.

    PubMed

    Paulis, J W; Bietz, J A; Bogyo, T P; Nelsen, T C; Darrah, L L; Zuber, M S

    1992-12-01

    Zeins, the major endosperm proteins in maize (Zea mays L.), are deficient in the essential amino acids lysine and tryptophan. Some mutant genes, like opaque-2 (o2) and floury-2 (fl2), reduce the levels of A- and B-zeins, thereby improving maize's nutritional value. Other mutants, such as amylose-extender (ae), floury-1 (fl1), soft starch (h), dull-1 (du), shrunken-1 (sh1), sugary-1 (su1), sugary-2 (su2), and waxy (wx), primarily affect starch composition, but also alter zein composition. We undertook this study to examine the effects of some of these mutant genes on A/B-zein composition and to study the interactions of these genes in double-mutant combinations. Endosperm prolamins were extracted from inbred B37, ten near-isogenic single mutants (ae, du, fl1, fl2, h, o2, sh1, su1, su2, and wx), and most double-mutant combinations. Zeins in these extracts were fractionated by reversed-phase highperformance liquid chromatography (RP-HPLC) into 22-24 peaks. Of the resulting 22 major peaks the areas of 16 (per milligram endosperm) were significantly affected by individual mutant genes relative to the zein composition of the normal inbred. In combination these genes exhibited significant epistatic interactions in regulating the expression of individual A/B zeins. Epistatic interactions were judged to be significant when the amount of a peak in a double mutant differed from the averages for the peak in the two respective single mutants. The o2 gene, alone and in combination with other mutant genes, significantly decreased the amounts of many individual zeins. The effect of the o2 gene was the greatest of all the genes examined. Various clustering techniques were used to see if mutant effects could be grouped; among these was principal component analysis, a multivariate statistical technique that analyzes all peak sizes simultaneously. Three-dimensional scatter graphs were constructed based on the first three principal components. For the single mutants, these showed no

  4. Mutant Analysis and Enzyme Subunit Complementation in Bacterial Bioluminescence in Photobacterium fischeri

    PubMed Central

    Nealson, Kenneth H.; Markovitz, Alvin

    1970-01-01

    Chemical mutagens were used to obtain mutants deficient in bioluminescence in the marine bacterium Photobacterium fischeri strain MAV. Acridine dyes were effective in the production of dark mutants but not in the production of auxotrophs. These dark mutants were all of one type and appeared to contain lesions blocking the synthesis of luciferase. ICR-191 was especially effective in the production of aldehyde mutants, i.e., dark strains that luminesce when a long-chain aldehyde such as n-decanal is added to them. However, other mutant types were isolated after treatment with ICR-191. N-methyl-N′-nitro-N-nitrosoguanidine induced many bioluminescence-deficient types with respect to both the site of the lesion and the quantitative effect on the luminescent system. We characterized the dark and dim mutants with respect to their response to exogenous decanal, levels of in vivo and in vitro luminescence, and their rates of reversion to wild type. In addition, the luciferases of the mutant strains were examined by subunit complementation. On the basis of these analyses, we identified mutants which synthesize altered luciferase, strains which are deficient in synthesis of luciferase, and aldehyde mutants. The results of analysis of luciferase from the aldehyde mutants and the complementation studies indicate that the lesions in these strains are in the luciferase itself. Results obtained with wild-type cells grown in minimal medium, and aldehyde mutant cells grown either in complete or minimal medium, indicate that a “natural aldehyde factor” is involved in in vivo light emission. These same studies showed that the long-chain aldehyde(s) could only partially substitute for the natural “aldehyde factor.” The possibility that the in vivo aldehyde factor is not a long-chain aldehyde is discussed. PMID:5479452

  5. Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes

    PubMed Central

    1987-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve

  6. Photoperiod Affects the Phenotype of Mitochondrial Complex I Mutants1[OPEN

    PubMed Central

    de Bont, Linda; Hao, Jingfang; Laureau, Constance; Rzigui, Touhami; Queval, Guillaume; Gilard, Françoise; Mauve, Caroline; Guérard, Florence; Lamothe-Sibold, Marlène; Marion, Jessica; Fresneau, Chantal; Tcherkez, Guillaume; Pineau, Bernard; De Paepe, Rosine

    2017-01-01

    Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8. Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD. We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis. PMID:27852950

  7. Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Sack, F. D.

    1989-01-01

    Gravitropism was studied in seedlings of Nicotiana sylvestris Speg. et Comes wild-type (WT) and mutant NS 458 which has a defective plastid phosphoglucomutase (EC 2.7.5.1.). Starch was greatly reduced in NS 458 compared to the WT, but small amounts of starch were detected in rootcap columella cells in NS 458 by light and electron microscopy. The roots of WT are more sensitive to gravity than mutant NS 458 roots since: (1) in mutant roots, curvature was reduced and delayed in the time course of curvature; (2) curvature of mutant roots was 24-56% that of WT roots over the range of induction periods tested; (3) in intermittent-stimulation experiments, curvature of mutant roots was 37% or less than that of WT roots in all treatments tested. The perception time, determined by intermittent-stimulation experiments, was < or = 5 s for WT roots and 30-60 s for mutant roots. The growth rates for WT and NS 458 roots were essentially equal. These results and our previous results with WT and starchless mutant Arabidopsis roots (Kiss et al. 1989, Planta 177, 198-206) support the conclusions that a full complement of starch is necessary for full gravitropic sensitivity and that amyloplasts function in gravity perception. Since a presumed relatively small increase in plastid buoyant mass (N. sylvestris mutant versus Arabidopsis mutant) significantly improves the orientation of the N. sylvestris mutant roots, we suggest that plastids are the likeliest candidates to be triggering gravity perception in roots of both mutants.

  8. The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids.

    PubMed

    Afitlhile, Meshack; Fry, Morgan; Workman, Samantha

    2015-02-01

    We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2) accumulates normal levels of fatty acids, and transcripts of fatty acid desaturases and galactolipid synthesis enzymes. The ppi2-2 mutant accumulates decreased pigments and total fatty acid content. The MGD1 gene was downregulated and the mutant accumulates decreased levels of monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3-ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. Interestingly, glycerolipids in the mutant accumulate increased levels of 18:0. A gene that encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to bilayer forming plastid lipids was decreased in the cold-acclimated wild type but not in the ppi2-2 mutant. This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of 18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty acids. We conclude that expression of the MGD1 gene and synthesis of MGDG are tightly linked to plastid biogenesis.

  9. Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role.

    PubMed

    Palenchar, Jennifer Brosius; Colman, Roberta F

    2003-02-25

    Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.

  10. Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy.

    PubMed

    Garcia, Eric L; Wen, Ying; Praveen, Kavita; Matera, A Gregory

    2016-08-01

    Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. Here, we used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies.

  11. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    SciTech Connect

    Gindt, Y.M.

    1993-04-01

    The role of the L{sub cm} (I), {beta}{sup 18} (II), and {alpha}{sup AP-B} (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, A{sub max} = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  12. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    SciTech Connect

    Gindt, Y.M.

    1993-04-01

    The role of the L[sub cm] (I), [beta][sup 18] (II), and [alpha][sup AP-B] (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, A[sub max] = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  13. A mouse B16 melanoma mutant deficient in glycolipids.

    PubMed Central

    Ichikawa, S; Nakajo, N; Sakiyama, H; Hirabayashi, Y

    1994-01-01

    Mouse B16 melanoma cell line, GM-95 (formerly designated as MEC-4), deficient in sialyllactosylceramide was examined for its primary defect. Glycolipids from the mutant cells were analyzed by high-performance TLC. No glycolipid was detected in GM-95 cells, even when total lipid from 10(7) cells was analyzed. In contrast, the content of ceramide, a precursor lipid molecule of glycolipids, was normal. Thus, the deficiency of glycolipids was attributed to the first glucosylation step of ceramide. The ceramide glucosyltransferase (EC 2.4.1.80) activity was not detected in GM-95 cells. There was no significant difference of sialyllactosylceramide synthase activity, however, between GM-95 and the parental cells. The deficiency of glycolipids in GM-95 cells was associated with changes of the cellular morphology and growth rate. The parental cells showed irregular shapes and tended to overlap each other. On the other hand, GM-95 cells exhibited an elongated fibroblastic morphology and parallel arrangement. The population-doubling times of GM-95 and the parental cells in serum-free medium were 28 hr and 19 hr, respectively. Images PMID:8146177

  14. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish

    PubMed Central

    Leong, Ivone U. S.; Skinner, Jonathan R.; Shelling, Andrew N.; Love, Donald R.

    2013-01-01

    Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients. PMID:27335675

  15. Plasmodium yoelii: induction of attenuated mutants by irradiation

    SciTech Connect

    Waki, S.; Yonome, I.; Suzuki, M.

    1986-12-01

    When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number of blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.

  16. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  17. Phenotypic analysis of Arabidopsis mutants: quantitative analysis of root growth.

    PubMed

    Doerner, Peter

    2008-03-01

    INTRODUCTIONThe growth of plant roots is very easy to measure and is particularly straightforward in Arabidopsis thaliana, because the increase in organ size is essentially restricted to one dimension. The precise measurement of root apical growth can be used to accurately determine growth activity (the rate of growth at a given time) during development in mutants, transgenic backgrounds, or in response to experimental treatments. Root growth is measured in a number of ways, the simplest of which is to grow the seedlings in a Petri dish and record the position of the advancing root tip at appropriate time points. The increase in root length is measured with a ruler and the data are entered into Microsoft Excel for analysis. When dealing with large numbers of seedlings, however, this procedure can be tedious, as well as inaccurate. An alternative approach, described in this protocol, uses "snapshots" of the growing plants, which are taken using gel-documentation equipment (i.e., a video camera with a frame-grabber unit, now commonly used to capture images from ethidium-bromide-stained electrophoresis gels). The images are analyzed using publicly available software (NIH-Image), which allows the user simply to cut and paste data into Microsoft Excel.

  18. Zebrafish atoh8 mutants do not recapitulate morpholino phenotypes

    PubMed Central

    Place, Elsie S.; Smith, James C.

    2017-01-01

    Atoh8 is a bHLH transcription factor expressed in pancreas, skeletal muscle, the nervous system, and cardiovascular tissues during embryological development. Although it has been implicated in the regulation of pancreatic and endothelial cell differentiation, the phenotypic consequences of Atoh8 loss are uncertain. Conclusions from knockout studies in the mouse differ widely depending on the targeting strategy used, while atoh8 knockdown by interfering morpholino oligonucleotides (morpholinos) in zebrafish has led to a range of developmental defects. This study characterised zebrafish embryos homozygous for atoh8sa1465, a loss-of-function allele of atoh8, in order to provide genetic evidence for the developmental role of Atoh8 in this species. Embryos homozygous for atoh8sa1465 present normal body morphology, swimbladder inflation, and heart looping, and survive to adulthood. These embryos do not develop pericardial oedema by 72 hpf and are not sensitised to the loss of Fog1 protein, suggesting that this previously described abnormality is not a specific phenotype. Vascular patterning and primitive haematopoiesis are unaffected in atoh8sa1465/sa1465 mutant embryos. Together, the data suggest that Atoh8 is dispensible for zebrafish development under standard laboratory conditions. PMID:28182631

  19. Fused 3-Hydroxy-3-trifluoromethylpyrazoles Inhibit Mutant Huntingtin Toxicity

    PubMed Central

    2013-01-01

    Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragment-based Huntington’s disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3-trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171–82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD. PMID:24900595

  20. Zymomonas mobilis mutants with an increased rate of alcohol production

    SciTech Connect

    Osman, Y.A.; Ingram, L.O.

    1987-07-01

    Two new derivatives of Zymomonas mobilis CP4 were isolated from enrichment cultures after 18 months of serial transfer. These new strains were selected for the ability to grow and produce ethanol rapidly on transfer into fresh broth containing ethanol and allyl alcohol. Ethanol production by these strains was examined in batch fermentations under three sets of conditions. Both new derivatives were found to be superior to the parent strain CP4 with respect to the speed and completeness of glucose conversion to ethanol. The best of these, strain YO2, produced 9.5% ethanol (by weight; 11.9% by volume) after 17.4 h compared with 31.8 h for the parent strain CP4. The addition of 1 mM magnesium sulfate improved ethanol production in all three strains. Two factors contributed to the decrease in fermentation time required by the mutants: more rapid growth with minimal lag on subculturing and the retention of higher rates of ethanol production as fermentation proceeded. Alcohol dehydrogenase isozymes were altered in both new strains and no longer catalyzed the oxidation of allyl alcohol into the toxic product acrolein. This loss of allyl alcohol-oxidizing capacity is proposed as a primary factor contributing to increased allyl alcohol resistance, although it is likely that other mutations affecting glycolysis also contribute to the improvement in ethanol production.

  1. Producing Conditional Mutants for Studying Plant Microtubule Function

    SciTech Connect

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

  2. Mutant p53 and ETS2, a Tale of Reciprocity.

    PubMed

    Martinez, Luis Alfonso

    2016-01-01

    TP53 is one of the most frequently inactivated tumor suppressor genes in human cancer. However, unlike other tumor suppressor genes whose expression is lost, TP53 is usually inactivated as a result of a single nucleotide change within the coding region. Typically, these single nucleotide mutations result in a codon change that creates an amino acid substitution. Thus, unlike other tumor suppressor genes whose expression is lost due to genetic or epigenetic changes, the p53 gene primarily suffers missense mutations, and therefore, the cells retain and express a mutant form of the p53 protein (mtp53). It is now well established that mtp53 contributes to tumor development through its gain-of-function (GOF) activities. These GOF activities can arise from novel protein-protein interactions that can either disable other tumor suppressors (e.g., p63 and p73) or enable oncogenes such as ETS2, an ETS family member. In this review, I will focus on the identification of the mtp53/ETS2 complex and outline the diverse activities that this transcriptional regulatory complex controls to promote cancer.

  3. Proton translocation in cytochrome-deficient mutants of Escherichia coli.

    PubMed Central

    Brookman, J J; Downie, J A; Gibson, F; Cox, G B; Rosenberg, H

    1979-01-01

    Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase. PMID:154508

  4. Serine 421 regulates mutant huntingtin toxicity and clearance in mice

    PubMed Central

    Kratter, Ian H.; Zahed, Hengameh; Lau, Alice; Daub, Aaron C.; Weiberth, Kurt F.; Gu, Xiaofeng; Humbert, Sandrine; Yang, X. William; Osmand, Alex; Steffan, Joan S.; Masliah, Eliezer

    2016-01-01

    Huntington’s disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (HTT). There are no cures or disease-modifying therapies for HD. HTT has a highly conserved Akt phosphorylation site at serine 421, and prior work in HD models found that phosphorylation at S421 (S421-P) diminishes the toxicity of mutant HTT (mHTT) fragments in neuronal cultures. However, whether S421-P affects the toxicity of mHTT in vivo remains unknown. In this work, we used murine models to investigate the role of S421-P in HTT-induced neurodegeneration. Specifically, we mutated the human mHTT gene within a BAC to express either an aspartic acid or an alanine at position 421, mimicking tonic phosphorylation (mHTT-S421D mice) or preventing phosphorylation (mHTT-S421A mice), respectively. Mimicking HTT phosphorylation strongly ameliorated mHTT-induced behavioral dysfunction and striatal neurodegeneration, whereas neuronal dysfunction persisted when S421 phosphorylation was blocked. We found that S421 phosphorylation mitigates neurodegeneration by increasing proteasome-dependent turnover of mHTT and reducing the presence of a toxic mHTT conformer. These data indicate that S421 is a potent modifier of mHTT toxicity and offer in vivo validation for S421 as a therapeutic target in HD. PMID:27525439

  5. Clinical significance of hepatitis B surface antigen mutants

    PubMed Central

    Coppola, Nicola; Onorato, Lorenzo; Minichini, Carmine; Di Caprio, Giovanni; Starace, Mario; Sagnelli, Caterina; Sagnelli, Evangelista

    2015-01-01

    Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the “a” determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications. PMID:26644816

  6. Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

    PubMed

    Asad, Sedigheh; Dabirmanesh, Bahareh; Khajeh, Khosro

    2014-01-01

    Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.

  7. Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold

    SciTech Connect

    Kalayanamitr, A.; Bhumiratana, A.; Flegel, T.W.; Glinsukon, T.; Shinmyo, A.

    1987-08-01

    A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production.

  8. Temperature-Sensitive, Photosynthesis-Deficient Mutants of Chlamydomonas reinhardtii1

    PubMed Central

    Spreitzer, Robert J.; Al-Abed, Souhail R.; Huether, Michael J.

    1988-01-01

    Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35°C. Whereas nonconditional mutants required acetate for growth at both 25 and 35°C, the conditional mutants have normal photoautotrophic growth at 25°C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35°C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35°C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebis-phosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis-deficient phenotypes was observed. PMID:16665986

  9. Correction of hair shaft defects through allele-specific silencing of mutant Krt75

    PubMed Central

    Liu, Ying; Snedecor, Elizabeth R.; Zhang, Xu; Xu, Yan-Feng; Huang, Lan; Jones, Evan; Zhang, Lianfeng; Clark, Richard A.; Roop, Dennis R.; Qin, Chuan; Chen, Jiang

    2015-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele specific siRNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific shRNA persistently suppressed this phenotype. The phenotypic correction was associated with significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of the mutant keratin genes. PMID:26763422

  10. Isolation of a recombination-deficient mutant of Streptococcus lactis ML3.

    PubMed

    Anderson, D G; McKay, L L

    1983-08-01

    A recombination-deficient mutant of Streptococcus lactis ML3 designated MMS36 was isolated on the basis of its sensitivity to methyl methanesulfonate. This mutant also displayed sensitivity to UV irradiation. The inability of MMS36 to mediate homologous recombination was demonstrated by transduction of plasmid-linked lactose fermenting ability but not chromosomally mediated streptomycin resistance.

  11. Characterization of a bi-pistil mutant in Medicago truncatula Gaertn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We propose the name bi-pistil, bip, for a floral organ mutant observed in transgenic Medicago truncatula plants. The mutant has two separate stigmas borne on two separate styles that emerge from a single superior carpel primordium. The bip plant was crossed to a previously reported male sterile mtap...

  12. Construction and characterization of an isogenic urease-negative mutant of Helicobacter mustelae.

    PubMed Central

    Solnick, J V; Josenhans, C; Suerbaum, S; Tompkins, L S; Labigne, A

    1995-01-01

    Helicobacter mustelae infects the ferret stomach and provides an opportunity to study pathogenic determinants of a Helicobacter species in its natural host. We constructed an isogenic urease-negative mutant of H. mustelae which produced no detectable urease and showed a reduced acid tolerance. This mutant provides an opportunity to further evaluate the role of urease in the pathogenesis of Helicobacter infection. PMID:7642313

  13. Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants.

    PubMed

    Arteca, Richard N; Arteca, Jeannette M

    2011-07-15

    The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.

  14. Gene-Enzyme Relations of Tryptophan Mutants in STREPTOMYCES COELICOLOR A3(2)

    PubMed Central

    Smithers, Charles M.; Engel, Paulinus P.

    1974-01-01

    Mutations in twenty-eight tryptophan mutants of S. coelicolor A3(2) were mapped relative to the nearest flanking markers. Mutants lacking single enzymatic activities for phosphoribosyltransferase, phosphoribosylanthranilate isomerase, indodeglycerol phosphate synthase, tryptophan synthase A and tryptophan synthase B were identified. PMID:4452474

  15. Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold.

    PubMed Central

    Kalayanamitr, A; Bhumiratana, A; Flegel, T W; Glinsukon, T; Shinmyo, A

    1987-01-01

    A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production. PMID:2444160

  16. Five carboxin-resistant mutants exhibited various responses to carboxin and related fungicides.

    PubMed

    Shima, Yoko; Ito, Yasuhiro; Hatabayashi, Hidemi; Koma, Akemi; Yabe, Kimiko

    2011-01-01

    Five carboxin-resistant mutants from Aspergillus oryzae were characterized by the sensitivities of their mycelial growth and succinate dehydrogenase (SDH) activity to carboxin and three related fungicides. Despite a significant resistance to carboxin, exhibited by all the mutants, their patterns of sensitivity to the other fungicides was distinct. This provides clues to the molecular interaction between SDH and these fungicides.

  17. A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

    SciTech Connect

    Hugly, S.; McCourt, P.; Somerville, C. ); Browse, J. ); Patterson, G.W. )

    1990-07-01

    A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18{degree}C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury - leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of {sup 14}CO{sub 2} into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury.

  18. Development and characterization of rice mutants for functional genomic studies and breeding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mutagenesis is a powerful tool for creating genetic materials for studying functional genomics, breeding, and understanding the molecular basis of disease resistance. Approximately 100,000 putative mutants of rice (Oryza sativa L.) have been generated with mutagens. Numerous mutant genes involved in...

  19. Genome Sequence of a Spontaneous Nonhemolytic Mutant of Mannheimia haemolytica 16041065 GH

    PubMed Central

    Ayalew, Sahlu; Confer, Anthony W.; Hansen, Richard D.

    2017-01-01

    ABSTRACT We report here the draft genome sequence of a spontaneous nonhemolytic mutant of Mannheimia haemolytica 16041065 GH. This mutant arose during routine passage and was devoid of hemolytic activity on standard blood agars. This genome sequence had a total size of 2.7 Mb with an N50 of 117 kb. PMID:28385858

  20. Some repair-deficient mutants of Dictyostelium discoideum display enhanced susceptibilities to bleomycin.

    PubMed Central

    Deering, R A; Guyer, R B; Stevens, L; Watson-Thais, T E

    1996-01-01

    Dictyostelium discoideum, a soil eukaryote, is highly resistant to DNA-damaging agents; repair mutants are more susceptible. Susceptibility to bleomycin, produced by Streptomyces verticillus, is greater for mutants which are susceptible to other agents than for resistant strains. The high potential for DNA repair may result from the need to cope with chemicals produced by other soil microorganisms. PMID:8834899

  1. Phenotypic and Transcriptomic Characterization of Bacillus subtilis Mutants with Grossly Altered Membrane Composition▿ †

    PubMed Central

    Salzberg, Letal I.; Helmann, John D.

    2008-01-01

    The Bacillus subtilis membrane contains diacylglycerol-based lipids with at least five distinct headgroups that together help to define the physical and chemical properties of the lipid bilayer. Here, we describe the phenotypic characterization of mutant strains lacking one or more of the following lipids: glycolipids (ugtP mutants), phosphatidylethanolamine (pssA and psd mutants), lysylphosphatidylglycerol (mprF), and cardiolipin (ywnE and ywjE). Alterations of membrane lipid headgroup composition are generally well-tolerated by the cell, and even severe alterations lead to only modest effects on growth proficiency. Mutants with decreased levels of positively charged lipids display an increased sensitivity to cationic antimicrobial compounds, and cells lacking glycolipids are more sensitive to the peptide antibiotic sublancin and are defective in swarming motility. A quadruple mutant strain (ugtP pssA mprF ywnE), with a membrane comprised predominantly of phosphatidylglycerol, is viable and grows at near-wild-type rates, although it forms long, coiled filaments. Transcriptome comparisons identified numerous regulons with altered expression in cells of the ugtP mutant, the pssA mprF ywnE triple mutant, and the ugtP pssA mprF ywnE quadruple mutant. These effects included a general decrease in expression of the SigD and FapR regulons and increased expression of cell envelope stress responses mediated by σM and the YvrGHb two-component system. PMID:18820022

  2. Abscisic acid content of a nondormant sunflower (Helianthus annuus L.) mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sunflower (Helianthus annuus L.) mutant was observed in the progeny of a cross between the sunflower cultivar HA 89 and an amphiploid of a H. divaricatus L. x P21 cross that exhibited loss of dormancy induction in the developing embryo. Seeds of this mutant frequently germinate on the head about 4...

  3. Characterization of a green nondormant sunflower (Helianthus annuus L.) mutant ndg

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sunflower (Helianthus annuus L.) mutant was observed in the progeny of a cross between the sunflower cultivar HA 89 and an amphiploid of a H. divaricatus L. x P21 cross that exhibited loss of dormancy induction in the developing embryo. Seeds of this mutant frequently germinate on the head about 4...

  4. A MUTANT OF YEAST APPARENTLY DEFECTIVE IN THE INITIATION OF PROTEIN SYNTHESIS*

    PubMed Central

    Hartwell, Leland H.; McLaughlin, Calvin S.

    1969-01-01

    A temperature-sensitive mutant of yeast, ts-187, which is apparently unable to initiate the synthesis of new polypeptide chains after a short incubation at the restrictive temperature, is described. The existence of this mutant demonstrates that in eucaryotic cells, as in procaryotic cells, there are processes unique to the initiation of polypeptide chains. PMID:5256225

  5. Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta.

    PubMed Central

    Jacobs, J. J.; Arroo, R. R.; De Koning, E. A.; Klunder, A. J.; Croes, A. F.; Wullems, G. J.

    1995-01-01

    Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type. The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants. Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagetes erecta. These experiments also showed that one mutant is deficient in demethylation of the monothiophene. PMID:12228405

  6. Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii.

    PubMed

    Tajima, Satoshi; Itoh, Yoko; Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y

    2009-10-01

    The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.

  7. Direct selection of Clostridium acetobutylicum fermentation mutants by a proton suicide method

    SciTech Connect

    Cueto, P.H.; Mendez, B.S. )

    1990-02-01

    Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

  8. Random Genetic Drift Determines the Level of Mutant mtDNA in Human Primary Oocytes

    PubMed Central

    Brown, D. T.; Samuels, D. C.; Michael, E. M.; Turnbull, D. M.; Chinnery, P. F.

    2001-01-01

    We measured the proportion of mutant mtDNA (mutation load) in 82 primary oocytes from a woman who harbored the A3243G mtDNA mutation. The frequency distribution of mutation load indicates that random drift is the principal mechanism that determines the level of mutant mtDNA within individual oocytes. PMID:11133360

  9. Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2.

    PubMed Central

    Maier, R J

    1981-01-01

    Rhizobium japonicum strain SR grows chemoautotrophically on a mineral salts medium when incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow or that grow very poorly chemoautotrophically with H2 have been isolated from strain SR. The mutant isolation procedure involved mutagenesis with ethyl methane sulfonate, penicillin selection under chemoautotrophic growth conditions, and plating of the survivors onto medium containing carbon. The resulting colonies were replica plated onto medium that did not contain carbon, and the plates were incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow under these conditions were chosen. Over 100 mutant strains with defects in chemoautotrophic metabolism were obtained. The phenotypes of the mutants fall into various classes. These include strains unable to oxidize H2 and strains deficient in CO2 uptake. Some of the mutant strains were capable of oxidizing H2 only when artificial electron acceptors were provided. Two mutant strains specifically lack activity of the key CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase. Other mutant strains lack both H2-oxidizing ability and ribulose 1,5-bisphosphate carboxylase activity. PMID:6780521

  10. Biofilm formation by exopolysaccharide mutants of Leuconostoc mesenteroides strain NRRL B-1355

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. A set of mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces ...

  11. Molecular mapping of three nuclear male sterility mutant genes in cultivated sunflower (Helianthus annuus L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nuclear male sterility (NMS) trait is a useful tool for sunflower (Helianthus annuus L.) breeding and genetic programs. Previously, we induced NMS mutants in cultivated line HA 89. The mutants possessed single recessive genes, ms6, ms7, and ms8, respectively, in NMS HA 89-872, NMS HA 89-552, and...

  12. Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants.

    PubMed Central

    Merberg, D; Maier, R J

    1984-01-01

    In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically. However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity. PMID:6384199

  13. Plastid distribution in columella cells of a starchless Arabidopsis mutant grown in microgravity

    NASA Technical Reports Server (NTRS)

    Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1997-01-01

    Wild-type and starchless Arabidopsis thaliana mutant seedlings (TC7) were grown and fixed in the microgravity environment of a U.S. Space Shuttle spaceflight. Computer image analysis of longitudinal sections from columella cells suggest a different plastid positioning mechanism for mutant and wild-type in the absence of gravity.

  14. Mutant p53 in cancer: Accumulation, gain-of-function and therapy.

    PubMed

    Yue, Xuetian; Zhao, Yuhan; Xu, Yang; Zheng, Min; Feng, Zhaohui; Hu, Wenwei

    2017-04-05

    Tumor suppressor p53 plays a central role in tumor suppression. p53 is the most frequently mutated gene in human cancer, and over half of human cancers contain p53 mutations. Majority of p53 mutations in cancer are missense mutations, leading to the expression of full-length mutant p53 protein. While the critical role of wild type p53 in tumor suppression has been firmly established, mounting evidence has demonstrated that many tumor-associated mutant p53 proteins not only lose tumor suppressive function of wild type p53, but also gain new activities to promote tumorigenesis independently of wild type p53, termed gain-of-function. Mutant p53 protein often accumulates to very high levels in tumors, contributing to malignant progression. Recently, mutant p53 has become an attractive target for cancer therapy. Further understanding of the mechanisms underlying mutant p53 protein accumulation and gain-of-function will accelerate the development of targeted therapies for human cancer harboring mutant p53. In this review, we summarize the recent advances in the studies on mutant p53 protein accumulation and gain-of-function as well as targeted therapies for mutant p53 in human cancer.

  15. PLK1 inhibition enhances temozolomide efficacy in IDH1 mutant gliomas.

    PubMed

    Koncar, Robert F; Chu, Zhengtao; Romick-Rosendale, Lindsey E; Wells, Susanne I; Chan, Timothy A; Qi, Xiaoyang; Bahassi, El Mustapha

    2017-02-28

    Despite multimodal therapy with radiation and the DNA alkylating agent temozolomide (TMZ), malignant gliomas remain incurable. Up to 90% of grades II-III gliomas contain a single mutant isocitrate dehydrogenase 1 (IDH1) allele. IDH1 mutant-mediated transformation is associated with TMZ resistance; however, there is no clinically available means of sensitizing IDH1 mutant tumors to TMZ. In this study we sought to identify a targetable mechanism of TMZ resistance in IDH1 mutant tumors to enhance TMZ efficacy. IDH1 mutant astrocytes rapidly bypassed the G2 checkpoint with unrepaired DNA damage following TMZ treatment. Checkpoint adaptation was accompanied by PLK1 activation and IDH1 mutant astrocytes were more sensitive to treatment with BI2536 and TMZ in combination (<20% clonogenic survival) than either TMZ (~60%) or BI2536 (~75%) as single agents. In vivo, TMZ or BI2536 alone had little effect on tumor size. Combination treatment caused marked tumor shrinkage in all mice and complete tumor regression in 5 of 8 mice. Mutant IDH1 promotes checkpoint adaptation which can be exploited therapeutically with the combination of TMZ and a PLK1 inhibitor, indicating PLK1 inhibitors may be clinically valuable in the treatment of IDH1 mutant gliomas.

  16. Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75.

    PubMed

    Liu, Ying; Snedecor, Elizabeth R; Zhang, Xu; Xu, Yanfeng; Huang, Lan; Jones, Evan C; Zhang, Lianfeng; Clark, Richard A; Roop, Dennis R; Qin, Chuan; Chen, Jiang

    2016-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes.

  17. A Laboratory Exercise for Isolation and Characterizing Microbial Mutants with Metabolic Defects.

    ERIC Educational Resources Information Center

    Doe, Frank J.; Leslie, John F.

    1993-01-01

    Describes science experiments for undergraduate biology instruction on the concepts of mutation and characterization of the resulting mutant strains. The filamentous fungi "Fusarium moniliforme" is used to illustrate the induction of mutants (mutagenesis), identification of the mutated gene, construction of a biochemical pathway, and…

  18. Canopy Light Interception of a Conventional and an Erect Leaf Mutant Sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two sorghum lines, an erect leafed mutant sorghum and the wild type from which the mutant was generated, were field grown in rectilinear arrays at low (23 plants per square meter) and high (10 plants per square meter) population densities. Canopy light interception, biomass accretion and yield were ...

  19. Identical substitutions in magnesium chelatase paralogs result in chlorophyll deficient soybean mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The soybean (Glycine max (L.) Merr.) chlorophyll deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a non-synonymous nucleotide substitution in the third exon of a Mg-chelat...

  20. Spontaneous white sectored-mutants in Streptomyces hygroscopicus 111-81: characterization and antibiotic productivity.

    PubMed

    Gesheva, Victoria

    2008-08-01

    Spontaneous white mutants from sectors of Streptomyces hygroscopicus 111-81 were isolated. The comparison of morphological, cultural, and biochemical properties of the mutants and ancestor showed the differences in colors of aerial, substrate mycelia, and sporulation. Changes in resistance to antibiotics and sensitivity to lysozyme indicated alterations in cell walls and cell membranes of the mutants. They showed antifungal activity close to that of the parent strain on fermentation medium FM2, with unchanged component composition of the AK-111-81 antibiotic complex. The cells of spontaneous white mutants are characterized with electron-transparent structures, vacuoles, aggregation of ribosomes, intrahyphal growth, and lack of multiple cell septa, which was established by transmission electron microscopy. The appearance of white sectored-mutants in S. hygroscopicus 111-81 is connected with exhausting of nutrients causing the substrate limitation and is a stress response to starvation.

  1. Isolation and characterization of Chinese hamster cell mutants resistant to the cytotoxic effects of chromate.

    PubMed

    Campbell, C E; Gravel, R A; Worton, R G

    1981-09-01

    Stable mutants resistant to the toxic anion chromate have been isolated from a variety of Chinese hamster cell lines. The mechanism of chromate toxicity is not known, but it must involve internalization via the sulfate transport pathway. All mutant lines had a defective sulfate transport system, showing a 10-fold reduction in the rate of uptake of radioactive sulfate into the cell. The chromate resistance phenotype in CHO cell mutants behave recessively in somatic cell hybrids; in other cell lines the Chr(r) phenotype was partially expressed (codominant) in cell hybrids. Complementation analysis in cell hybrids between 18 different mutant pairs failed to reveal any complementation, indicating that chromate selects mutants primarily, if not exclusively, at a single gene locus.

  2. Reduced mycorrhizal colonization (rmc) tomato mutant lacks expression of SymRK signaling pathway genes

    PubMed Central

    Nair, Aswathy; Bhargava, Sujata

    2012-01-01

    Comparison of the expression of 13 genes involved in arbuscular mycorrhizal (AM) symbiosis was performed in a wild type tomato (Solanum lycopersicum cv 76R) and its reduced mycorrhizal colonization mutant rmc in response to colonization with Glomus fasiculatum. Four defense-related genes were induced to a similar extent in the mutant and wild type AM colonized plants, indicating a systemic response to AM colonization. Genes related to nutrient exchange between the symbiont partners showed higher expression in the AM roots of wild type plants than the mutant plants, which correlated with their arbuscular frequency. A symbiosis receptor kinase that is involved in both nodulation and AM symbiosis was not expressed in the rmc mutant. The fact that some colonization was observed in rmc was suggestive of the existence of an alternate colonization signaling pathway for AM symbiosis in this mutant. PMID:23221680

  3. A non-cell autonomous mouse model of CNS haemangioblastoma mediated by mutant KRAS

    PubMed Central

    Bao, Leyuan; Al-Assar, Osama; Drynan, Lesley F.; Arends, Mark J.; Tyers, Pam; Barker, Roger A.; Rabbitts, Terence H.

    2017-01-01

    Haemangioblastoma is a rare malignancy of the CNS where vascular proliferation causes lesions due to endothelial propagation. We found that conditionally expressing mutant Kras, using Rag1-Cre, gave rise to CNS haemangioblastoma in the cortex and cerebellum in mice that present with highly vascular tumours with stromal cells similar to human haemangioblastomas. The aberrant haemangioblastoma endothelial cells do not express mutant Kras but rather the mutant oncogene is expressed in CNS interstitial cells, including neuronal cells and progeny. This demonstrates a non-cell autonomous origin of this disease that is unexpectedly induced via Rag1-Cre expression in CNS interstitial cells. This is the first time that mutant RAS has been shown to stimulate non-cell autonomous proliferation in malignancy and suggests that mutant RAS can control endothelial cell proliferation in neo-vascularisation when expressed in certain cells. PMID:28322325

  4. Distinct innate responses are induced by attenuated Salmonella enterica serovar Typhimurium mutants.

    PubMed

    Powell, Daniel A; Roberts, Lydia M; Ledvina, Hannah E; Sempowski, Gregory D; Curtiss, Roy; Frelinger, Jeffrey A

    2016-01-01

    Upon bacterial infection the host cells generate a wide variety of cytokines. Genetic attenuation of bacterial physiological pathogens can be accomplished not only by disruption of normal bacterial processes, but also by the loss of the ability to redirect the host immune system. We examined nine attenuated Salmonella Typhimurium mutants for their ability to replicate as well as the cytokines produced after infection of Bone Marrow Derived Macrophages (BMDM). Infection of BMDM with attenuated Salmonella mutants led to host cytokine patterns distinct from those that followed WT infection. Surprisingly, each bacterial mutant had a unique cytokine signature. Because some of the mutants induced an IL-10 response not seen in WT, we examined the role of IL-10 on Salmonella replication. Surprisingly, addition of IL-10 before or concurrent with infection restricted growth of WT Salmonella in BMDM. Bacterial attenuation is not a single process and results in attenuated host responses, which result in unique patterns for each attenuated mutants.

  5. Distinct Innate Responses are Induced by Attenuated Salmonella enterica serovar Typhimurium Mutants

    PubMed Central

    Powell, Daniel A.; Roberts, Lydia M.; Ledvina, Hannah E.; Sempowski, Gregory D.; Curtiss, Roy; Frelinger, Jeffrey A.

    2015-01-01

    Upon bacterial infection the host cells generate a wide variety of cytokines. Genetic attenuation of bacterial physiological pathogens can be accomplished not only by disruption of normal bacterial processes, but also by the loss of the ability to redirect the host immune system. We examined nine attenuated Salmonella Typhimurium mutants for their ability to replicate as well as the cytokines produced after infection of Bone Marrow Derived Macrophages (BMDM). Infection of BMDM with attenuated Salmonella mutants led to host cytokine patterns distinct from those that followed WT infection. Surprisingly, each bacterial mutant had a unique cytokine signature. Because some of the mutants induced an IL-10 response not seen in WT, we examined the role of IL-10 on Salmonella replication. Surprisingly, addition of IL-10 before or concurrent with infection restricted growth of WT Salmonella in BMDM. Bacterial attenuation is not a single process and results in attenuated host responses, which result in unique patterns for each attenuated mutants. PMID:26546408

  6. Elevated 3-hydroxypropionaldehyde production from glycerol using a Citrobacter freundii mutant.

    PubMed

    West, Thomas P; Peterson, Jessica L

    2014-01-01

    A mutant strain of Citrobacter freundii capable of elevated 3-hydroxypropionaldehyde production from glycerol was isolated using chemical mutagenesis and a screening protocol. The protocol involved screening mutagenized bacterial cells on solid minimal medium containing 5 % (v/v) glycerol. Colonies were picked onto duplicate solid minimal medium plates and one plate was stained with 1 % (w/v) phloroglucinol. Those colonies staining red were further screened and a mutant, HPAO-1, was identified. The mutant strain produced a several-fold higher 3-hydroxypropionaldehyde concentration than did the parent strain when grown on 5 % (v/v) glycerol. The ratio of culture volume to flask volume influenced 3-hydroxypropionaldehyde production by the mutant cells compared to the parent cells. Aldehyde production was highest when the mutant strain was grown on 5 % (v/v) glycerol at a ratio of culture volume to flask volume of 1:3 or 1:12.5.

  7. Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization.

    PubMed Central

    Chinchar, V G; Granoff, A

    1986-01-01

    Nineteen frog virus 3 temperature-sensitive mutants were isolated after mutagenesis with nitrosoguanidine and assayed for viral DNA, RNA, and protein synthesis, as well as assembly site formation at permissive (25 degrees C) and nonpermissive (30 degrees C) temperatures. In addition, mutants were characterized for complementation by both quantitative and qualitative assays. Based on the genetic and biochemical data, the 19 mutants, along with 9 mutants isolated earlier, were ordered into four phenotypic classes which define defects in virion morphogenesis (class I), late mRNA synthesis (class II), viral assembly site formation (class III), and viral DNA synthesis (class IV). In addition, we used two-factor crosses to order 11 mutants, comprising 7 complementation groups, onto a linkage map spanning 77 recombination units. Images PMID:3951023

  8. Purine transport by malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster.

    PubMed

    Sullivan, D T; Bell, L A; Paton, D R; Sullivan, M C

    1979-06-01

    Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (pp) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.

  9. Studies on cellulase production by a mutant - Penicillium funiculosum UV-49

    SciTech Connect

    Joglekar, A.V.; Karanth, N.G.

    1984-01-01

    Rapidly depleting reserves and the rising cost of petroleum has focused worldwide attention on the development of alternate sources of energy. One of the most promising potential solutions to this problem is utilization of cellulosic materials through bioconversion. In search of hypercelluloytic microorganisms, ultraviolet irradiation carried out with Penicillium funiculosum has yielded a superior mutant. The investigations reported in this article are shake flask studies on some important nutritional requirements of the mutant, namely, nitrogen source, carbon source, and inducers. The mutant shows an ability to metabolize inorganic nitrogen sources like urea and sodium nitrate both for growth and enzyme production. A comparison of the long-term saccharification ability and the utilization efficiency of the mutant enzyme with those reported in the literature is also carried out, showing the superior performance of the mutant enzyme.

  10. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    PubMed

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-12-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

  11. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    PubMed Central

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-01-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity. PMID:2687252

  12. Misfolded opsin mutants display elevated β -sheet structure

    SciTech Connect

    Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; Park, Paul S. -H.

    2015-09-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.

  13. Elevated curdlan production by a mutant of Agrobacterium sp. ATCC 31749.

    PubMed

    West, Thomas P

    2009-12-01

    A mutant strain of the curdlan-producing bacterium Agrobacterium sp. ATCC 31749, isolated by ethylmethane sulfonate mutagenesis and resistance to ampicillin, was capable of elevated curdlan synthesis. Using 2.5% corn syrup, glucose or maltose as a carbon source, the mutant strain was shown to produce a 1.5-fold, 1.5-fold or 1.5-fold higher level of curdlan, respectively, than its parent strain after 120 h of growth. The mutant strain produced higher curdlan levels after 96 or 120 h of growth on glucose or maltose as a carbon source than it did on corn syrup. Biomass production by the mutant strain grown on the carbon sources studied was slightly elevated compared to its parent strain. It was concluded that the elevated curdlan production observed for the mutant strain grown on corn syrup or glucose was not due to an increase in biomass production.

  14. Early-blocked asporogenous mutants of Bacillus subtilis are lysogenized at reduced frequency by temperate bacteriophages.

    PubMed Central

    Ikeuchi, T; Kurahashi, K

    1978-01-01

    The establishment of lysogeny in early-blocked asporogenous (Spo-) mutants of Bacillus subtilis 168, which were also defective in the production of antibiotics (Abs-), by temperate phage phi105 or SPO2 was studied. It was found that the frequency of lysogenization of Spo-Abs-mutants was 10 to 20% that of the wild-type bacteria. There was no difference in the efficiency of plating and the burst size of phi105 between wild-type and mutant strains. Phi105 lysogens of mutant strains were as stable as those of the wild type. Several rifampin-resistant mutants defective in the production of antibiotics were isolated. They were also defective in spore formation and lysogenized by phi105 at reduced frequency. PMID:96089

  15. Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

    PubMed

    Grygoryev, Dmytro; Dan, Cristian; Gauny, Stacey; Eckelmann, Bradley; Ohlrich, Anna P; Connolly, Marissa; Lasarev, Michael; Grossi, Gianfranco; Kronenberg, Amy; Turker, Mitchell S

    2014-05-01

    High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

  16. Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism.

    PubMed

    Cushman, John C; Agarie, Sakae; Albion, Rebecca L; Elliot, Stewart M; Taybi, Tahar; Borland, Anne M

    2008-05-01

    Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

  17. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

    PubMed

    Amorim-Vaz, Sara; Delarze, Eric; Ischer, Françoise; Sanglard, Dominique; Coste, Alix T

    2015-01-01

    The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.

  18. Transposon-Derived Brucella abortus Rough Mutants Are Attenuated and Exhibit Reduced Intracellular Survival

    PubMed Central

    Allen, Chris A.; Adams, L. Garry; Ficht, Thomas A.

    1998-01-01

    The O antigen of Brucella abortus has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species, Brucella ovis and Brucella canis, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate B. abortus rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the B. abortus rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen. B. abortus O antigen appears to be essential

  19. Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

    PubMed Central

    Ramaswamy, S; Dworkin, M; Downard, J

    1997-01-01

    Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates

  20. Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

    PubMed

    Ramaswamy, S; Dworkin, M; Downard, J

    1997-05-01

    Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.