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1

Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli.  

PubMed Central

A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc. Natl. Acad. Sci. U.S.A. 74:3485-3489, 1977). At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures. This suggests that the mutant may be among the most ligase-deficient strains yet characterized.

Dermody, J J; Robinson, G T; Sternglanz, R

1979-01-01

2

Isolation and Characterization of Conditional Lethal Mutants of Escherichia coli Defective in Transcription Termination Factor rho  

Microsoft Academic Search

Polarity suppressor mutants that are conditional lethal for growth have been isolated in E. coli K12. The mutations map between the ilv and cya loci of the E. coli chromosome. Rho factor isolated from one of these ts mutants does not show transcription termination activity at any temperature tested; however, it is found to be temperature sensitive for its poly(C)-dependent

Asis Das; Don Court; Sankar Adhya

1976-01-01

3

A new simple method for isolating multistress-tolerant semidominant mutants of Saccharomyces cerevisiae by one-step selection under lethal hydrogen peroxide stress condition.  

PubMed

Tolerance of microorganisms to diverse stresses (i.e., multistress tolerance) is a very useful property with industrial applications. We have developed a simple method for isolating multistress-tolerant semidominant mutants of the budding yeast Saccharomyces cerevisiae by one-step selection under lethal hydrogen peroxide (H(2)O(2)) stress condition, which we named the lethal concentration of H(2)O(2) (LCH) method. This method involves simply isolating colonies after plating of mutagenized S. cerevisiae cells, which are cultivated overnight in liquid media, on agar plates containing a lethal concentration of H(2)O(2) for the wild-type strain. Phenotypic and genetic analyses of the ten strains isolated by this method revealed that two strains exhibiting stress tolerance to H(2)O(2), ethanol, heat shock, salt, organic solvent, freeze-thaw, chronological aging, and high concentrations of glucose possess semidominant and distinct single-gene mutations designated as MLT1-1 (multistress tolerance) and MLT2-1, which are responsible for multistress tolerance. From these results, we expect this method to confer multistress tolerance on industrial yeasts. PMID:23391901

Nakagawa, Youji; Seita, Junya; Komiyama, Shohei; Yamamura, Hideki; Hayakawa, Masayuki; Iimura, Yuzuru

2013-02-07

4

The Saccharomyces cerevisiae Hyperrecombination Mutant hpr1? Is Synthetically Lethal with Two Conditional Alleles of the Acetyl Coenzyme A Carboxylase Gene and Causes a Defect in Nuclear Export of Polyadenylated RNA  

PubMed Central

In a screen for mutants that display synthetic lethal interaction with hpr1?, a hyperrecombination mutant of Saccharomyces cerevisiae, we have isolated a novel cold-sensitive allele of the acetyl coenzyme A (CoA) carboxylase gene, acc1cs, encoding the rate-limiting enzyme of fatty acid synthesis. The synthetic lethal phenotype of the acc1cs hpr1? double mutant was only partially complemented by exogenous fatty acids. hpr1? was also synthetically lethal with a previously isolated, temperature-sensitive allele of ACC1, mtr7 (mRNA transport), indicating that the lethality of the acc1cs hpr1? double mutant was not allele specific. The basis for the interaction between conditional acc1 alleles and hpr1? was investigated in more detail. In the hpr1? mutant background, acetyl-CoA carboxylase enzyme activity was reduced about 15-fold and steady-state levels of biotinylated Acc1p and ACC1 mRNA were reduced 2-fold. The reduced Acc1p activity in hpr1? cells, however, did not result in an altered lipid or fatty acid composition of the mutant membranes but rendered cells hypersensitive to soraphen A, an inhibitor of Acc1p. Similar to mtr7, hpr1? and acc1cs mutant cells displayed a defect in nuclear export of polyadenylated RNA. Oversized transcripts were detected in hpr1?, and rRNA processing was disturbed, but pre-mRNA splicing appeared wild type. Surprisingly, the transport defect of hpr1? and acc1cs mutant cells was accompanied by an altered ring-shaped structure of the nucleolus. These observations suggest that the basis for the synthetic lethal interaction between hpr1? and acc1 may lie in a functional overlap of the two mutations in nuclear poly(A)+ RNA production and export that results in an altered structure of the nucleolus.

Schneiter, Roger; Guerra, Cesar E.; Lampl, Manfred; Gogg, Gabriela; Kohlwein, Sepp D.; Klein, Hannah L.

1999-01-01

5

Lethal acrodysgenital dwarfism: a severe lethal condition resembling Smith-Lemli-Opitz syndrome.  

PubMed Central

We report eight cases of a lethal association of failure to thrive, facial dysmorphism, ambiguous genitalia, syndactyly, postaxial polydactyly, and internal developmental anomalies (Hirschsprung's disease, cardiac and renal malformation). This syndrome is likely to be autosomal recessive and resembles Smith-Lemli-Opitz (SLO) syndrome. However, the lethality, the common occurrence of polydactyly, and the sexual ambiguity distinguishes this condition from SLO syndrome. A review of published reports supports the separate classification of this syndrome for which we propose the name lethal acrodysgenital dwarfism. Images

Merrer, M L; Briard, M L; Girard, S; Mulliez, N; Moraine, C; Imbert, M C

1988-01-01

6

Transcription of viral late genes is dependent on expression of the viral intermediate gene G8R in cells infected with an inducible conditional-lethal mutant vaccinia virus.  

PubMed Central

There are three temporal classes of vaccinia virus genes: early, intermediate, and late. The object of this study was to determine the effects on virus replication of regulating the expression of G8R, an intermediate gene that encodes a late transcription factor. We inserted the lac operator adjacent to the RNA start site of the G8R gene in a recombinant vaccinia virus that constitutively expresses the Escherichia coli lac repressor to make expression of the G8R gene dependent on the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). In case repression would not be complete, we also weakened the promoter of the G8R gene by making a single-nucleotide substitution designed to reduce its basal level of transcription. The mutant virus replicated well in the presence of the inducer, although synthesis of the G8R-encoded 30,000-M(r) protein was only 10% of that of the wild-type virus. In the absence of IPTG, (i) synthesis of the G8R protein was inhibited by more than 99% relative to that of the wild-type virus, (ii) synthesis of early and intermediate mRNAs appeared to be unaffected, (iii) intermediate proteins accumulated to higher than normal levels, (iv) synthesis of late mRNA and protein was reduced by about 90%, (v) viral DNA was replicated but incompletely resolved concatemeric molecules accumulated, (vi) not even the earliest stages of virion assembly were detectable by transmission electron microscopy, and (vii) virus yield under one-step growth conditions and plaque formation were 10(-3) and 10(-4) times the wild-type values, respectively. The defect in late gene expression could be overcome by transfection of a G8R gene that was not under lac operator control, as well as by addition of IPTG, further demonstrating the specificity of the repression. The correlation between decreased expression of the G8R intermediate gene and inhibition of late mRNA synthesis is consistent with the notion that the G8R product serves as an essential late transcription factor and supports a cascade mechanism of vaccinia virus gene regulation. In addition, the inducer-dependent vaccinia virus mutant provided a tool for selective inhibition of late gene expression while allowing synthesis of early and intermediate mRNAs and proteins. Images

Zhang, Y; Keck, J G; Moss, B

1992-01-01

7

Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda  

Microsoft Academic Search

Termination of transcription at bacteriophage ? terminators as well as at the Escherichia coli trp a attenuator was examined in the conditionally lethal mutant (nusAts11) defective in the NusA protein of E. coli. Experiments using terminator-assay ? vectors revealed that the efficiency of termination at both ?-dependent (?tL1) and ?-independent (?tL2 and trp a) terminators decreases in the mutant. The

Yoshikazu Nakamura; Saeko Mizusawa; Akiko Tsugawa; Mutsuo Imai

1986-01-01

8

Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineered mutant pro-alpha1(I) collagen gene  

Microsoft Academic Search

Substitutions of single glycine residues of alpha1(I) collagen have previously been associated with the inherited disease osteogenesis imperfecta type II. Transgenic mice bearing a mutant alpha1(I) collagen gene into which specific glycine substitutions have been engineered show a dominant lethal phenotype characteristic of the human disease, and demonstrate that as little as 10% mutant gene expression can disrupt normal collagen

Alex Stacey; John Bateman; Ted Choi; Tom Mascara; William Cole; Rudolf Jaenisch

1988-01-01

9

Regulators of the Actin Cytoskeleton Mediate Lethality in a Caenorhabditis elegans dhc-1 Mutant  

PubMed Central

Functional analysis of cytoplasmic dynein in Caenorhabditis elegans has revealed a wide range of cellular functions for this minus-end–directed motor protein. Dynein transports a variety of cargos to diverse cellular locations, and thus cargo selection and destination are likely regulated by accessory proteins. The microtubule-associated proteins LIS-1 and dynein interact, but the nature of this interaction remains poorly understood. Here we show that both LIS-1 and the dynein heavy-chain DHC-1 are required for integrity of the actin cytoskeleton in C. elegans. Although both dhc-1(or195ts) and lis-1 loss-of-function disrupt the actin cytoskeleton and produce embryonic lethality, a double mutant suppresses these defects. A targeted RNA interference screen revealed that knockdown of other actin regulators, including actin-capping protein genes and prefoldin subunit genes, suppresses dhc-1(or195ts)–induced lethality. We propose that release or relocation of the mutant dynein complex mediates this suppression of dhc-1(or195ts)--induced phenotypes. These results reveal an unexpected direct or indirect interaction between the actin cytoskeleton and dynein activity.

Gil-Krzewska, Aleksandra J.; Farber, Erica; Buttner, Edgar A.

2010-01-01

10

Genetic Characterization of the Drosophila jaguar322 Mutant Reveals That Complete Myosin VI Loss of Function Is Not Lethal  

PubMed Central

Myosin VI is an actin-based motor that has been implicated in many cellular processes. Studies in vertebrates have demonstrated that animals lacking this ubiquitously expressed myosin are viable. However in Drosophila, myosin VI loss of function has been thought to be lethal. We show here that complete loss of myosin VI is not lethal in flies and that the previously reported lethality of the null mutation (jar322) is most likely due to deletion of a neighboring gene. Maternally provided myosin VI does not account for the survival of myosin VI null animals. Mutant animals are recovered at a lower than expected Mendelian frequency, suggesting that myosin VI participates in processes which contribute to normal development, but its participation is not essential.

Morrison, Julie K.; Miller, Kathryn G.

2008-01-01

11

Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway.  

PubMed

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway. PMID:10500155

Osmond, B C; Specht, C A; Robbins, P W

1999-09-28

12

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet.  

PubMed

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2-3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention. PMID:22106289

Krebs, Philippe; Fan, Weiwei; Chen, Yen-Hui; Tobita, Kimimasa; Downes, Michael R; Wood, Malcolm R; Sun, Lei; Li, Xiaohong; Xia, Yu; Ding, Ning; Spaeth, Jason M; Moresco, Eva Marie Y; Boyer, Thomas G; Lo, Cecilia Wen Ya; Yen, Jeffrey; Evans, Ronald M; Beutler, Bruce

2011-11-21

13

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet  

PubMed Central

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2–3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.

Krebs, Philippe; Fan, Weiwei; Chen, Yen-Hui; Tobita, Kimimasa; Downes, Michael R.; Wood, Malcolm R.; Sun, Lei; Xia, Yu; Ding, Ning; Spaeth, Jason M.; Moresco, Eva Marie Y.; Boyer, Thomas G.; Lo, Cecilia Wen Ya; Yen, Jeffrey; Evans, Ronald M.; Beutler, Bruce

2011-01-01

14

Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa  

PubMed Central

Here the first reproductive sterility system for the tephritid fruit fly pest, Anastrepha suspensa, is presented, based on lethality primarily limited to embryos heterozygous for a conditional lethal transgene combination. This tetracycline (Tet)-suppressible system uses a driver construct having the promoter from the newly isolated embryo-specific A. suspensa serendipity ? gene linked to the Tet-transactivator. This was used to drive expression of a phosphomutated variant of the pro-apoptotic cell death gene, hid, from A. ludens, that was isolated, based on its identity to A. suspensa hid. The AlhidAla2 variant was shown to have the highest cell death activity in an in vitro A. suspensa cell death assay compared to the orthologous genes Ashid, Dmhid, and the variant DmhidAla5. These cell death assays also allowed a determination of the most-efficient driver-effector cassette combinations for use in A. suspensa transformants, resulting in two hybrid strains exhibiting 100% lethality. One strain was 96% lethal in embryos in the absence of tetracycline, with none surviving past the first larval instar, which is critical for pests that are most damaging in late-larval stages. We demonstrate that the isolation and in vitro validation of species-specific promoters and lethal effector genes can greatly improve the efficiency of creating high-performance conditional lethality strains that may be extended to other insect pest species.

Schetelig, Marc F.; Handler, Alfred M.

2012-01-01

15

Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa.  

PubMed

Here the first reproductive sterility system for the tephritid fruit fly pest, Anastrepha suspensa, is presented, based on lethality primarily limited to embryos heterozygous for a conditional lethal transgene combination. This tetracycline (Tet)-suppressible system uses a driver construct having the promoter from the newly isolated embryo-specific A. suspensa serendipity ? gene linked to the Tet-transactivator. This was used to drive expression of a phosphomutated variant of the pro-apoptotic cell death gene, hid, from A. ludens, that was isolated, based on its identity to A. suspensa hid. The Alhid(Ala2) variant was shown to have the highest cell death activity in an in vitro A. suspensa cell death assay compared to the orthologous genes Ashid, Dmhid, and the variant Dmhid(Ala5). These cell death assays also allowed a determination of the most-efficient driver-effector cassette combinations for use in A. suspensa transformants, resulting in two hybrid strains exhibiting 100% lethality. One strain was 96% lethal in embryos in the absence of tetracycline, with none surviving past the first larval instar, which is critical for pests that are most damaging in late-larval stages. We demonstrate that the isolation and in vitro validation of species-specific promoters and lethal effector genes can greatly improve the efficiency of creating high-performance conditional lethality strains that may be extended to other insect pest species. PMID:22647610

Schetelig, Marc F; Handler, Alfred M

2012-05-30

16

Conditional Lethal Amber Mutations in Essential Escherichia coli Genes  

Microsoft Academic Search

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia

Christopher D. Herring; Frederick R. Blattner

2004-01-01

17

Perinatal synthetic lethality and hematopoietic defects in compound mafG::mafK mutant mice.  

PubMed

Prior studies exploring the mechanisms controlling erythroid gene regulation implicated MARE (Maf recognition element) cis-elements as crucial to the transcriptional activity of many erythroid genes. Numerous transcription factors can elicit responses through MAREs, including not only the AP-1 family proteins, but also a growing list of factors composed of Cap-N-Collar (CNC)-small Maf heterodimers. While these factors can activate transcription from MAREs in co-transfection assays, mouse germline mutations in cnc genes tested to date have failed to reveal primary erythroid phenotypes. Here we report that after combining the mafK and mafG targeted null alleles, mutant animals display several synthetic phenotypes, including erythroid deficiencies. First, compound homozygous small maf gene mutants survive embryogenesis, but die postnatally. Secondly, compound mutant animals develop severe neurological disorders. Thirdly, they exhibit an exacerbated mafG deficiency in megakaryopoiesis, specifically in proplatelet formation, resulting in profound thrombocytopenia. Finally, the compound mutant animals develop severe anemia accompanied by abnormal erythrocyte morphology and membrane protein composition. These data provide direct evidence that the small Maf transcription factors play an important regulatory role in erythropoiesis. PMID:10716933

Onodera, K; Shavit, J A; Motohashi, H; Yamamoto, M; Engel, J D

2000-03-15

18

Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance  

PubMed Central

Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) ?vma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like ?vma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance.

White, W. H.; Johnson, D. I.

1997-01-01

19

Taste-aversion conditioning of house mice ( Mus domesticus) using the non-lethal repellent, cinnamamide  

Microsoft Academic Search

The potential of cinnamamide, a non-lethal repellent, to induce a conditioned taste aversion in house mice (Mus domesticus) was investigated. Mice were presented with saccharin solution, then assigned to one of four groups, each of six individuals, receiving lithium chloride, cinnamamide or blank carrier by oral intubation. The fourth group were handled but not intubated. Animals treated with cinnamamide developed

R. W. Watkins; J. E. Gurney; D. P. Cowan

1998-01-01

20

When a Fly Has to Fly to Reproduce: Selection against Conditional Recessive Lethals in "Drosophila"  

ERIC Educational Resources Information Center

|We propose an experimental model suitable for demonstrating allele frequency change in Drosophila melanogaster populations caused by selection against an easily scorable conditional lethal, namely recessive flightless alleles such as apterous and vestigial. Homozygotes for these alleles are excluded from reproduction because the food source used…

Plunkett, Andrea D.; Yampolsky, Lev Y.

2010-01-01

21

The Process of Family Management When a Baby Is Diagnosed With a Lethal Congenital Condition  

Microsoft Academic Search

The Family Management Style Framework (FMSF) was used as a conceptual basis for secondary data analysis of 55 previously conducted interviews with mothers and fathers of children with a lethal congenital condition from two surgical treatment eras. The directed content analysis was guided by a coding structure developed from family management dimensions identified in prior research of family response to

Gwen R. Rempel; Catriona Blythe; Laura G. Rogers; Vinitha Ravindran

2012-01-01

22

Embryonic Lethality of Fortilin-null mutant mice by BMP-pathway overactivation  

PubMed Central

Background Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. Methods & Results In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin+/? mice have the ability to survive and exhibit normal growth, while fortilin?/? mice are embryonically lethal around the 3.5 days post-coitum (dpc). Cultured blastocysts from fortilin+/? embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin?/? embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin+/? embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin’s message severely compromises the formation of neural tissue, including the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. Conclusion We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. General Significance Abnormalities in the fortilin gene may be associated with early pregnancy loss.

Koide, Yuichi; Kiyota, Tomomi; Tonganunt, Moltira; Pinkaew, Decha; Liu, Zhihe; Kato, Yoichi; Hutadilok-Towantana, Nongporn; Phongdara, Amornrat; Fujise, Ken

2009-01-01

23

Pathogenesis of lethal cardiac arrhythmias in Mecp2 mutant mice: implication for therapy in Rett syndrome.  

PubMed

Rett syndrome is a neurodevelopmental disorder typically caused by mutations in methyl-CpG-binding protein 2 (MECP2) in which 26% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction, we characterized the electrocardiograms in 379 people with Rett syndrome and found that 18.5% show prolongation of the corrected QT interval (QTc), an indication of a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function, Mecp2(Null/Y), also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice, Mecp2(Null/+), show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia, implicating neuronally mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome, ?-adrenergic receptor blockers, did not prevent ventricular tachycardia in Mecp2(Null/Y) mice. To determine whether an alternative therapy would be more appropriate, we characterized cardiomyocytes from Mecp2(Null/Y) mice and found increased persistent sodium current, which was normalized when cells were treated with the sodium channel-blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2(Null/Y) mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control, which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current to prevent lethal cardiac arrhythmias. PMID:22174313

McCauley, Mark D; Wang, Tiannan; Mike, Elise; Herrera, Jose; Beavers, David L; Huang, Teng-Wei; Ward, Christopher S; Skinner, Steven; Percy, Alan K; Glaze, Daniel G; Wehrens, Xander H T; Neul, Jeffrey L

2011-12-14

24

Pathogenesis of Lethal Cardiac Arrhythmias in Mecp2 Mutant Mice: Implication for Therapy in Rett Syndrome  

PubMed Central

Rett Syndrome is a neurodevelopmental disorder typically caused by mutations in Methyl-CpG-Binding Protein 2 (MECP2) in which 26% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction, we characterized the electrocardiograms (ECGs) in 379 people with Rett syndrome and found that 18.5% show prolongation of the corrected QT interval (QTc), indicating a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function, Mecp2Null/Y, also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice, Mecp2Null/+, show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia, implicating neuronally-mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome, ?-adrenergic receptor blockers, did not prevent ventricular tachycardia in Mecp2Null/Y mice. To determine whether an alternative therapy would be more appropriate, we characterized cardiomyocytes from Mecp2Null/Y mice and found increased persistent sodium current, which was normalized when cells were treated with the sodium channel-blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2Null/Y mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control, which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current in order to prevent lethal cardiac arrhythmias.

McCauley, Mark D.; Wang, Tiannan; Mike, Elise; Herrera, Jose; Beavers, David L.; Huang, Teng-Wei; Ward, Christopher S.; Skinner, Steven; Percy, Alan K.; Glaze, Daniel G.; Wehrens, Xander H. T.; Neul, Jeffrey L.

2013-01-01

25

Mutant loxP vectors for selectable marker recycle and conditional knock-outs  

PubMed Central

Background Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP. Results We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell. Conclusions Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved ?-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines.

Arakawa, Hiroshi; Lodygin, Dimitry; Buerstedde, Jean-Marie

2001-01-01

26

Cancer susceptibility and embryonic lethality in Mob1a/1b double-mutant mice.  

PubMed

Mps one binder 1a (MOB1A) and MOB1B are key components of the Hippo signaling pathway and are mutated or inactivated in many human cancers. Here we show that intact Mob1a or Mob1b is essential for murine embryogenesis and that loss of the remaining WT Mob1 allele in Mob1a(?/?)1b(tr/+) or Mob1a(?/+)1b(tr/tr) mice results in tumor development. Because most of these cancers resembled trichilemmal carcinomas, we generated double-mutant mice bearing tamoxifen-inducible, keratinocyte-specific homozygous-null mutations of Mob1a and Mob1b (kDKO mice). kDKO mice showed hyperplastic keratinocyte progenitors and defective keratinocyte terminal differentiation and soon died of malnutrition. kDKO keratinocytes exhibited hyperproliferation, apoptotic resistance, impaired contact inhibition, enhanced progenitor self renewal, and increased centrosomes. Examination of Hippo pathway signaling in kDKO keratinocytes revealed that loss of Mob1a/b altered the activities of the downstream Hippo mediators LATS and YAP1. Similarly, YAP1 was activated in some human trichilemmal carcinomas, and some of these also exhibited MOB1A/1B inactivation. Our results clearly demonstrate that MOB1A and MOB1B have overlapping functions in skin homeostasis, and exert their roles as tumor suppressors by regulating downstream elements of the Hippo pathway. PMID:23143302

Nishio, Miki; Hamada, Koichi; Kawahara, Kohichi; Sasaki, Masato; Noguchi, Fumihito; Chiba, Shuhei; Mizuno, Kensaku; Suzuki, Satoshi O; Dong, Youyi; Tokuda, Masaaki; Morikawa, Takumi; Hikasa, Hiroki; Eggenschwiler, Jonathan; Yabuta, Norikazu; Nojima, Hiroshi; Nakagawa, Kentaro; Hata, Yutaka; Nishina, Hiroshi; Mimori, Koshi; Mori, Masaki; Sasaki, Takehiko; Mak, Tak W; Nakano, Toru; Itami, Satoshi; Suzuki, Akira

2012-11-12

27

Vaccination with plasmid DNA encoding a mutant toxic shock syndrome toxin-1 ameliorates toxin-induced lethal shock in mice.  

PubMed

Staphylococcal toxic shock syndrome toxin-1 (TSST-1), a superantigenic toxin produced by Staphylococcus (S.) aureus, is a major cause of septic shock and toxic shock syndrome. To investigate whether vaccination with a plasmid DNA encoding a non-toxic mutant TSST-1 (mTSST-1) can protect mice against wild-type TSST-1-induced lethal shock, the mice were intranasally immunized with the plasmid DNA (named pcDNA-mTSST-1) plus a mucosal adjuvant, a non-toxic mutant labile toxin (mLT). After the immunization, the mice were challenged with TSST-1 and lipopolysaccharide (LPS). The survival rate of mice immunized with pcDNA-mTSST-1 plus mLT was higher than that of the control mice immunized with PBS alone, mLT alone, pcDNA-mTSST-1 alone, or a parent plasmid plus mLT. The titers of interferon-? (IFN-?) in the sera of mice immunized with pcDNA-mTSST-1 plus mLT were significantly lower than those of the mLT control mice. Immunization with pcDNA-mTSST-1 plus mLT increased the serum levels of TSST-1-specific antibodies, especially immunoglobulin G1 (IgG1) and IgG2a subclasses. Furthermore, the sera obtained from mice immunized with pcDNA-mTSST-1 plus mLT significantly inhibited the TSST-1-induced secretion of IFN-? and tumor necrosis factor-? (TNF-?) in murine spleen cells in vitro. These results indicate that immunization with pcDNA-mTSST-1 plus mLT provides protection against the lethal toxic shock of mice induced by wild-type TSST-1. The protective effect could be due to TSST-1-specific neutralizing antibodies as well as the inhibition of IFN-? and TNF-? secretions. Since TSST-1 is commonly released by invasive S. aureus, the pcDNA-mTSST-1 should be useful in preventing toxin-induced shock resulting from S. aureus infection. PMID:23985881

Feng, Mao-Hui; Cui, Jing-Chun; Nakane, Akio; Hu, Dong-Liang

2013-01-01

28

Mutual correction of faulty PCNA subunits in temperature-sensitive lethal mus209 mutants of Drosophila melanogaster.  

PubMed Central

Proliferating cell nuclear antigen (PCNA) functions in DNA replication as a processivity factor for polymerases delta and epsilon, and in multiple DNA repair processes. We describe two temperature-sensitive lethal alleles (mus209(B1) and mus209(2735)) of the Drosophila PCNA gene that, at temperatures permissive for growth, result in hypersensitivity to DNA-damaging agents, suppression of position-effect variegation, and female sterility in which ovaries are underdeveloped and do not produce eggs. We show by mosaic analysis that the sterility of mus209(B1) is partly due to a failure of germ-line cells to proliferate. Strikingly, mus209(B1) and mus209(2735) interact to restore partial fertility to heteroallelic females, revealing additional roles for PCNA in ovarian development, meiotic recombination, and embryogenesis. We further show that, although mus209(B1) and mus209(2735) homozygotes are each defective in repair of transposase-induced DNA double-strand breaks in somatic cells, this defect is substantially reversed in the heteroallelic mutant genotype. These novel mutations map to adjacent sites on the three-dimensional structure of PCNA, which was unexpected in the context of this observed interallelic complementation. These mutations, as well as four others we describe, reveal new relationships between the structure and function of PCNA.

Henderson, D S; Wiegand, U K; Norman, D G; Glover, D M

2000-01-01

29

Low-temperature conditional cell division mutants of Escherichia coli.  

PubMed Central

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome. Images

Sturgeon, J A; Ingram, L O

1978-01-01

30

Lethality of high linear energy transfer cosmic radiation to Escherichia coli DNA repair-deficient mutants during the `SL-J\\/FMPT' space experiment  

Microsoft Academic Search

We investigated the lethal and mutagenic effects of high linear energy transfer cosmic radiation on 11 strains of Escherichia coli, including DNA repair-deficient mutants, using the Radiation Monitoring Container and Dosimeter in the space shuttle `Endeavour' as part of the `SL-J\\/FMPT' space experiment, the `Fuwatto '92' project. After the return to earth of the shuttle, we evaluated survival and mutations

Kazuki Harada; Shunji Nagaoka; Mamoru Mohri; Takeo Ohnishi; Tsutomu Sugahara

1998-01-01

31

BRCA1, PARP, and 53BP1: conditional synthetic lethality and synthetic viability  

PubMed Central

BRCA1 plays a critical role in the regulation of homologous recombination (HR)-mediated DNA double-strand break repair. BRCA1-deficient cancers have evolved to tolerate loss of BRCA1 function. This renders them vulnerable to agents, such as PARP inhibitors, that are conditionally ‘synthetic lethal' with their underlying repair defect. Recent studies demonstrate that BRCA1-deficient cells may acquire resistance to these agents by partially correcting their defect in HR-mediated repair, either through reversion mutations in BRCA1 or through ‘synthetic viable' loss of 53BP1. These findings and their clinical implications will be reviewed in this article.

Aly, Amal; Ganesan, Shridar

2011-01-01

32

A conditional lethal mutation in Rana grylio virus ORF 53R resulted in a marked reduction in virion formation.  

PubMed

Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, a recombinant RGV (i53R-RGV-lacIO) containing the inducible lac repressor/operator system was constructed. i53R-RGV-lacIO was a conditional lethal mutant in which the expression of envelope protein 53R was regulated by IPTG. i53R-RGV-lacIO shared characteristics similar to RGV in the presence of IPTG. However, the expression level of 53R, the ability of plaques formation, and the virus titers were strongly reduced in the absence of IPTG. Electron microscopy showed that the number of progeny virus produced by i53R-RGV-lacIO was remarkably reduced without IPTG. Furthermore, over-expression of 53R in vitro could increase titers of i53R-RGV-lacIO in the absence of IPTG. Therefore, the current data suggested that the lac repressor/operator system could regulate gene expression in the recombinant iridovirus. Our study was thought to be the first report of the system in aquatic virus. PMID:23911634

He, Li-Bo; Gao, Xiao-Chan; Ke, Fei; Zhang, Qi-Ya

2013-07-30

33

Evolution of antibiotic resistance at non-lethal drug concentrations.  

PubMed

Human use of antimicrobials in the clinic, community and agricultural systems has driven selection for resistance in bacteria. Resistance can be selected at antibiotic concentrations that are either lethal or non-lethal, and here we argue that selection and enrichment for antibiotic resistant bacteria is often a consequence of weak, non-lethal selective pressures - caused by low levels of antibiotics - that operates on small differences in relative bacterial fitness. Such conditions may occur during antibiotic therapy or in anthropogenically drug-polluted natural environments. Non-lethal selection increases rates of mutant appearance and promotes enrichment of highly fit mutants and stable mutators. PMID:22516308

Andersson, Dan I; Hughes, Diarmaid

2012-04-18

34

Protection against lethal vaccinia virus challenge by using an attenuated matrix protein mutant vesicular stomatitis virus vaccine vector expressing poxvirus antigens.  

PubMed

Recombinant vesicular stomatitis viruses (VSV) are excellent candidate vectors for vaccination against human diseases. The neurovirulence of VSV in animal models requires the attenuation of the virus for use in humans. Previous efforts have focused on attenuating virus replication. Studies presented here test an alternative approach for attenuation that uses a matrix (M) protein mutant (rM51R) VSV as a vaccine vector against respiratory infection. This mutant is attenuated for viral virulence by its inability to suppress the innate immune response. The ability of rM51R VSV vectors to protect against lethal respiratory challenge was tested using a vaccinia virus intranasal challenge model. Mice immunized intranasally with rM51R vectors expressing vaccinia virus antigens B5R and L1R were protected against lethal vaccinia virus challenge. A single immunization with the vectors provided protection against vaccinia virus-induced mortality; however, a prime-boost strategy reduced the severity of the vaccinia virus-induced disease progression. Antibody titers measured after the prime and boost were low despite complete protection against lethal challenge. However, immunized animals had higher antibody titers during the challenge, suggesting that memory B-cell responses may be important for the protection. Depletion experiments demonstrated that B cells but not CD8 T cells were involved in the protection mediated by rM51R vaccine vectors that express B5R and L1R. These results demonstrate the potential of M protein mutant VSVs as candidate vaccine vectors against human diseases. PMID:20089648

Braxton, Cassandra L; Puckett, Shelby H; Mizel, Steven B; Lyles, Douglas S

2010-01-20

35

Identification of Novel Temperature-sensitive Lethal Alleles in Essential ?-Tubulin and Nonessential ?2-Tubulin Genes as Fission Yeast Polarity Mutants  

PubMed Central

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two ?-tubulin genes) and alp12 to nda3 (the single ?-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between ?/?-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Radcliffe, Pippa; Hirata, Dai; Childs, Dylan; Vardy, Leah; Toda, Takashi

1998-01-01

36

Lethal phenotype in conditional late-onset arginase 1 deficiency in the mouse.  

PubMed

Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia, which lead to neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation; uncommonly, patients suffer early death from this disorder. In a murine targeted knockout model, onset of the phenotypic abnormality is heralded by weight loss at around day 15, and death occurs typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. This discrepancy between the more attenuated juvenile-onset human disease and the lethal neonatal murine model has remained suboptimal for studying and developing therapy for the more common presentation of arginase deficiency. These investigations aimed to address this issue by creating an adult conditional knockout mouse to determine whether later onset of arginase deficiency also resulted in lethality. Animal survival and ammonia levels, body weight, circulating amino acids, and tissue arginase levels were examined as outcome parameters after widespread Cre-recombinase activation in a conditional knockout model of arginase 1 deficiency. One hundred percent of adult female and 70% of adult male mice died an average of 21.0 and 21.6days, respectively, after the initiation of tamoxifen administration. Animals demonstrated elevated circulating ammonia and arginine at the onset of phenotypic abnormalities. In addition, brain and liver amino acids demonstrated abnormalities. These studies demonstrate that (a) the absence of arginase in adult animals results in a disease profile (leading to death) similar to that of the targeted knockout and (b) the phenotypic abnormalities seen in the juvenile-onset model are not exclusive to the age of the animal but instead to the biochemistry of the disorder. This adult model will be useful for developing gene- and cell-based therapies for this disorder that will not be limited by the small animal size of neonatal therapy and for developing a better understanding of the characteristics of hyperargininemia. PMID:23920045

Kasten, Jennifer; Hu, Chuhong; Bhargava, Ragini; Park, Hana; Tai, Denise; Byrne, James A; Marescau, Bart; De Deyn, Peter P; Schlichting, Lisa; Grody, Wayne W; Cederbaum, Stephen D; Lipshutz, Gerald S

2013-07-06

37

Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional-lethal mutants of barley  

Microsoft Academic Search

Three plants, R9201 and R11301 (from cv. Maris Mink) and R12202 (from cv. Golden Promise), were selected by screening M2 populations of barley (Hordeum vulgare L.) seedlings (mutagenised with azide in the M1) for resistance to 10 mM potassium chlorate. Selections R9201 and R11301 were crossed with the wild-type cv. Maris Mink and analysis of the F2 progeny showed that

Barbara J. Steven; Dennis W. Kirk; Simon W. J. Bright; John L. Wray

1989-01-01

38

Mitochondrial degeneration and not apoptosis is the primary cause of embryonic lethality in ceramide transfer protein mutant mice  

PubMed Central

Ceramide transfer protein (CERT) functions in the transfer of ceramide from the endoplasmic reticulum (ER) to the Golgi. In this study, we show that CERT is an essential gene for mouse development and embryonic survival and, quite strikingly, is critical for mitochondrial integrity. CERT mutant embryos accumulate ceramide in the ER but also mislocalize ceramide to the mitochondria, compromising their function. Cells in mutant embryos show abnormal dilation of the ER and degenerating mitochondria. These subcellular changes manifest as heart defects and cause severely compromised cardiac function and embryonic death around embryonic day 11.5. In spite of ceramide accumulation, CERT mutant mice do not die as a result of enhanced apoptosis. Instead, cell proliferation is impaired, and expression levels of cell cycle–associated proteins are altered. Individual cells survive, perhaps because cell survival mechanisms are activated. Thus, global compromise of ER and mitochondrial integrity caused by ceramide accumulation in CERT mutant mice primarily affects organogenesis rather than causing cell death via apoptotic pathways.

Wang, Xin; Rao, Raghavendra Pralhada; Kosakowska-Cholody, Teresa; Masood, M. Athar; Southon, Eileen; Zhang, Helin; Berthet, Cyril; Nagashim, Kunio; Veenstra, Timothy K.; Tessarollo, Lino; Acharya, Usha; Acharya, Jairaj K.

2009-01-01

39

Synthetic Lethality of Retinoblastoma Mutant Cells in the Drosophila Eye by Mutation of a Novel Peptidyl Prolyl Isomerase Gene  

Microsoft Academic Search

Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such muta- tions promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, we performed a clonal screen in the Drosophila eye to identify second-site mutations that

Kyle A. Edgar; Marcia Belvin; Annette L. Parks; Kellie Whittaker; Matt B. Mahoney; Monique Nicoll; Christopher C. Park; Christopher G. Winter; Feng Chen; Kim Lickteig; Ferhad Ahmad; Hanife Esengil; Matthew V. Lorenzi; Amanda Norton; Brent A. Rupnow; Laleh Shayesteh; Mariano Tabios; Lynn M. Young; Pamela M. Carroll; Casey Kopczynski; Gregory D. Plowman; Lori S. Friedman; Helen L. Francis-Lang

2005-01-01

40

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae)  

PubMed Central

Background The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness. Results Here, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests. Conclusion The high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs.

Schetelig, Marc F; Caceres, Carlos; Zacharopoulou, Antigone; Franz, Gerald; Wimmer, Ernst A

2009-01-01

41

Studies on radiosensitive lines of Drosophila. IX. Analysis of fertility and frequency of dominant lethal mutations in the gamma-irradiated females of the mutant line rad(2)201\\/sup G1  

Microsoft Academic Search

Fertility and frequency of dominant lethal mutations (DLM) induced by gamma rays in females at the age of 0-5 h and 5-7 days were studied in the radiosensitive mutant rad(2)201\\/sup G1\\/ of Drosophila. It has been found that the oocytes of mutant lines are more radiosensitive as compared to those of the wild type flies when compared on the basis

E. R. Varentsova; V. I. Sharygin; Yu. M. Khromykh

1986-01-01

42

Mutations at pipX Suppress Lethality of PII-Deficient Mutants of Synechococcus elongatus PCC 7942?  

PubMed Central

The PII proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of PII proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which PII proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where PII forms complexes with the NtcA coactivator PipX, attempts to engineer PII-deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that PII is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated.

Espinosa, Javier; Castells, Miguel Angel; Laichoubi, Karim Boumediene; Contreras, Asuncion

2009-01-01

43

Isolation and partial characterization of conditional cell division cycle mutants in Chlamydomonas  

Microsoft Academic Search

Summary We have isolated a number of temperature conditional cell division cycle mutants of the unicellular plantChlamydomonas reinhardtii that are defective in single nuclear genes. Cells grow and divide normally at the permissive temperature (21 °C), but arrest in division at the restrictive temperature (33 °C). We have characterized these mutants using DNA probes and immunofluorescence techniques to localize cytoskeletal

J. D. I. Harper; L. Wu; S. Sakuanrungsirikul; P. C. L. John

1995-01-01

44

Comparison of the Behavior of Epiphytic Fitness Mutants of Pseudomonas syringae under Controlled and Field Conditions  

PubMed Central

The epiphytic fitness of four Tn5 mutants of Pseudomonas syringae that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established, demonstrating that their fitness was reduced more under field conditions than in the laboratory. Under both conditions, the four mutants exhibited distinctive behaviors. One mutant exhibited particularly large population decreases and short half-lives following inoculation but grew epiphytically at near-wild-type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by B728a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of P. syringae, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes.

Beattie, Gwyn A.; Lindow, Steven E.

1994-01-01

45

[Characterization of attenuated Salmonella C500 strain with a delta asd mutant and use as an Asd+ balanced-lethal host-vector system].  

PubMed

Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. The objective of this study was to evaluate the potential of attenuated Salmonella enterica serovar Choleraesuis C500 strain with a delta asd mutant as an effective live vaccine vector by the Asd+ balanced-lethal host-vector system. Here, we compared the characteristics of S. enterica serovar Choleraesuis delta asdC500 strain with the parent C500 strain, including phenotype, growth rate, virulence, safety, and expression for heterologous antigen. The mean generation times of delta asdC500 mutant, the vector control delta asdC500 (pYA3493), and the parent avirulent C500 vaccine strain in Luria broth were 30.7, 28.1, and 27.9 min, respectively. The fermentation patterns of theses three strains on different carbohydrates, and the levels of production of H2S, were similar. The O and H antigens of delta asdC500 mutant, delta asdC500 (pYA3493) and delta asdC500 (pYA-F1P2) were 6,7:C:1,5, identical to the parent strain C500. By the method of Reed and Muench, groups of mice were challenged by the intraperitoneal route with different amounts of delta asdC500 (pYA3493) or the parent C500 strain, and the virulence of delta asdC500 (pYA3493) with LD50 of 1.1 x 10(7) CFU was a little lower than C500 with LD50 of 4.4 x 10(6) CFU. All piglets inoculated with delta asdC500 (pYA3493) or C500 survived, and no signs of disease were observed during the entire experimental period. No major differences were found in these two groups. In addition, the recombinant pYA-F1P2 plasmid was very stable in the recombinant delta asdC500 (pYA-F1P2) strain, which expressed secretorily a large amount of the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen (rF1P2) of Bordetella bronchiseptica. In this study, we have shown that the delta asdC500 mutant had a series of biological characteristics similar to the parent vaccine strain C500. Furthermore, the strain could express secretorily a large amount of heterologous antigen. It is likely that this Salmonella expression and delivery system could be easily adapted to develop multivalent recombinant Salmonella vaccines against infectious agents using the Asd+ balanced-lethal host-vector system. PMID:19441223

Zhao, Zhanqin; Xu, Yindi; Wu, Bin; Cheng, Xiangchao; Li, Yinju; Tang, Xibiao; Zhang, Chunjie; Chen, Huanchun

2009-01-01

46

Identification of two DNA helicases UvrD and DinG as suppressors for lethality caused by mutant cspA mRNAs.  

PubMed

CspA is a major cold shock-inducible protein (70 aa), and its major role in the cold shock response was shown to be as an RNA chaperone destabilizing secondary structure of mRNAs at low temperature. Previously, we showed that the overexpression of mutant cspA containing premature non-sense codons at various positions led to stalled ribosomes on mutant cspA transcripts, ultimately leading to cell death. This lethality is primarily due to the highly translatable cspA 5'-UTR that recruits most of the ribosomes from other mRNAs, which are then stalled at the abnormal stop codon. This was called the 'LACE' effect. We show here that non-sense mutation even at the 67th position as well as substitutions of aromatic amino acid residues present on the RNA-binding surface of CspA protein to alanine caused the LACE effect by trapping a substantial amount of ribosomes on cspA mRNAs. In an attempt to identify a suppressor(s), which may help the cells to recover from the inhibitory LACE effect, genetic screening of an Escherichia coli genomic library was performed. We isolated suppressors that contained the genomic fragments encoding uvrD and dinG, respectively, whose gene products are ATP-dependent DNA helicases. The nucleic acid-binding and ATPase activities of these two helicases were found to be essential for their suppression activity. This genomic screening offers an approach to shed light on the mechanistic of 5'-UTR of cspA mRNA and novel roles of E. coli helicases that function in DNA repair. PMID:22832783

Hwang, Jihwan; Lee, Kangseok; Phadtare, Sangita; Inouye, Masayori

2012-07-24

47

Comparison of the behavior of epiphytic fitness mutants of pseudomonas syringae under controlled and field conditions  

SciTech Connect

The epiphytic fitness of four Tn5 of Pseudomonas syringae that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions, in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established at near-wild type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by By28a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of P. syringae, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes. 58 refs., 5 figs., 3 tabs.

Beattie, G.A.; Lindow, S.E. (Univ. of California, Berkeley, CA (United States))

1994-10-01

48

Combination Therapy Using Chimeric Monoclonal Antibodies Protects Mice from Lethal H5N1 Infection and Prevents Formation of Escape Mutants  

PubMed Central

Background Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. Methods/Principal Findings We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. Conclusions/Significance Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.

He, Fang; Hongliang, Qian; Ho, Hui-Ting; Qiang, Jia; TaoMeng; Goutama, Michael; Kwang, Jimmy

2009-01-01

49

The zebrafish early arrest mutants.  

PubMed

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. PMID:9007229

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

50

Classical conditioning and retention in normal and mutant Drosophila melanogaster  

Microsoft Academic Search

Summary By changing the conditioned discrimination paradigm of Quinn et al. (1974) from an instrumental procedure to a classical (Pavlovian) one, we have demonstrated strong learning in type flies. About 150 flies were sequestered in a closed chamber and trained by explosing them sequentially to two odors in air currents. Flies received twelve electric shock pulses in the presence of

Tim Tully; William G. Quinn

1985-01-01

51

Burkholderia cenocepacia conditional growth mutant library created by random promoter replacement of essential genes.  

PubMed

Identification of essential genes by construction of conditional knockouts with inducible promoters allows the identification of essential genes and creation of conditional growth (CG) mutants that are then available as genetic tools for further studies. We used large-scale transposon delivery of the rhamnose-inducible promoter, PrhaB followed by robotic screening of rhamnose-dependent growth to construct a genomic library of 106 Burkholderia cenocepacia CG mutants. Transposon insertions were found where PrhaB was in the same orientation of widely conserved, well-characterized essential genes as well as genes with no previous records of essentiality in other microorganisms. Using previously reported global gene-expression analyses, we demonstrate that PrhaB can achieve the wide dynamic range of expression levels required for essential genes when the promoter is delivered randomly and mutants with rhamnose-dependent growth are selected. We also show specific detection of the target of an antibiotic, novobiocin, by enhanced sensitivity of the corresponding CG mutant (PrhaB controlling gyrB expression) within the library. Modulation of gene expression to achieve 30-60% of wild-type growth created conditions for specific hypersensitivity demonstrating the value of the CG mutant library for chemogenomic experiments. In summary, CG mutants can be obtained on a large scale by random delivery of a tightly regulated inducible promoter into the bacterial chromosome followed by a simple screening for the CG phenotype, without previous information on gene essentiality. PMID:23389959

Bloodworth, Ruhi A M; Gislason, April S; Cardona, Silvia T

2013-02-07

52

A yeast mutant conditionally defective only for reentry into the mitotic cell cycle from stationary phase.  

PubMed Central

We report the isolation of a cold-sensitive mutant of the yeast Saccharomyces cerevisiae that is conditionally defective only for reentry into the mitotic cell cycle from stationary phase. Although actively dividing mutant cells shifted to the restrictive temperature continued to divide, stationary-phase mutant cells placed in fresh medium at the restrictive temperature failed to divide or even perform the cell cycle regulatory step "start" but did lose the characteristic stationary-phase properties of thermotolerance, accumulation of storage carbohydrates, and resistance to cell-wall-lytic enzymes. Order-of-function analysis indicated that the cold-sensitive defect blocked cells during reentry before start of the first mitotic cell cycle. Genetic analysis showed that the mutant phenotype is due to the interaction between two mutations, a cold-sensitive mutation gcs1 and a suppressor mutation sed1. These mutations thus provide the genetic basis for further analysis of stationary phase and the G0 state.

Drebot, M A; Johnston, G C; Singer, R A

1987-01-01

53

Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena.  

PubMed

Derepressed mutant PR-22 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg(-1) and cellulolytic activity from 1.9 to 4 U mg(-1); this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20-32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l(-1) of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22. PMID:20803244

Rojas-Rejón, Oscar A; Poggi-Varaldo, Héctor M; Ramos-Valdivia, Ana C; Martínez-Jiménez, Alfredo; Cristiani-Urbina, Eliseo; de la Torre Martínez, Mayra; Ponce-Noyola, Teresa

2010-08-29

54

Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa.  

PubMed

The Phi CTX-based integration vector pYM101 harboring a tightly controlled modified phage T7 early gene promoter/LacI(q) repressor (T7/LacI) system was constructed for the generation of unmarked conditional mutants in Pseudomonas aeruginosa. Promoter activity of the T7/LacI system was demonstrated to be dependent on the presence of the inducer isopropyl -beta-D-1-thiogalactopyranoside (IPTG), as evaluated by measuring beta-galactosidase activity. In the absence of the inducer, the promoter was silent as its activity was lower than those of a promoter-less lacZ control. Unmarked conditional mutants of four predicted essential genes (lolCDE (PA2988-86), lpxC (PA4406), rho (PA5239), and def (PA0019)) were successfully constructed using this recombination system. In the absence of IPTG, the growth of all mutants was repressed; however, the addition of either 0.1 or 1mM IPTG restored growth rates to levels nearly identical to wild-type cells. It was therefore demonstrated that the inducible integration vector pYM101 is suitable for the creation of unmarked conditional mutants of P. aeruginosa, and is particularly useful for examining the function of essential genes. PMID:20538017

Morita, Yuji; Narita, Shin-ichiro; Tomida, Junko; Tokuda, Hajime; Kawamura, Yoshiaki

2010-06-09

55

Characterization of the Two Maize Embryo-Lethal Defective Kernel Mutants rgh*-1210 andfl*-1253B: Effects on Embryo and Gametophyte Development  

Microsoft Academic Search

We have examined the effects on embryonic and gametophytic development of two nonallelic defective-kernel mutants of maize. Earlier studies indicated that both mutants are abnormal in embryonic morphogenesis as well as in the formation of their endosperm. Mutant rgh*-1210 embryos depart from the normal embryogenic pathway at the proembryo and transition stage, by developing meristematic lobes and losing bilateral symmetry.

Janice K. Clark; William F. Sheridan

56

Swimming Behaviour and Otolith Characteristics of wildtype and mutant Zebrafish (AIE) under diminished Gravity Conditions  

NASA Astrophysics Data System (ADS)

During microgravity humans often suffer from sensorimotor disorders e g motion sickness a kinetosis Using fish as vertebrate model systems we could previously provide ample evidence that the individually different susceptibility to such disorders is based on an individually differently pronounced asymmetric mineralisation calcification of inner ear stones otoliths In the course of a preliminary study we subjected mutant zebrafish Danio rerio due to malformation of the inner ear - see below - this mutant was termed Asymmetric Inner Ear AIE to diminished gravity conditions during parabolic aircraft flight PF As compared to wildtype WT animals the mutants showed a pronounced kinetotic behaviour The gross-morphology of the inner ears of AIE and WT animals strikingly differed In WT specimens the saccular otoliths were located at the periphery of the inner ear whereas the utricular stones were positioned mediad as it is usually the case in teleosts in most AIE animals dissected however the respective otoliths were positioned in an opposite arrangement Moreover the mutants sported transparent otoliths whereas the otoliths of WT specimens had an opaque appearance This finding clearly indicates that mutant otoliths differed from wildtype ones in their lattice structure i e the calcium carbonate polymorph and thus the compostion of the proteinacious matrix which is a template for calcium carbonate deposition In the course of the present study the PF experiment is scheduled to be carried out in March 2006 we intend to statistically verify

Weigele, J.; Anken, R.; Hilbig, R.

57

Mutants of Anabaena strain CA altered in their ability to grow under nitrogen-fixing conditions.  

PubMed Central

Mutants of Anabaena strain CA impaired in nitrogenase activity and growth on N2 were isolated and characterized. One mutant was selected for resistance to L-methionine-DL-sulfoximine, and others were selected for resistance to DL-7-azatryptophan or for requirements for combined nitrogen. The mutants varied in sensitivity of growth and nitrogenase activity to atmospheric 02. Several of the mutants whose growth on N2 was impaired under aerobic conditions could grow and reduce acetylene at rates comparable to the wild type when grown microaerobically under N2-CO2 (99:1). The acetylene reduction activity of some of the strains grown under N2-CO2 was immediately and completely lost upon exposure to atmospheric O2, but in at least one strain this loss was reversed when the O2 concentration was lowered, even after 10 h of exposure to air. The characteristics of the O2-sensitive mutants suggest that there may be several sites sensitive to O2 and that the protective mechanism involves several different phenomena.

Gotto, J W; Tabita, F R; Van Baalen, C

1979-01-01

58

Physiological and genetic analysis of Arabidopsis thaliana anthocyanin biosynthesis mutants under chronic adverse environmental conditions  

PubMed Central

Anthocyanin production is a characteristic response of flowering plants to unfavourable environmental conditions. The potential roles of flavonoids and anthocyanins in plant growth were investigated by growing Arabidopsis thaliana anthocyanin production mutants (transparent testa) under limiting nitrogen and high light conditions. Inability to produce kaempferol or subsequent intermediate compounds by some transparent testa lines was correlated with less biomass accumulation in mature plants compared with wild-type control plants under all growth conditions tested. However, under both limiting nitrogen and high light chronic stress conditions, mutant lines defective in later steps of the anthocyanin production pathway produced the same or more biomass than wild-type plants. No difference in senescence between transparent testa and wild-type plants was found using chlorophyll catabolism and SAG12 expression measurements, and no mutants were impaired in the ability to remobilize nutrients from the vegetative to reproductive tissues. Moreover, the absence of anthocyanin and/or upstream flavonoids does not affect the ability of plants to respond to limiting nitrogen by reducing photosynthetic capacity. These results support a role for kaempferol and quercetin accumulation in normal plant growth and development. Further, the absence of anthocyanins has no effect on plant growth under the chronic stress conditions tested.

Rothstein, Steven J.

2013-01-01

59

Reduced ethanol-induced conditioned taste aversion and conditioned place preference in GIRK2 null mutant mice  

Microsoft Academic Search

Rationale  Previous studies have shown that GIRK2 channel function is enhanced by ethanol and that GIRK2 null mutant mice are less sensitive\\u000a to some of ethanol's effects, including anxiolysis, habituated locomotor stimulation, and acute handling-induced convulsions\\u000a than wild types. Under some conditions, GIRK2 knockout mice consume more ethanol than wild types, but it is unclear whether\\u000a they do so because they

Katherine G. Hill; Herminia Alva; Yuri A. Blednov; Christopher L. Cunningham

2003-01-01

60

A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat.  

PubMed

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele. PMID:17490633

Mbikay, Majambu; Croissandeau, Gilles; Sirois, Francine; Anini, Younes; Mayne, Janice; Seidah, Nabil G; Chrétien, Michel

2007-04-01

61

Studies on radiosensitive lines of Drosophila. IX. Analysis of fertility and frequency of dominant lethal mutations in the gamma-irradiated females of the mutant line rad(2)201/sup G1/  

SciTech Connect

Fertility and frequency of dominant lethal mutations (DLM) induced by gamma rays in females at the age of 0-5 h and 5-7 days were studied in the radiosensitive mutant rad(2)201/sup G1/ of Drosophila. It has been found that the oocytes of mutant lines are more radiosensitive as compared to those of the wild type flies when compared on the basis of DLM frequency obtained through the entire maturation period. The early oocytes of stages 2-7, i.e., at the stages corresponding to the recombination-defective properties of mutation rad(2)201/sup g1/ are the most sensitive. It has also been demonstrated that the gamma-ray doses exceeding 10 Gy cause a strong sterilizing effect in the mutant females as a result of destruction and resorption of the egg chamber, irradiated at the stages of previtellogenic growth of oocytes. In the radiosensitive mutant females, the sensitivity of the oocytes for DLM induction does not correlate with the sensitivity of the ovarian follicles toward the resorbing effect of gamma rays. The possible involvement of the mutant locus in the genetic processes in different specialized cells of the sexual pathway in Drosophila is discussed.

Varentsova, E.R.; Sharygin, V.I.; Khromykh, Yu.M.

1986-03-01

62

Characterization of the Two Maize Embryo-Lethal Defective Kernel Mutants Rgh*-1210 and Fl*-1253b: Effects on Embryo and Gametophyte Development  

PubMed Central

We have examined the effects on embryonic and gametophytic development of two nonallelic defective-kernel mutants of maize. Earlier studies indicated that both mutants are abnormal in embryonic morphogenesis as well as in the formation of their endosperm. Mutant rgh*-1210 embryos depart from the normal embryogenic pathway at the proembryo and transition stage, by developing meristematic lobes and losing bilateral symmetry. They continue growth as irregular cell masses that enlarge and become necrotic. Somatic embryos arising in rgh*-1210 callus cultures display the rgh*-1210 mutant phenotype. Mutant fl*-1253B embryos are variably blocked from the coleoptilar stage through stage 2. Following formation of the shoot apex in the mutant embryos the leaf primordia and tissues surrounding the embryonic axis continue growth and cell division, while the scutellum ceases development and becomes hypertrophied. Mutant fl*-1253B embryos are unable to germinate, either in mutant kernels or as immature embryos in culture, and the mutant scutellar tissue does not produce regenerable callus. Expression of the fl*-1253B locus during male gametophytic development is revealed by a marked reduction in pollen transmission as a result of mutant expression during the interval between meiosis and the initiation of pollen tube growth. In both mutants, there is considerable proliferation of the aleurone cells of the endosperm. Mutant expression of rgh*-1210 in the female gametophyte is revealed by the abnormal antipodal cells of the embryo sac. These results show that these two gene loci play unique and crucial roles in normal morphogenesis of the embryo. In addition, it is evident that both mutants are pleiotropic in affecting the development of the endosperm and gametophyte as well as the embryo. These pleiotropisms suggest some commonality in the gene regulation of development in these three tissues.

Clark, J. K.; Sheridan, W. F.

1988-01-01

63

Mutant Amyloid Precursor Protein Differentially Alters Adipose Biology under Obesogenic and Non-Obesogenic Conditions  

PubMed Central

Mutations in amyloid precursor protein (APP) have been most intensely studied in brain tissue for their link to Alzheimer’s disease (AD) pathology. However, APP is highly expressed in a variety of tissues including adipose tissue, where APP is also known to exhibit increased expression in response to obesity. In our current study, we analyzed the effects of mutant APP (E693Q, D694N, K670N/M671L) expression toward multiple aspects of adipose tissue homeostasis. These data reveal significant hypoleptinemia, decreased adiposity, and reduced adipocyte size in response to mutant APP, and this was fully reversed upon high fat diet administration. Additionally, mutant APP was observed to significantly exacerbate insulin resistance, triglyceride elevations, and macrophage infiltration of adipose tissue in response to a high fat diet. Taken together, these data have significant implications for linking mutant APP expression to adipose tissue dysfunction and global changes in endocrine and metabolic function under both obesogenic and non-obesogenic conditions.

Freeman, Linnea R.; Zhang, Le; Dasuri, Kalavathi; Fernandez-Kim, Sun-Ok; Bruce-Keller, Annadora J.; Keller, Jeffrey N.

2012-01-01

64

Selection and characterization of mutants of Phanerochaete chrysosporium exhibiting ligninolytic activity under nutrient-rich conditions  

SciTech Connect

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin{yields}CO{sub 2}) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U{center dot}liter{sup {minus}1}. The mutant PSBL-1 was also able to degrade ({sup 14}C)lignin to {sup 14}CO{sub 2}, indicating that the complete ligninolytic system is deregulated.

Tien, Ming; Myer, S.B. (Pennsylvania State Univ., University Park (USA))

1990-08-01

65

The Live Attenuated Actinobacillus pleuropneumoniae Triple-Deletion Mutant ?apxIC ?apxIIC ?apxIV-ORF1 Strain, SLW05, Immunizes Pigs against Lethal Challenge with Haemophilus parasuis  

PubMed Central

Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (?apxIC ?apxIIC ?apxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-?) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine.

Fu, Shulin; Ou, Jiwen; Zhang, Minmin; Xu, Juan; Liu, Huazhen; Liu, Jinlin; Yuan, Fangyan; Chen, Huanchun

2013-01-01

66

The live attenuated Actinobacillus pleuropneumoniae triple-deletion mutant ?apxIC ?apxIIC ?apxIV-ORF1 strain, SLW05, Immunizes pigs against lethal challenge with Haemophilus parasuis.  

PubMed

Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (?apxIC ?apxIIC ?apxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-?) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine. PMID:23220998

Fu, Shulin; Ou, Jiwen; Zhang, Minmin; Xu, Juan; Liu, Huazhen; Liu, Jinlin; Yuan, Fangyan; Chen, Huanchun; Bei, Weicheng

2012-12-05

67

Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase II? Mutant Human Cell LineD?V?  

PubMed Central

DNA Topoisomerase II? (topoII?) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoII? expression can be silenced in all cells by the addition of tetracycline. TopoII?-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoII? fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoII? as an indicator for endogenous topoII? dynamics, our analyses provide new evidence that topoII? plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoII? and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

Carpenter, Adam J.; Porter, Andrew C.G.

2004-01-01

68

[Construction and characterization of delta crp delta asd mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain].  

PubMed

Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid, attenuated by chemical method. Although the vaccine has good immunogenicity, it remains some residual virulence. In order to develop a safer vaccine strain and exploit C500 as a live vaccine vector for mucosal immunization, delta crp delta asd double deletion mutant was constructed. Firstly, the recombination suicide vector with 320 bp-deleted crp (cAMP receptor protein) gene and sacB (sucrose-sensitive gene) gene was constructed and conjugated with C500. The unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR. Then the asd (beta-aspartic semialdehyde dehydrogenase) gene was further deleted in the delta crp strain by the same method. Foreign DAP (diaminopimelic acid) must be supplied for delta crp delta asd mutant to grow. The phenotype, growth properties and virulence in mice of delta crp mutant were further characterized. In conclusion, the delta crp delta asd double-deletion mutant was successfully constructed. The delta crp delta asd mutant can be used as a live vector to express foreign genes and to develop potential oral multivalent vaccines. PMID:16755912

Xu, Yin-Di; Guo, Ai-Zhen; Liu, Wei-Hong; Jia, Ai-Qing; Chen, Huan-Chun

2006-05-01

69

Retinal isomer composition in some bacteriorhodopsin mutants under light and dark adaptation conditions  

SciTech Connect

The isomeric composition of retinal was measured in a number of bacteriorhodopsin (bR) mutants (D85N, D212N, R82A, Y185F, and D115N) under various conditions, using a rapid retinal extraction technique followed by HPLC analysis. Besides the 13-cis and the all-trans retinal isomers observed in wild type (wt) bR under physiological conditions, the 11-cis and 9-cis retinal isomers were observed in variable but minor amounts in the bR mutants. In addition, the values of the equilibrium constant at two temperatures and the enthalpy change for the all-trans to 13-cis isomerization process in the dark-adapted state of D212N, D85N, deionized blue bR, and wt bR were determined. We find that perturbation of the retinal cavity (pocket) by residue replacement changes the relative thermal stability of the different retinal isomers, allowing for thermal-and/or photoisomerization of the retinal chromophore along C{sub 9}-C{sub 10} and C{sub 11}-C{sub 12} bonds to moderately compete with the isomerization around the C{sub 13}-C{sub 14} bond. The bR mutants expressed in Halobacterium salinarium studied in the present work showed normal 13-cis to all-trans light adaptation, in contrast with abnormal all-trans to 13-cis light adaptation observed for D212E, D212A, and D212N expressed in Escherichia coli, suggesting an influence of the purple membrane lattice and/or the lipids on the stability of the different retinal isomers within the protein. 38 refs., 2 tabs.

Song, L.; Yang, D.; El-Sayed, M.A. [Georgia Inst. of Technology, Atlanta, GA (United States); Lanyi, J.K. [Univ. of California, Irvine, CA (United States)

1995-06-15

70

Improved trehalose production from biodiesel waste using parent and osmotically sensitive mutant of Propionibacterium freudenreichii subsp. shermanii under aerobic conditions.  

PubMed

Trehalose is an important nutraceutical of wide commercial interest in the food processing industry. Recently, crude glycerol was reported to be suitable for the production of trehalose using a food microbe, Propionibacterium freudenreichii subsp. shermanii, under static flask conditions. Similarly, enhanced trehalose yield was reported in an osmotically sensitive mutant of the same strain under anaerobic conditions. In the present study, an effort was made to achieve higher production of trehalose, propionic acid, and lactic acid using the parent and an osmotically sensitive mutant of P. freudenreichii subsp. shermanii under aeration conditions. Under aeration conditions (200 rpm in shake flasks and 30 % air saturation in a batch reactor), biomass was increased and approximately 98 % of crude glycerol was consumed. In the parent strain, a trehalose titre of 361 mg/l was achieved, whereas in the mutant strain a trehalose titre of 1.3 g/l was produced in shake flask conditions (200 rpm). In the mutant strain, propionic and lactic acid yields of 0.53 and 0.21 g/g of substrate were also achieved with crude glycerol. Similarly, in controlled batch reactor culturing conditions a final trehalose titre of approximately 1.56 g/l was achieved with the mutant strain using crude glycerol as the substrate. Enhanced production of trehalose using P. freudenreichii subsp. shermanii from waste under aeration conditions is reported here. Higher production of trehalose was not due to a higher yield of trehalose but to a higher final biomass concentration. PMID:22526328

Ruhal, Rohit; Choudhury, Bijan

2012-04-18

71

Biochemical dissection of diageotropica and Never ripe tomato mutants to Cd-stressful conditions.  

PubMed

In order to further address the modulation of signaling pathways of stress responses and their relation to hormones, we used the ethylene-insensitive Never ripe (Nr) and the auxin-insensitive diageotropica (dgt) tomato mutants. The two mutants and the control Micro-Tom (MT) cultivar were grown over a 40-day period in the presence of Cd (0.2 mM CdCl? and 1 mM CdCl?). Lipid peroxidation, leaf chlorophyll, proline content, Cd content and antioxidant enzyme activities in roots, leaves and fruits were determined. The overall results indicated that the MT genotype had the most pronounced Cd damage effects while Nr and dgt genotypes might withstand or avoid stress imposed by Cd. This fact may be attributed, at least in part, to the fact that the known auxin-stimulated ethylene production is comprised in dgt plants. Conversely, the Nr genotype was more affected by the Cd imposed stress than dgt, which may be explained by the fact that Nr retains a partial sensitivity to ethylene. These results add further information that should help unraveling the relative importance of ethylene in regulating the cell responses to stressful conditions. PMID:22609458

Gratão, Priscila L; Monteiro, Carolina C; Carvalho, Rogério F; Tezotto, Tiago; Piotto, Fernando A; Peres, Lázaro E P; Azevedo, Ricardo A

2012-04-25

72

Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila.  

PubMed Central

The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.

Bowman, G R; Turkewitz, A P

2001-01-01

73

Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.  

PubMed

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity. PMID:1748674

Malmström, K; Krieger, M

1991-12-15

74

Response of X-ray-sensitive CHO mutant cells to gamma radiation. I. Effects of low dose rates and the process of repair of potentially lethal damage in G1 phase.  

PubMed

X-ray-sensitive CHO mutants (xrs-5 and xrs-6) were exposed to isoleucine-deficient (IL-) medium for 24-36 h to accumulate G1-phase cells. Cells exposed to IL- medium for up to 5 days did not show significant changes in plating efficiency when returned to normal medium. Nearly confluent cultures of IL- -treated cells were irradiated with either 60Co gamma rays (75 cGy/min) or 137Cs gamma rays (2.7, 6.0, or 15.3 cGy/h). A significant reduction (approximately 2.5-fold) in the radiation sensitivity of the parental CHO K-1 cells was observed for chronic low-dose-rate radiation exposure compared to the results obtained for acute high-dose-rate exposure. However, no noticeable differences were observed in the survival curves of either xrs-5 or xrs-6 cells when low-dose-rate and acute exposures were compared. CHO K-1 cells exhibited potentially lethal damage repair while held in IL- medium after gamma irradiation, whereas no repair was observed in either of the radiation-sensitive mutant lines examined at similar survival levels. PMID:2727276

Nagasawa, H; Chen, D J; Strniste, G F

1989-06-01

75

A physically altered leucyl-tRNA synthetase complex in a CHO cell mutant  

Microsoft Academic Search

CONDITIONALLY lethal mutants in animal cells have been of great value in advancing our knowledge of genetics, biochemistry and molecular biology. Mammalian cells with temperature sensitive (ts) mutant phenotypes have been isolated by several laboratories, however no thorough characterisations of the altered gene products are available1. Selection procedures have been devised by Thompson et al. to isolate ts aminoacyl-tRNA synthetase

Arnold E. Hampel

1978-01-01

76

Genetic relationships among three chlorophyll-deficient mutants in peanut, Arachis hypogaea L  

Microsoft Academic Search

The genetic relationships of three chlorophyll-deficient mutant peanuts, lutescens (lu), aureus (au), and virescent (v) were studied under field and greenhouse conditions. The F1 plants from crosses between these mutants produced phenotypically normal green. In F2, aureus X virescent segregated 675 normal green : 225 virescent : 45 aureus : 15 virescent aureus : 64 seedling lethal, and lutescens X

P. Y. P. Tai; R. O. Hammons; R. S. Matlock

1977-01-01

77

Conditioning protects C. elegans from lethal effects of enteropathogenic E. coli through activation of genes that regulate lifespan and innate immunity  

PubMed Central

SUMMARY Caenorhabditis elegans exhibit avoidance behavior when presented with diverse bacterial pathogens. We hypothesized that exposure to pathogens might not only cause worms to move away but also simultaneously activate pathways that promote resistance to the pathogen. We show that brief exposure to the virulent or avirulent strains of the bacterial pathogen enteropathogenic E. coli (EPEC) “conditions” or “immunizes” C. elegans to survive a subsequent exposure that would otherwise prove lethal. Conditioning requires dopaminergic neurons. Conditioning also requires the p38 MAP Kinase pathway, which regulates innate immunity, and the insulin/IGFR pathway, which regulates lifespan. Our findings suggest that the molecular pathways that regulate innate immunity and lifespan and provide protection may, in nature, be regulated or “conditioned” by exposure to pathogens, and perhaps allow survival in noxious environments.

Anyanful, Akwasi; Easley, Kirk A.; Benian, Guy M.; Kalman, Daniel

2010-01-01

78

Conditional infectivity of a human immunodeficiency virus matrix domain deletion mutant.  

PubMed Central

We constructed a human immunodeficiency virus (HIV) matrix (MA) deletion mutant by deletion of about 80% of the HIV type 1 Gag MA domain but retaining myristylation and proteolytic processing signals. The effects of this deletion matrix (dl.MA) mutant on HIV particle assembly, processing, and infectivity were analyzed. Surprisingly, the dl.MA mutant still could assemble and process virus particles, had a wild-type (wt) retrovirus particle density, and possessed wt reverse transcriptase activity. RNase protection experiments showed that dl.MA mutant particles preferentially packaged viral genomic RNA. When both mutant and wt particles were pseudotyped with an amphotropic murine leukemia virus envelope protein, mutant infectivity was about 10% of wt level. In contrast, infectivity of the dl.MA mutant was 1,000-fold less than that of wild-type when mutant and wt particles were pseudotyped with the HIV envelope protein. Protein analyses of pseudotyped virions indicated that there were no major differences between mutant and wt viruses in the efficiency of amphotropic murine leukemia virus envelope protein incorporation. In contrast, there was a reduction in the amount of mutant particle-associated HIV envelope protein gp120. Our results suggest that an intact HIV matrix domain is not absolutely required for reverse transcription, nuclear localization, or integration but is necessary for appropriate HIV envelope protein function. Images

Wang, C T; Zhang, Y; McDermott, J; Barklis, E

1993-01-01

79

Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae.  

PubMed Central

Genes CDC24 and CDC42 are required for the establishment of cell polarity and for bud formation in Saccharomyces cerevisiae. Temperature-sensitive (Ts-) mutations in either of these genes cause arrest as large, unbudded cells in which the nuclear cycle continues. MSB1 was identified previously as a multicopy suppressor of Ts- cdc24 and cdc42 mutations. We have now sequenced MSB1 and constructed a deletion of this gene. The predicted amino acid sequence does not closely resemble any other in the available data bases, and the deletion does not produce any readily detectable phenotype. However, we have used a colony-sectoring assay to identify additional genes that appear to interact with MSB1 and play a role in bud emergence. Starting with a strain deleted for the chromosomal copy of MSB1 but containing MSB1 on a high-copy-number plasmid, mutants were identified in which MSB1 had become essential for viability. The new mutations defined two genes, BEM1 and BEM2; both the bem1 and bem2 mutations are temperature sensitive and are only partially suppressed by MSB1. In bem1 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 or 30 degrees C, but even multiple copies of MSB1 do not fully suppress the growth defect at 37 degrees C. In bem2 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 degrees C, multiple copies are necessary for viability at 30 degrees C, and even multiple copies of MSB1 do not suppress the growth defect at 37 degrees C. In a wild-type background (i.e., a single chromosomal copy of MSB1), both bem1 and bem2 mutations cause cells to become large and multinucleate even during growth at 23 degrees C, suggesting that these genes are involved in bud emergence. This suggestion is supported for BEM1 by other evidence obtained in a parallel study (J. Chant, K. Corrado, J. Pringle, and I. Herskowitz, submitted for publication). BEM1 maps centromere distal to TYR1 on chromosome II, and BEM2 maps between SPT15 and STP2 on chromosome V. Images

Bender, A; Pringle, J R

1991-01-01

80

Paramylon (?-1,3-glucan) content in wild type and WZSL mutant of Euglena gracilis . Effects of growth conditions  

Microsoft Academic Search

The effects of growth conditions on paramylon (a ß-1,3-glucanreserve carbohydrate) content were examined in the photosyntheticwild-type and a spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. This carbohydrate is known toshow interesting applications in human and veterinary medicine, asimmunostimulant and immunopotentiator. For both strains, the synthesisof reserve depends mainly on the growth conditions, i.e. light or dark. Thehighest amount

Laura Barsanti; Rosa Vismara; Vincenzo Passarelli; Paolo Gualtieri

2001-01-01

81

Manipulating individual decisions and environmental conditions reveal individual quality in decision-making and non-lethal costs of predation risk.  

PubMed

Habitat selection is a crucial decision for any organism. Selecting a high quality site will positively impact survival and reproductive output. Predation risk is an important component of habitat quality that is known to impact reproductive success and individual condition. However, separating the breeding consequences of decision-making of wild animals from individual quality is difficult. Individuals face reproductive decisions that often vary with quality such that low quality individuals invest less. This reduced reproductive performance could appear a cost of increased risk but may simply reflect lower quality. Thus, teasing apart the effects of individual quality and the effect of predation risk is vital to understand the physiological and reproductive costs of predation risk alone on breeding animals. In this study we alter the actual territory location decisions of pied flycatchers by moving active nests relative to breeding sparrowhawks, the main predators of adult flycatchers. We experimentally measure the non-lethal effects of predation on adults and offspring while controlling for effects of parental quality, individual territory choice and initiation of breeding. We found that chicks from high predation risk nests (<50 m of hawk) were significantly smaller than chicks from low risk nests (>200 m from hawk). However, in contrast to correlative results, females in manipulated high risk nests did not suffer decreased body condition or increased stress response (HSP60 and HSP70). Our results suggest that territory location decisions relative to breeding avian predators cause spatial gradients in individual quality. Small adjustments in territory location decisions have crucial consequences and our results confirm non-lethal costs of predation risk that were expressed in terms of smaller offspring produced. However, females did not show costs in physiological condition which suggests that part of the costs incurred by adults exposed to predation risk are quality determined. PMID:23272226

Thomson, Robert L; Tomás, Gustavo; Forsman, Jukka T; Mönkkönen, Mikko

2012-12-13

82

Manipulating Individual Decisions and Environmental Conditions Reveal Individual Quality in Decision-Making and Non-Lethal Costs of Predation Risk  

PubMed Central

Habitat selection is a crucial decision for any organism. Selecting a high quality site will positively impact survival and reproductive output. Predation risk is an important component of habitat quality that is known to impact reproductive success and individual condition. However, separating the breeding consequences of decision-making of wild animals from individual quality is difficult. Individuals face reproductive decisions that often vary with quality such that low quality individuals invest less. This reduced reproductive performance could appear a cost of increased risk but may simply reflect lower quality. Thus, teasing apart the effects of individual quality and the effect of predation risk is vital to understand the physiological and reproductive costs of predation risk alone on breeding animals. In this study we alter the actual territory location decisions of pied flycatchers by moving active nests relative to breeding sparrowhawks, the main predators of adult flycatchers. We experimentally measure the non-lethal effects of predation on adults and offspring while controlling for effects of parental quality, individual territory choice and initiation of breeding. We found that chicks from high predation risk nests (<50 m of hawk) were significantly smaller than chicks from low risk nests (>200 m from hawk). However, in contrast to correlative results, females in manipulated high risk nests did not suffer decreased body condition or increased stress response (HSP60 and HSP70). Our results suggest that territory location decisions relative to breeding avian predators cause spatial gradients in individual quality. Small adjustments in territory location decisions have crucial consequences and our results confirm non-lethal costs of predation risk that were expressed in terms of smaller offspring produced. However, females did not show costs in physiological condition which suggests that part of the costs incurred by adults exposed to predation risk are quality determined.

Thomson, Robert L.; Tomas, Gustavo; Forsman, Jukka T.; Monkkonen, Mikko

2012-01-01

83

Combining Inhibitor Resistance-conferring Mutations in Cytochrome b Creates Conditional Synthetic Lethality in Saccharomyces cerevisiae*S?  

PubMed Central

The mitochondrial cytochrome bc1 complex is an essential respiratory enzyme in oxygen-utilizing eukaryotic cells. Its core subunit, cytochrome b, contains two sites, center P and center N, that participate in the electron transfer activity of the bc1 complex and that can be blocked by specific inhibitors. In yeast, there are various point mutations that confer inhibitor resistance at center P or center N. However, there are no yeast strains in which the bc1 complex is resistant to both center P and center N inhibitors. We attempted to create such strains by crossing yeast strains with inhibitor-resistant mutations at center P with yeast strains with inhibitor-resistant mutations at center N. Characterization of yeast colonies emerging from the cross revealed that there were multiple colonies resistant against either inhibitor alone but that the mutational changes were ineffective when combined and when the yeast were grown in the presence of both inhibitors. Inhibitor titrations of bc1 complex activities in mitochondrial membranes from the various yeast mutants showed that a mutation that confers resistance to an inhibitor at center P, when combined with a mutation that confers resistance to an inhibitor at center N, eliminates or markedly decreases the resistance conferred by the center N mutation. These results indicate that there is a pathway for structural communication between the two active sites of cytochrome b and open new possibilities for the utilization of center N as a potential drug target.

Ding, Martina G.; di Rago, Jean-Paul; Trumpower, Bernard L.

2009-01-01

84

Levan production using mutant strains of Zymomonas mobilis in different culture conditions  

Microsoft Academic Search

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l-1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l-1 and 0.5 g (NH4)2SO4 l-1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.

Ana Carolina Muro; Emilio Rodríguez; Carlos M. Abate; Faustino Siñeriz

2000-01-01

85

Culture conditions of protoplast-derived cells of nicotiana sylvestris for mutant selection  

Microsoft Academic Search

Large numbers of protoplasts showing reproducible high plating efficiency can be isolated from in vitro propagated, haploid and diploid, plants of Nicotiana sylvestris. Their successful use in the selection of biochemical mutants depends on the establishment of suitable selection parameters: culture medium, cell density, age of cells at selection etc. Plating of protoplasts at low densities as well as simulation

I. Negrutiu; J. F. Muller

1981-01-01

86

Characteristics of Wines Made by Saccharomyces Mutants Which Produce a Polygalacturonase under Wine-Making Conditions  

Microsoft Academic Search

Wines by yeast mutants producing polygalacturonase in high glucose concentration, from Saccharomyces wine-making strains, had higher filterability and more concentrated anthocyanin contents than that of their parent strains. These results suggest that the clarifica- tion process was improved at a lower cost by the low viscosity and that high-quality wines result from the increase in the anthocyanin contents.

Florentina RADOI; Masao KISHIDA; Haruhiko KAWASAKI

2005-01-01

87

RBE of ? particles and postradiation recovery in a temperature conditional mutant of diploid yeast for various criteria of cell death  

Microsoft Academic Search

The influence of linear energy transfer (LET) on relative biological effectiveness (RBE) and the liquid-holding recovery (LHR)\\u000a ability was studied for a temperature conditional mutant of diploid yeast. Both the RBE and the rate of LHR were much lower\\u000a for reproductive killing at 36C and cell killing without division at both 23 and 36C. However, the probability of recovery\\u000a was

V. G. Petin; N. M. Kabakova; V. I. Wrigglesworth; J. K. Kim

2006-01-01

88

Conditions supporting repair of potentially lethal damage cause a significant reduction of ultraviolet light-induced division delay in synchronized and plateau-phase Ehrlich ascites tumor cells  

SciTech Connect

Repair of potentially lethal damage (PLD) induced by uv light in synchronized and in plateau-phase cultures of Ehrlich ascites tumor cells was studied by measuring cell survival. In particlar the influence of conditions supporting repair of PLD on growth kinetics was investigated. In synchronized G/sub 1/, S, or G/sub 2/ + M cells as well as in plateau-phase cells, uv light induced, almost exclusively, delay in the next S phase. A significant decrease of this delay was observed when the cells were incubated for 24 hr in balanced salt solution. Repair of PLD after uv irradiation was found to occur in plateau-phase cells and in cells in different phases of the cell cycle provided that after irradiation these were kept under conditions inhibiting cell multiplication (incubation in balanced salt solution or in conditioned medium). The repair time constant t/sub 50/ was significantly higher than those found for X irradiation (5-10 hr compared to 2 hr), and repair was not significantly inhibited by either 20 ..mu..g/ml cycloheximide or 2 mM caffeine in 24 hr.

Iliakis, G.; Nusse, M.

1982-09-01

89

Conditional inactivation of TGF-? type II receptor in smooth muscle cells and epicardium causes lethal aortic and cardiac defects  

Microsoft Academic Search

To understand the role of TGF-? signaling in cardiovascular development, we generated mice with conditional deletion of the\\u000a TGF-? type II receptor (T?RII) gene (Tgfbr2) in cells expressing the smooth muscle cell-specific protein SM22?. The SM22? promoter was active in tissues involved in cardiovascular development: vascular smooth muscle cells (VSMCs), epicardium and\\u000a myocardium. All SM22-Cre+\\/?\\/Tgfbr2\\u000a flox\\/flox embryos died during the

Dominique Langlois; Mohammad Hneino; Lamia Bouazza; Ara Parlakian; Takako Sasaki; Giampiero Bricca; Jacques Yuan Li

2010-01-01

90

Bright Mutants of Vibrio fischeri ES114 Reveal Conditions and Regulators That Control Bioluminescence and Expression of the lux Operon?  

PubMed Central

Vibrio fischeri ES114, an isolate from the Euprymna scolopes light organ, produces little bioluminescence in culture but is ?1,000-fold brighter when colonizing the host. Cell-density-dependent regulation alone cannot explain this phenomenon, because cells within colonies on solid medium are much dimmer than symbiotic cells despite their similar cell densities. To better understand this low luminescence in culture, we screened ?20,000 mini-Tn5 mutants of ES114 for increased luminescence and identified 28 independent “luminescence-up” mutants with insertions in 14 loci. Mutations affecting the Pst phosphate uptake system led to the discovery that luminescence is upregulated under low-phosphate conditions by PhoB, and we also found that ainS, which encodes an autoinducer synthase, mediates repression of luminescence during growth on plates. Other novel luminescence-up mutants had insertions in acnB, topA, tfoY, phoQ, guaB, and two specific tRNA genes. Two loci, hns and lonA, were previously described as repressors of bioluminescence in transgenic Escherichia coli carrying the light-generating lux genes, and mutations in arcA and arcB were consistent with our report that Arc represses lux. Our results reveal a complex regulatory web governing luminescence and show how certain environmental conditions are integrated into regulation of the pheromone-dependent lux system.

Lyell, Noreen L.; Dunn, Anne K.; Bose, Jeffrey L.; Stabb, Eric V.

2010-01-01

91

Hypomorphic Lethal Mutations and Their Implications for the Interpretation of Lethal Complementation Studies in Drosophila  

PubMed Central

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.

Nash, David; Janca, Frank C.

1983-01-01

92

Excretion of pyruvate by mutants of Alcaligenes eutrophus , which are impaired in the accumulation of poly(?-hydroxybutyric acid) (PHB), under conditions permitting synthesis of PHB  

Microsoft Academic Search

Mutants of Alcaligenes eutrophus, which are defective in the intracellular accumulation of poly(ß-hydroxybutyric acid), PHB, were cultivated in the presence of excess carbon source after growth had ceased due to depletion of ammonium, sulphate, phosphate, potassium, magnesium, or iron. Under these conditions all mutants excreted large amounts of pyruvate into the medium. Excretion of pyruvate occurred with lactate, gluconate or

Alexander Steinbiichel; Hans G. Schlegel

1989-01-01

93

LT(R192G), a non-toxic mutant of the heat-labile enterotoxin of Escherichia coli, elicits enhanced humoral and cellular immune responses associated with protection against lethal oral challenge with Salmonella spp  

Microsoft Academic Search

In the current study we examined the ability of a novel mucosal adjuvant, LT(R192G), to enhance the humoral and cellular immune responses against killed Salmonella spp. and to affect protection against lethal oral challenge with wild-type organisms. Mice orally immunized with killed S. dublin in conjunction with LT(R192G) were protected against lethal oral challenge and had higher IFN-?, IL-2 and

Celeste Chong; Maria Friberg; John D Clements

1998-01-01

94

Identification of Drosophila mutant with memory defects after acquisition of conditioned reflex suppression of courtship  

Microsoft Academic Search

Four lines were selected from a collection of 33 lines prepared by P insertion mutagenesis using a single-copy P-element system;\\u000a the males of these four lines showed memory defects after acquisition of conditioned reflex suppression of courting. In two\\u000a lines (P171 and P95), the dynamics of retention of the conditioned reflex in the repeated impregnated-female courting test\\u000a were similar to

N. G. Kamyshev; K. G. Iliadi; Yu. V. Bragina; E. V. Savvateeva-Popova; E. V. Tokmacheva; T. Preat

2000-01-01

95

Construction and characterization of herpes simplex virus type 1 mutants with conditional defects in immediate early gene expression.  

PubMed

The herpes simplex virus type 1 (HSV-1) mutant in 1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICPO, to yield mutant in 1820. In almost all cell types tested, in 1820 initiated infection less efficiently than in 1814, behaving as if lacking both VP16 and ICPO functions, but in BHK cells in 1820 was less impaired than in 1814. A rescuant of in 1820 at the VP16 locus, in 1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types. In 1825 was unable to complement an ICPO null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICPO protein in functionally significant amounts. The novel host range properties of in 1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1 tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, into in 1820 to produce in 1820K. This mutant could be propagated effectively in BHK cells at 31 degrees but was effectively devoid of the major regulators ICPO, ICP4, and VP16 in other cells types at 38.5 degrees. Cultures could withstand infection with 5 PFU of in 1820K per cell without detectable cytopathology and could be reseeded to form colonies at approximately 90% efficiency. A derivative of in 1820K containing the Escherichia coli lacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of beta-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5 degrees. Cultures infected with 5 PFU per cell of an in 1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based on in 1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-1-derived vectors. PMID:9123865

Preston, C M; Mabbs, R; Nicholl, M J

1997-03-01

96

Dynein Modifiers in C. elegans: Light Chains Suppress Conditional Heavy Chain Mutants  

Microsoft Academic Search

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis

Sean M ORourke; Marc D Dorfman; J. Clayton Carter; Bruce Bowerman

2007-01-01

97

Gene expression profiles in developing nephrons using Lim1 metanephric mesenchyme-specific conditional mutant mice  

Microsoft Academic Search

BACKGROUND: Lim1 is a homeobox gene that is essential for nephrogenesis. During metanephric kidney development, Lim1 is expressed in the nephric duct, ureteric buds, and the induced metanephric mesenchyme. Conditional ablation of Lim1 in the metanephric mesenchyme blocks the formation of nephrons at the nephric vesicle stage, leading to the production of small, non-functional kidneys that lack nephrons. METHODS: In

You-Tzung Chen; Akio Kobayashi; Kin Ming Kwan; Randy L Johnson; Richard R Behringer

2006-01-01

98

Lethal and sublethal effects of endosulfan, imidacloprid and indoxacarb on first instar larvae of Chrysoperla carnea (Neu.: Chrysopidae) under laboratory conditions  

Microsoft Academic Search

The common green lacewing is an important natural enemy used for pest control in greenhouses. It is also very common in many agricultural systems. Hence, studying lethal and sublethal effects of insecticides on this predator would be useful. Toxicity of endosulfan, imidacloprid and indoxacarb was assessed on 1 st instar larvae of Chrysoperla carnea (Stephens) in laboratory. Residual bioassays were

M. Hejazi; S. A. Mohammadi

99

Failure of chimerism formation and tolerance induction from Fas ligand mutant bone marrow donors after nonmyeloablative conditioning.  

PubMed

Formation of donor-recipient mixed chimerism after nonmyeloablative conditioning allows co-existence of donor and recipient hematopoietic stem cells, with solid organ allograft tolerance and less likeliness of graft versus host development. Using a post-transplant bronchiolitis obliterans murine model, we aimed to test the hypothesis that allograft preservation after mixed chimerism formation is dependent on the presence of a functional Fas ligand (FasL) on donor hematopoietic cells. To form mixed chimerism, two aliquots of 30 × 10(6) whole bone marrow cells (BMC) from either wild-type C57BL/6 in one group, or transgenic gld mice with mutant FasL (d = 0 and 2+) in the other were used, with both groups receiving intravenous busulfan (10mg/kg) on d-1 and intraperitoneal cyclophosphamide (200mg/kg) on d+1. Tracheal allografts obtained from C57BL/6 mice were implanted into recipient BALB/c mice subcutaneously on d = 0. Tracheal allografts were harvested at d+28 post-transplant and were evaluated by histopathology. Mixed chimerism formation was detected in wild type C57BL/6 whole BMC recipients, which was accompanied by tracheal allograft acceptance with near normal structure at d+28 post implantation. However, in recipients of FasL mutant whole BMC, neither mixed chimerism formation nor tracheal allograft acceptance was obtained. We thus conclude that bone marrow cells lacking functional FasL molecules could not be engrafted in allogeneic recipients to form stable mixed chimerism after nonmyeloablative conditioning, thus not allowing tracheal allograft acceptance. PMID:22801052

Nusair, Samir; Gincberg, Galit; Almogi-Hazan, Osnat; Breuer, Raphael; Or, Reuven; Wallach-Dayan, Shulamit B

2012-07-16

100

Condition stabilization for Aspergillus niger FCBP-198 and its hyperactive mutants to yield high titres of alpha-amylase.  

PubMed

A number of substrates were tested for the cultivation of microorganisms to produce a host of enzymes. The effect of different substrates (wheat and rice straw, sugar cane waste, wood waste), incubation temperatures (20-40 degrees C), initial pH levels (3.5-9.0), incubation periods (0-72 hours) and nitrogen sources (ammonium sulfate, urea, peptone, yeast extract, sodium nitrate) on growth and alpha-amylase activity was studied for the native and mutant strains. Maximum enzyme activity was observed at 1.5% wheat straw for Aspergillus niger FCBP-198 and An-Ch-4.7 and at 2% wheat straw for An-UV-5.6, with sodium nitrate as a principle nitrogen source. The optimum temperature for maximum enzyme activity was 30 degrees C for the parental strain, while An-UV-5.6 and An-Ch-4.7 thrived well at 32.5 degrees C. The best conditions of pH and incubation duration were 4.5 and 48 hours, respectively, for all the strains. Mass production under preoptimized growth conditions demonstrated the suitability of wheat straw for swift mycelial colonization and viability. PMID:20734811

Shafique, Sobiya; Bajwa, Rukhsana; Shafique, Shazia

101

Model-driven analysis of experimentally determined growth phenotypes for 465 yeast gene deletion mutants under 16 different conditions  

PubMed Central

Background Understanding the response of complex biochemical networks to genetic perturbations and environmental variability is a fundamental challenge in biology. Integration of high-throughput experimental assays and genome-scale computational methods is likely to produce insight otherwise unreachable, but specific examples of such integration have only begun to be explored. Results In this study, we measured growth phenotypes of 465 Saccharomyces cerevisiae gene deletion mutants under 16 metabolically relevant conditions and integrated them with the corresponding flux balance model predictions. We first used discordance between experimental results and model predictions to guide a stage of experimental refinement, which resulted in a significant improvement in the quality of the experimental data. Next, we used discordance still present in the refined experimental data to assess the reliability of yeast metabolism models under different conditions. In addition to estimating predictive capacity based on growth phenotypes, we sought to explain these discordances by examining predicted flux distributions visualized through a new, freely available platform. This analysis led to insight into the glycerol utilization pathway and the potential effects of metabolic shortcuts on model results. Finally, we used model predictions and experimental data to discriminate between alternative raffinose catabolism routes. Conclusions Our study demonstrates how a new level of integration between high throughput measurements and flux balance model predictions can improve understanding of both experimental and computational results. The added value of a joint analysis is a more reliable platform for specific testing of biological hypotheses, such as the catabolic routes of different carbon sources.

Snitkin, Evan S; Dudley, Aimee M; Janse, Daniel M; Wong, Kaisheen; Church, George M; Segre, Daniel

2008-01-01

102

The conditional connexin43G138R mouse mutant represents a new model of hereditary oculodentodigital dysplasia in humans  

PubMed Central

Oculodentodigital dysplasia (ODDD) is a dominant negatively inherited disorder with variable but characteristic anomalies of the fingers and toes, eyes, face and teeth, which are caused by mutations in the connexin 43 (Cx43) gene. All mutations analyzed so far have a negative influence on the conductance through gap junctional channels and hemichannels, as well as trafficking of Cx43 protein in transfected cells. In this study, we inserted the human Cx43G138R point mutation into the mouse Cx43 gene and generated mice conditionally expressing this mutation. All ODDD phenotypic manifestations observed in humans, including syndactyly and enamel hypoplasia as well as craniofacial, bone and heart anomalies, were also observed with significant penetrance in Cx43G138R mice. When this mutation was specifically expressed in cardiomyocytes, characteristic alterations in the electrocardiogram and spontaneous arrhythmias were recorded. In vitro studies with Cx43G138R-expressing cells revealed loss of the Cx43 P2 phosphorylation state, which was also absent in the mutated hearts. This loss has previously been associated with gap junctional dysfunction and increased cellular ATP release. The Cx43G138R mutated mice show significantly increased arrhythmogeneity ex vivo in Langendorff experiments with explanted hearts and in vivo in particular under hypoxic conditions. Our results suggest that the increased activity of ATP-releasing channels in Cx43G138R mutated cardiomyocytes may further reduce the already decreased gap junctional communication and thus aggravate arrhythmogenesis in the mouse mutant.

Dobrowolski, Radoslaw; Sasse, Philipp; Schrickel, Jan W.; Watkins, Marcus; Kim, Jung-Sun; Rackauskas, Mindaugas; Troatz, Clemens; Ghanem, Alexander; Tiemann, Klaus; Degen, Joachim; Bukauskas, Feliksas F.; Civitelli, Roberto; Lewalter, Thorsten; Fleischmann, Bernd K.; Willecke, Klaus

2010-01-01

103

Simultaneous Gain and Loss of Functions Caused by a Single Amino Acid Substitution in the ? Subunit of Escherichia Coli RNA Polymerase: Suppression of Nusa and Rho Mutations and Conditional Lethality  

PubMed Central

Transcript elongation and termination in Escherichia coli is modulated, in part, by the nusA gene product, an acidic protein that interacts not only with RNA polymerase itself but also with ancillary factors, namely the host termination protein Rho and phage ? antitermination protein, N. The E. coli nusA1 mutant fails to support ? development due to a specific defect in N-mediated antitermination. Certain rifampicin-resistant (rif(R)) variants of the nusA1 host support ? growth. We report here the isolation and pleiotropic properties of one such rif(R) mutant, ts8, resulting from a single amino acid substitution mutation in rpoB, the structural gene for polymerase ? subunit. ts8 is a recessive lethal mutation that blocks cell growth at 42°. Pulse-labeling and analysis of newly synthesized proteins indicate that the mutant cell is proficient in RNA synthesis at high temperature. Apparently, ts8 causes a loss of some specialized function of RNA polymerase without a gross defect in general transcription activities. ts8 is an allele-specific suppressor of nusA1. It does not suppress nusAsal, nusB5 and nusE71 mutations nor does it bypass the requirement for a functional N gene and the nut site for antitermination and ? growth. A mutation in the N gene, punA1, that restores ? growth in the nusA1 mutant host but not in the nusAsal host, compensates for the nusAsal allele in the ts8 mutant. This combined effect of two allele-specific suppressors suggests that they enhance some aspect of polymerase-NusA-N interaction and function. ts8 suppresses the rho15 mutation, but not the rho112 mutation, indicating that it might render RNA polymerase susceptible to the action of a defective Rho protein. Marker rescue analysis has localized ts8 to a 910-bp internal segment of rpoB that encodes the Rif domain. By amplification, cloning and sequencing of this segment of the mutant chromosome we have determined that ts8 contains Phe in place of Ser522, caused by a C to T transition. By gene conversion, we have established that the simultaneous gain and loss of three functions of polymerase is caused by this single amino acid substitution. Clearly, a site in the ? subunit critical for the functioning of both termination and antitermination factors is altered by ts8. The alteration, we imagine, might make this site on polymerase receptive to some factors but repulsive to others.

Sparkowski, J.; Das, A.

1992-01-01

104

Optimization of flask culture medium and conditions for hyaluronic acid production by a Streptococcus equisimilis mutant nc2168.  

PubMed

A mutant designated NC2168, which was selected from wild-type Streptococcus equisimilis CVCC55116 by ultraviolet ray combined with(60)Co-? ray treatment and does not produce streptolysin, was employed to produce hyaluronic acid (HA). In order to increase the output of HA in a flask, the culture medium and conditions for NC2168 were optimized in this study. The influence of culture medium ingredients including carbon sources, nitrogen sources and metal ions on HA production was evaluated using factional factorial design. The mathematical model, which represented the effect of each medium component and their interaction on the yield of HA, was established by the quadratic rotary combination design and response surface method. The model estimated that, a maximal yield of HA could be obtained when the concentrations of yeast extract, peptone, glucose, and MgSO4 were set at 3 g/100 mL, 2 g/100 mL, 0.5 g/100 mL and 0.15 g/100 mL, respectively. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a remarkable increase in the output of HA and the maximum of the predicted HA production was 174.76 mg/L. The model developed was accurate and reliable for predicting the production of HA by NC2168.Cultivation conditions were optimized by an orthogonal experimental design and the optimal conditions were as follows: temperature 33°C, pH 7.8, agitation speed 200 rpm, medium volume 20 mL. PMID:24031987

Chen, Yong-Hao; Li, Jun; Liu, Li; Liu, Hong-Zhi; Wang, Qiang

2012-06-01

105

Plaque Formation by a Host Range Mutant of Vaccinia Virus in Non-permissive Cells Co-infected with Yaba Virus  

Microsoft Academic Search

SUMMARY A host-dependent conditional lethal mutant of vaccinia virus strain Dis was rescued to form plaques in a non-permissive cell line JINET co-infected with Yaba virus, a poxvirus serologically distant from the rescued virus. The efficiency of plaque formation under optimal conditions in this system was comparable to that in Vero ceils which were permissive for the mutant. The plaque

Y. Tsuchiya; I. Tagaya

1979-01-01

106

Multiple Alterations in Metabolite Uptake in a Mutant of Neurospora crassa  

PubMed Central

A temperature-conditional lethal mutant of Neurospora crassa, un-t (55701), was resistant to neutral amino acid analogues by virtue of a decreased ability to transport these analogues and their natural congeners across the cell membrane. The uptake of acidic, but not basic, amino acids was also impaired, as was the uptake of potassium ions. After preincubation above the tolerated temperature, the ability to take up a still wider variety of metabolites was greatly reduced. Protoplasts of the mutant were more osmotically fragile than those of wild type. The possibility that the mutant has a generalized membrane defect is discussed.

Kappy, Michael S.; Metzenberg, Robert L.

1967-01-01

107

Functional Analysis of the Interaction between VPg-Proteinase (NIa) and RNA Polymerase (NIb) of Tobacco Etch Potyvirus, Using Conditional and Suppressor Mutants  

PubMed Central

The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.

Daros, Jose-Antonio; Schaad, Mary C.; Carrington, James C.

1999-01-01

108

Construction of Listeria monocytogenes Mutants with In-Frame Deletions in the Phosphotransferase Transport System (PTS) and Analysis of Their Growth under Stress Conditions.  

PubMed

Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365_0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365_0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444) and the downstream gene LMOf2365_0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of ?LMOf2365_0445 was increased under nisin (125 ?g/mL) treatments compared to the wild-type (P < 0.01). The growth of ?LMOf2365_0442 in salt (brain-heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and ?LMOf2365_0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants ?LMOf2365_0442 and ?LMOf2365_0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food. PMID:23909479

Liu, Yanhong; Ceruso, Marina; Jiang, Yuji; Datta, Atin R; Carter, Laurenda; Strain, Errol; Pepe, Tiziana; Anastasi, Aniello; Fratamico, Pina

2013-08-02

109

LT(R192G), a non-toxic mutant of the heat-labile enterotoxin of Escherichia coli, elicits enhanced humoral and cellular immune responses associated with protection against lethal oral challenge with Salmonella spp.  

PubMed

In the current study we examined the ability of a novel mucosal adjuvant, LT(R192G), to enhance the humoral and cellular immune responses against killed Salmonella spp. and to affect protection against lethal oral challenge with wild-type organisms. Mice orally immunized with killed S. dublin in conjunction with LT(R192G) were protected against lethal oral challenge and had higher IFN-gamma, IL-2 and IgG responses than did mice orally immunized with killed S. dublin alone which were not protected. This study demonstrates that the function of LT(R192G) in protection against typhoid-like disease is to upregulate/enhance the Th1 arm of the immune response against killed organisms. When used as a mucosal adjuvant, LT(R192G) enables the use of killed bacteria or viruses as vaccines by enhancing the overall humoral and cellular host immune response to these organisms, especially the Th1 arm of the immune response. These findings have significant implications for vaccine development and further support the potential of LT(R192G) to function as a safe, effective adjuvant for mucosally administered vaccines. PMID:9562694

Chong, C; Friberg, M; Clements, J D

1998-04-01

110

Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator (ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain.  

PubMed

The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts. PMID:16112258

Zhu, Chengru; Feng, Shuzhang; Thate, Timothy E; Kaper, James B; Boedeker, Edgar C

2005-07-28

111

Generation of new Notch2 mutant alleles.  

PubMed

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. We previously described construction and analysis of a hypomorphic allele of the Notch2 gene. Homozygosity for this allele leads to embryonic and perinatal lethality due to cardiovascular and kidney defects. We report here novel Notch2 mutant alleles generated by gene targeting in embryonic stem cells, including a conditional null allele in which exon 3 of the Notch2 gene is flanked by loxP sequences. These new Notch2 mutant alleles expand the set of tools available for studying the myriad roles of the Notch pathway during mammalian development and will enable analysis of Notch2 function at additional stages of embryogenesis and in adult mice. PMID:16397869

McCright, Brent; Lozier, Julie; Gridley, Thomas

2006-01-01

112

Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiella oxytoca under microaerobic conditions  

Microsoft Academic Search

Klebsiella oxytoca M5al is an excellent 1,3-propanediol (1,3-PD) producer, but too much lactic acid yielded greatly lessened the fermentation\\u000a efficiency for 1,3-PD. To counteract the disadvantage, four lactate deficient mutants were obtained by knocking out the ldhA gene of lactate dehydrogenase (LDH) of K. oxytoca M5al. The LDH activities of the four mutants were from 3.85 to 6.92% of the

Guang Yang; Jiesheng Tian; Jilun Li

2007-01-01

113

A targeted deletion\\/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat  

Microsoft Academic Search

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of

Majambu Mbikay; Gilles Croissandeau; Francine Sirois; Younes Anini; Janice Mayne; Nabil G. Seidah; Michel Chrétien

2007-01-01

114

NON-LETHAL INCAPACITATION  

Microsoft Academic Search

IN SIMPLE TERMS, NON-LETHAL (OR LESS-THAN-LETHAL) INCAPACITATION means 'to render a suspect incapable of action by means of force which is highly unlikely to cause death or serious injury when properly applied'. In America, this process requires that the suspect fall to the ground as a result of the force. An important caveat at this point is to emphasise that

Robert Hamdorf

115

Modeling synthetic lethality  

PubMed Central

Background Synthetic lethality defines a genetic interaction where the combination of mutations in two or more genes leads to cell death. The implications of synthetic lethal screens have been discussed in the context of drug development as synthetic lethal pairs could be used to selectively kill cancer cells, but leave normal cells relatively unharmed. A challenge is to assess genome-wide experimental data and integrate the results to better understand the underlying biological processes. We propose statistical and computational tools that can be used to find relationships between synthetic lethality and cellular organizational units. Results In Saccharomyces cerevisiae, we identified multi-protein complexes and pairs of multi-protein complexes that share an unusually high number of synthetic genetic interactions. As previously predicted, we found that synthetic lethality can arise from subunits of an essential multi-protein complex or between pairs of multi-protein complexes. Finally, using multi-protein complexes allowed us to take into account the pleiotropic nature of the gene products. Conclusions Modeling synthetic lethality using current estimates of the yeast interactome is an efficient approach to disentangle some of the complex molecular interactions that drive a cell. Our model in conjunction with applied statistical methods and computational methods provides new tools to better characterize synthetic genetic interactions.

Le Meur, Nolwenn; Gentleman, Robert

2008-01-01

116

Acetyl-CoA Carboxylase 2?/? Mutant Mice are Protected against Fatty Liver under High-fat, High-carbohydrate Dietary and de Novo Lipogenic Conditions*  

PubMed Central

Hepatic fat accumulation resulting from increased de novo fatty acid synthesis leads to hepatic steatosis and hepatic insulin resistance. We have shown previously that acetyl-CoA carboxylase 2 (Acc2?/?) mutant mice, when fed a high-fat (HF) or high-fat, high-carbohydrate (HFHC) diet, are protected against diet-induced obesity and maintained whole body and hepatic insulin sensitivity. To determine the effect of an ACC2 deletion on hepatic fat metabolism, we studied the regulation of the enzymes involved in the lipogenic pathway under Western HFHC dietary and de novo lipogenic conditions. After completing the HFHC regimen, Acc2?/? mutant mice were found to have lower body weight, smaller epididymal fat pads, lower blood levels of nonesterified fatty acids and triglycerides, and higher hepatic cholesterol than wild-type mice. Significant up-regulation of lipogenic enzymes and an elevation in hepatic peroxisome proliferator-activated receptor-? (PPAR-?) protein were found in Acc2?/? mutant mice under de novo lipogenic conditions. The increase in lipogenic enzyme levels was accompanied by up-regulation of the transcription factors, sterol regulatory element-binding proteins 1 and 2, and carbohydrate response element-binding protein. In contrast, hepatic levels of the PPAR-? and PPAR-? proteins were significantly lower in the Acc2?/? mutant mice fed an HFHC diet. When compared with wild-type mice fed the same diet, Acc2?/? mutant mice exhibited a similar level of AKT but with a significant increase in pAKT. Hence, deleting ACC2 ameliorates the metabolic syndrome and protects against fatty liver despite increased de novo lipogenesis and dietary conditions known to induce obesity and diabetes.

Abu-Elheiga, Lutfi; Wu, Hongmei; Gu, Ziwei; Bressler, Rubin; Wakil, Salih J.

2012-01-01

117

Perinatal-lethal Gaucher disease.  

PubMed

Gaucher disease is a lysosomal storage disease caused by glucocerebrosidase deficiency. Although purely visceral in most cases, some Gaucher disease patients have neurological signs. Signs of Gaucher disease appear after a symptom-free period, except in rare cases with fetal onset. The description of such cases was based mainly on single reports and siblings. We report here a series of perinatal-lethal Gaucher disease cases highlighting the specificity of this phenotype. We retrospectively studied eight original cases of proven Gaucher disease with fetal onset. Non-immune hydrops fetalis was present in all cases but one, and associated with hepatosplenomegaly, ichthyosis, arthrogryposis, and facial dysmorphy. The similarities between our cases and 33 previously described cases allow us to better delineate the perinatal-lethal Gaucher disease phenotype. Hydrops fetalis, in utero fetal death and neonatal distress are prominent features. When hydrops is absent, neurological involvement begins in the first week and leads to death within three months. Hepatosplenomegaly is a major sign, and associated with ichthyosis, arthrogryposis, and facial dysmorphy in some 35-43% of cases. Perinatal-lethal Gaucher disease is a specific entity defined by its particular course and signs that are absent in classical type 2 Gaucher disease. Our study provides clues to the diagnosis of this likely underdiagnosed condition, which must be biochemically confirmed in order to propose appropriate genetic counselling. PMID:12838552

Mignot, C; Gelot, A; Bessières, B; Daffos, F; Voyer, M; Menez, F; Fallet Bianco, C; Odent, S; Le Duff, D; Loget, P; Fargier, P; Costil, J; Josset, P; Roume, J; Vanier, M T; Maire, I; Billette de Villemeur, T

2003-07-30

118

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes.  

PubMed

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci. PMID:21390299

O'Rourke, Sean M; Carter, Clayton; Carter, Luke; Christensen, Sara N; Jones, Minh P; Nash, Bruce; Price, Meredith H; Turnbull, Douglas W; Garner, Aleena R; Hamill, Danielle R; Osterberg, Valerie R; Lyczak, Rebecca; Madison, Erin E; Nguyen, Michael H; Sandberg, Nathan A; Sedghi, Noushin; Willis, John H; Yochem, John; Johnson, Eric A; Bowerman, Bruce

2011-03-01

119

Euglena gracilis as source of the antioxidant vitamin E. Effects of culture conditions in the wild strain and in the natural mutant WZSL  

Microsoft Academic Search

The photosynthetic wild type and the spontaneous non-photosynthetic WZSL mutant of the unicellular flagellate Euglena gracilis\\u000a were grown to investigate the influence of photoheterotrophic and heterotrophic conditions on ?-tocopherol (vitamin E) content.\\u000a HPLC analysis demonstrated a marked enhancement (almost 100%) of tocopherol content in the light in both strains, independent\\u000a of the presence of chloroplasts. These findings indicate that the

C. Kusmic; R. Barsacchi; L. Barsanti; P. Gualtieri; V. Passarelli

1998-01-01

120

Molecular Foundations of Reproductive Lethality in Arabidopsis thaliana  

PubMed Central

The SeedGenes database (www.seedgenes.org) contains information on more than 400 genes required for embryo development in Arabidopsis. Many of these EMBRYO-DEFECTIVE (EMB) genes encode proteins with an essential function required throughout the life cycle. This raises a fundamental question. Why does elimination of an essential gene in Arabidopsis often result in embryo lethality rather than gametophyte lethality? In other words, how do mutant (emb) gametophytes survive and participate in fertilization when an essential cellular function is disrupted? Furthermore, why do some mutant embryos proceed further in development than others? To address these questions, we first established a curated dataset of genes required for gametophyte development in Arabidopsis based on information extracted from the literature. This provided a basis for comparison with EMB genes obtained from the SeedGenes dataset. We also identified genes that exhibited both embryo and gametophyte defects when disrupted by a loss-of-function mutation. We then evaluated the relationship between mutant phenotype, gene redundancy, mutant allele strength, gene expression pattern, protein function, and intracellular protein localization to determine what factors influence the phenotypes of lethal mutants in Arabidopsis. After removing cases where continued development potentially resulted from gene redundancy or residual function of a weak mutant allele, we identified numerous examples of viable mutant (emb) gametophytes that required further explanation. We propose that the presence of gene products derived from transcription in diploid (heterozygous) sporocytes often enables mutant gametophytes to survive the loss of an essential gene in Arabidopsis. Whether gene disruption results in embryo or gametophyte lethality therefore depends in part on the ability of residual, parental gene products to support gametophyte development. We also highlight here 70 preglobular embryo mutants with a zygotic pattern of inheritance, which provide valuable insights into the maternal-to-zygotic transition in Arabidopsis and the timing of paternal gene activation during embryo development.

Muralla, Rosanna; Lloyd, Johnny; Meinke, David

2011-01-01

121

Metabolic flux analysis of Escherichia coli creB and arcA mutants reveals shared control of carbon catabolism under microaerobic growth conditions.  

PubMed

Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of (13)C-labeling experiments in chemostat cultures of a wild-type strain, DeltacreB and DeltaarcA single mutants, and a DeltacreB DeltaarcA double mutant. Continuous cultures were conducted at D = 0.1 h(-1) under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its DeltaarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the DeltaarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains. PMID:19561129

Nikel, Pablo I; Zhu, Jiangfeng; San, Ka-Yiu; Méndez, Beatriz S; Bennett, George N

2009-06-26

122

Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions?  

PubMed Central

Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of 13C-labeling experiments in chemostat cultures of a wild-type strain, ?creB and ?arcA single mutants, and a ?creB ?arcA double mutant. Continuous cultures were conducted at D = 0.1 h?1 under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its ?arcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ?arcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.

Nikel, Pablo I.; Zhu, Jiangfeng; San, Ka-Yiu; Mendez, Beatriz S.; Bennett, George N.

2009-01-01

123

Physiological and biochemical responses of fruit exocarp of tomato (Lycopersicon esculentum Mill.) mutants to natural photo-oxidative conditions  

Microsoft Academic Search

Photo-oxidative stress was imposed under natural solar radiation on exposed and shaded sections of detached fruit of immature green tomato (Lycopersicon esculentum Miller5Solanum lycopersicum L.) mutants (anthocyanin absent, b-carotene, Delta, and high pigment-1) and their nearly isogenic parents ('Ailsa Craig' and 'Rutgers'). After 5 h exposure to high solar irradiance, either with or without ultraviolet (UV) radi- ation, surface colour

Carolina A. Torres; Preston K. Andrews; Neal M. Davies

2006-01-01

124

Lethality of suicide methods  

Microsoft Academic Search

Objectives: To (a) quantify the lethality of suicide methods used in Australia in the period 1 July 1993 to 30 June 2003, (b) examine method-specific case fatality by age and sex, and (c) identify changes in case fatality during the study period. Methods: Two sources of data on episodes of self-harm in Australia were used, mortality and hospital separation data.

A A Elnour; J Harrison

2008-01-01

125

Non?Lethal Weapons  

Microsoft Academic Search

This article gives an overview of non?lethal weapons (NLWs) programmes, the technologies involved, and their present and future deployment. It also looks at the risks and dangers involved with their use, and comments on possible chemical and biological warfare treaty violations. Increasing interest is being paid by military and related research communities to NLWs, and pressure is being put on

Nick Lewer

1995-01-01

126

Heat Shock Transcriptional Responses in an MC-Producing Cyanobacterium (Planktothrix agardhii) and Its MC-Deficient Mutant under High Light Conditions  

PubMed Central

Microcystins (MCs) are the most commonly-reported hepatotoxins produced by various cyanobacterial taxa in fresh waters to constitute a potential threat to human and animal health. The biological role of MCs in the producer organisms is not known, and it would be very useful to understand the driving force behind the toxin production. Recent studies have suggested that MCs may have a protective function in cells facing environmental stress. Following this starting premise, we speculate that under adverse conditions the expression of stress-related genes coding for Heat Shock Proteins (Hsp) might be different in an MC-producing strain and its MC-deficient mutant. We therefore used RT-qPCR to compare the expression of 13 hsp genes of an MC-producing strain of Planktothrix agardhii (CYA126/8) and its MC-deficient ?mcyD mutant over different periods of exposure to high light stress (HL). Three reference genes (RGs) were selected from six candidates to normalize the RT-qPCR data. Of these three RGs (rsh, rpoD, and gltA), gltA is used here for the first time as an RG in prokaryotes. Under HL stress, five genes were found to be strongly up-regulated in both strains (htpG, dnaK, hspA, groES, and groEL). Unexpectedly, we found that the MC-producing wild type strain accumulated higher levels of htpG and dnaK transcripts in response to HL stress than the MC-deficient mutant. In addition, a significant increase in the mcyE transcript was detected in the mutant, suggesting that MCs are required under HL conditions. We discuss several possible roles of MCs in the response to HL stress through their possible involvement in the protective mechanisms of the cells.

Tran, Thi Du Chi; Bernard, Cecile; Ammar, Myriam; Chaouch, Soraya; Comte, Katia

2013-01-01

127

Conditional intercellular cohesion in a Dictyostelium discoideum mutant which is temperature sensitive for correct processing of asparagine-linked oligosaccharides.  

PubMed

Mutants of Dictyostelium discoideum have been isolated by a selection for cells with temperature-sensitive defects in the maturation of glycoprotein N-linked oligosaccharides. Here we describe a mutant, HT7, which is unable to aggregate at the restrictive temperature, but which aggregates and makes fruiting bodies at the permissive temperature. HT7 shows normal early developmental intercellular cohesion, but is temperature sensitive for expression of the ethylenediamine-tetraacetic acid (EDTA)-resistant cohesion characteristic of aggregation. The mutant initiates aggregation, but forms only loose cell mounds which later disperse. Metabolic labelling studies indicate that the thermolabile defect is not in protein synthesis, assembly of the lipid-linked precursor of N-linked oligosaccharides or transfer of the precursor to proteins. However, the defect does prevent assembly of fully processed N-linked oligosaccharides. Further, two glycopeptides, obtained from exhaustive Pronase digests of wild-type plasma membrane glycoproteins, inhibit intercellular cohesion of aggregation-stage wild-type cells. HT7 produces only approximately 50% of the wild-type level of these glycopeptides at the restrictive temperature and one of the glycopeptides has reduced cohesion inhibition ability. A revertant of HT7 was found to aggregate normally, to have restored EDTA-resistant cohesion, to have normal profiles of N-linked oligosaccharides and to express the two cohesion-inhibiting glycopeptides normally. These data strongly support a model in which cohesion during late aggregation is at least in part due to recognition between surface glycans and receptors on neighbouring cells. PMID:1665373

Boose, J A; Henderson, E J

1991-06-01

128

Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy.  

PubMed

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) ? Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) ? Leu and Thr(8) ? Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) ? Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy. PMID:22427649

Ceholski, Delaine K; Trieber, Catharine A; Young, Howard S

2012-03-16

129

A proteomic approach to analyzing responses of Arabidopsis thaliana root cells to different gravitational conditions using an agravitropic mutant, pin2 and its wild type  

PubMed Central

Background Root gravitropsim has been proposed to require the coordinated, redistribution of the plant signaling molecule auxin within the root meristem, but the underlying molecular mechanisms are still unknown. PIN proteins are membrane transporters that mediate the efflux of auxin from cells. The PIN2 is important for the basipetal transport of auxin in roots and plays a critical role in the transmission of gravity signals perceived in the root cap to the root elongation zone. The loss of function pin2 mutant exhibits a gravity-insensitive root growth phenotype. By comparing the proteomes of wild type and the pin2 mutant root tips under different gravitational conditions, we hope to identify proteins involved in the gravity-related signal transduction. Results To identify novel proteins involved in the gravity signal transduction pathway we have carried out a comparative proteomic analysis of Arabidopsis pin2 mutant and wild type (WT) roots subjected to different gravitational conditions. These conditions included horizontal (H) and vertical (V) clinorotation, hypergravity (G) and the stationary control (S). Analysis of silver-stained two-dimensional SDS-PAGE gels revealed 28 protein spots that showed significant expression changes in altered gravity (H or G) compared to control roots (V and S). Whereas the majority of these proteins exhibited similar expression patterns in WT and pin2 roots, a significant number displayed different patterns of response between WT and pin2 roots. The latter group included 11 protein spots in the H samples and two protein spots in the G samples that exhibited an altered expression exclusively in WT but not in pin2 roots. One of these proteins was identified as annexin2, which was induced in the root cap columella cells under altered gravitational conditions. Conclusions The most interesting observation in this study is that distinctly different patterns of protein expression were found in WT and pin2 mutant roots subjected to altered gravity conditions. The data also demonstrate that PIN2 mutation not only affects the basipetal transport of auxin to the elongation zone, but also results in an altered expression of proteins in the root columella.

2011-01-01

130

Lethal and mutagenic effects of elevated temperature on haploid yeast  

Microsoft Academic Search

The lethal and cytoplasmic mutagenic effects of 52°C incubation during the cell cycle of a haploid strain of Saccharomyces cerevisiae were examined. Both effects varied periodically in a rather parallel pattern: the maximum thermosensitivity was seen at budding time, corresponding to the S period (Williamson, 1965). The 52°C induction of a nuclear forward mutation was also examined: canavanine-resistant mutants were

Ana Schenberg-Frascino; Ethel Moustacchi

1972-01-01

131

The effects of body weight, temperature, salinity, pH, light intensity and feeding condition on lethal DO levels of whiteleg shrimp, Litopenaeus vannamei (Boone, 1931)  

Microsoft Academic Search

Tolerance of whiteleg shrimp Litopenaeus vannamei exposed to different temperatures (14.5, 21.5, 24.8, 27.8, 30.8, and 35.0 °C), salinities (9, 15, 26, 35, and 40‰), pH (3.3, 6.5, 7.7, 8.1, and 9.2), and light intensities (strong 2100 lx and weak 60 lx) at various body weights (3.0, 3.7, 4.3, 5.7, 7.8, 9.0, 9.5, 10.7, 11.9, and 13.3 g) and feeding conditions (fed

Peidong Zhang; Xiumei Zhang; Jian Li; Guoqiang Huang

2006-01-01

132

P-lacW Insertional Mutagenesis on the Second Chromosome of Drosophila melanogaster: Isolation of Lethals With Different Overgrowth Phenotypes  

Microsoft Academic Search

A single P-element insertional mutagenesis experiment was carried out for the second chromosome of Drosophila melanogaster using the P-lacW transposon. Out of 15,475 insertions on the second chromosome, 2,308 lethal and 403 semilethal mutants (altogether 2,711) were recovered. After eliminating clusters, 72% of the mutants represent independent insertions. Some of the mutants with larval, prepupal or pupal lethal phases have

Tibor Torok; Gabriella Tick; Martha Alvarado; Istvan Kiss

1993-01-01

133

Strategy for enhanced transgenic strain development for embryonic conditionnal lethality in Anastrepha suspensa  

Technology Transfer Automated Retrieval System (TEKTRAN)

Here the first reproductive sterility system for the tephritid pest, Anastrepha suspensa, is presented, based on lethality primarily in embryos heterozygous for a lethal conditional transgene combination. The tetracycline-suppressible system uses the cellularization-specific A. suspensa serendipity...

134

Electroshock weapons can be lethal!  

NASA Astrophysics Data System (ADS)

Electroshock weapons (EWs)-stun guns, tasers, riot shields-are electroconductive devices designed to safely incapacitate healthy men neuromuscularly, so they are called nonlethal or less-lethal. EW firms seeking large nonmilitary markets targeted law enforcement and corrections personnel, who began using EWs in prisons/jails and on public patrol in 1980 in the USA. This shifted the EW-shocked population from healthy soldiers to a heterogeneous mix of both sexes, ages 6-92, in a wide variety of health conditions! An EW operates by disrupting normal physiological processes, producing transient effects in healthy people. But if a person's health is sufficiently compromised, the margin of safety can be lost, resulting in death or permanent health problems. 325 people have died after EW shock since 1980. Did the EW cause these deaths? Evidence indicates that EWs do play a causal role in most such deaths. EWs can be lethal for people in diabetic shock^1 (hypoglycemia), which may be why Robert Dziekanski-a Polish immigrant to Canada-died so quickly after he was tasered at Vancouver Airport: not having eaten for over 10 hours, he likely was severely hypoglycemic. The EW death rate in North America is 30 times higher than need be, because EW users have not been properly trained to use EWs on a heterogeneous population safely! ^1J. Clinical Engineering 30(3):111(2005).

Lundquist, Marjorie

2008-03-01

135

Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8?-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition  

PubMed Central

Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8?-hydroxyase gene which was highly expressed during seed development (TaABA8?OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8?OH1 on the D genome (TaABA8?OH1-D) was identified in Japanese cultivars including ‘Tamaizumi’. However, a single mutation in TaABA8?OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8?OH1 on the A genome (TaABA8?OH1-A), TM1833, was identified from gamma-ray irradiation lines of ‘Tamaizumi’. TM1833 (a double mutant in TaABA8?OH1-A and TaABA8?OH1-D) showed lower TaABA8?OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in ‘Tamaizumi’ (a single mutant in TaABA8?OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8?OH1 may be effective in germination inhibition in field-grown wheat.

Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

2013-01-01

136

Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8'-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition.  

PubMed

Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8'-hydroxyase gene which was highly expressed during seed development (TaABA8'OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8'OH1 on the D genome (TaABA8'OH1-D) was identified in Japanese cultivars including 'Tamaizumi'. However, a single mutation in TaABA8'OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8'OH1 on the A genome (TaABA8'OH1-A), TM1833, was identified from gamma-ray irradiation lines of 'Tamaizumi'. TM1833 (a double mutant in TaABA8'OH1-A and TaABA8'OH1-D) showed lower TaABA8'OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in 'Tamaizumi' (a single mutant in TaABA8'OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8'OH1 may be effective in germination inhibition in field-grown wheat. PMID:23641187

Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

2013-03-01

137

Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: evaluation of reversible unfolding conditions.  

PubMed

Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles. To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)]. sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles. PMID:10563824

Chen, G Q; Gouaux, E

1999-11-16

138

Perinatal lethal Gaucher disease.  

PubMed

Perinatal Lethal Gaucher Disease (PLGD) is a rare form of Gaucher disease and is often considered a distinct form of type 2 Gaucher disease. The authors report on an infant who presented with progressive hepatosplenomegaly, ichthyosis, generalized skin edema and neonatal encephalopathy and died at 6 h of age. Autopsy revealed massive hepatosplenomegaly, ichthyosis, a diffuse collodion picture and histological evidence of infiltration by Gaucher cells in the liver, spleen, lung, thymus, lymph node and bone marrow. Genetic testing of the parents revealed both to be carriers of Gaucher disease. PMID:20924719

Plakkal, Nishad; Soraisham, Amuchou Singh; Jirapradittha, Junya; Pinto-Rojas, Alfredo

2010-10-06

139

Tasers - Less than Lethal!  

PubMed Central

We report a case of potentially lethal injury associated with the use of Taser. A 42-year-old man was stopped by police for potential detention. He held a large carving knife over his epigasrium threatening to stab himself.With a view to achieving immobilisation, a Taser gun was used. On activation of the Taser, the subject suffered a 7-cm wide and 10-cm deep stab injury to the upper abdomen. In this case, activation of the Taser resulted in the contraction of skeletal muscles, flexors more intensely than extensors, resulting in the stab injury.

Sharma, Abiram; Theivacumar, Nada S; Souka, Hesham M

2009-01-01

140

Stem cell expansion during carcinogenesis in stem cell-depleted conditional telomeric repeat factor 2 null mutant mice.  

PubMed

To examine the role of telomeric repeat-binding factor 2 (TRF2) in epithelial tumorigenesis, we characterized conditional loss of TRF2 expression in the basal layer of mouse epidermis. These mice exhibit some characteristics of dyskeratosis congenita, a human stem cell depletion syndrome caused by telomere dysfunction. The epidermis in conditional TRF2 null mice exhibited DNA damage response and apoptosis, which correlated with stem cell depletion. The stem cell population in conditional TRF2 null epidermis exhibited shorter telomeres than those in control mice. Squamous cell carcinomas induced in conditional TRF2 null mice developed with increased latency and slower growth due to reduced numbers of proliferating cells as the result of increased apoptosis. TRF2 null epidermal stem cells were found in both primary and metastatic tumors. Despite the low-grade phenotype of the conditional TRF2 null primary tumors, the number of metastatic lesions was similar to control cancers. Basal cells from TRF2 null tumors demonstrated extreme telomere shortening and dramatically increased numbers of telomeric signals by fluorescence in situ hybridization due to increased genomic instability and aneuploidy in these cancers. DNA damage response signals were detected at telomeres in TRF2 null tumor cells from these mice. The increased genomic instability in these tumors correlated with eightfold expansion of the transformed stem cell population compared with that in control cancers. We concluded that genomic instability resulting from loss of TRF2 expression provides biological advantages to the cancer stem cell population. PMID:23178498

Bojovic, B; Ho, H-Y; Wu, J; Crowe, D L

2012-11-26

141

Conditional loss of ErbB3 delays mammary gland hyperplasia induced by mutant PIK3CA without affecting mammary tumor latency, gene expression, or signaling.  

PubMed

Mutations in PIK3CA, the gene encoding the p110? catalytic subunit of phosphoinositide 3-kinase (PI3K), have been shown to transform mammary epithelial cells (MEC). Studies suggest this transforming activity requires binding of mutant p110? via p85 to phosphorylated YXXM motifs in activated receptor tyrosine kinases (RTK) or adaptors. Using transgenic mice, we examined if ErbB3, a potent activator of PI3K, is required for mutant PIK3CA-mediated transformation of MECs. Conditional loss of ErbB3 in mammary epithelium resulted in a delay of PIK3CA(H1047R)-dependent mammary gland hyperplasia, but tumor latency, gene expression, and PI3K signaling were unaffected. In ErbB3-deficient tumors, mutant PI3K remained associated with several tyrosyl phosphoproteins, potentially explaining the dispensability of ErbB3 for tumorigenicity and PI3K activity. Similarly, inhibition of ErbB RTKs with lapatinib did not affect PI3K signaling in PIK3CA(H1047R)-expressing tumors. However, the p110?-specific inhibitor BYL719 in combination with lapatinib impaired mammary tumor growth and PI3K signaling more potently than BYL719 alone. Furthermore, coinhibition of p110? and ErbB3 potently suppressed proliferation and PI3K signaling in human breast cancer cells harboring PIK3CA(H1047R). These data suggest that PIK3CA(H1047R)-driven tumor growth and PI3K signaling can occur independently of ErbB RTKs. However, simultaneous blockade of p110? and ErbB RTKs results in superior inhibition of PI3K and mammary tumor growth, suggesting a rational therapeutic combination against breast cancers harboring PIK3CA activating mutations. PMID:23633485

Young, Christian D; Pfefferle, Adam D; Owens, Philip; Kuba, María G; Rexer, Brent N; Balko, Justin M; Sánchez, Violeta; Cheng, Hailing; Perou, Charles M; Zhao, Jean J; Cook, Rebecca S; Arteaga, Carlos L

2013-04-30

142

Lethal intrauterine fetal trauma.  

PubMed

Eight cases of lethal intrauterine fetal trauma secondary to motor vehicle accidents are retrospectively studied. In each instance the mother survived, usually sustaining only minor injuries. Some degree of placental abruption or infarction occurred in each case, but fetal abnormalities were more varied. Significant fetal injuries were limited to the head and included two instances of skull fracture associated with cortical lacerations and contusions. Six of the eight fetuses were stillborn, and the other two died during the first postnatal day. At least five of the mothers were unrestrained at the time of the accident, three of whom experienced abdominal impact against the steering wheel but no external abdominal injuries. Although two mothers were wearing seat belts, in only one instance could the seat belt be implicated in contributing to the fetal injury. This study shows that lethal placental or direct fetal injury can occur even though maternal injuries are minor or insignificant. The findings also support current recommendations for use of three-point restraints. PMID:3407709

Stafford, P A; Biddinger, P W; Zumwalt, R E

1988-08-01

143

Repair of potentially lethal damage following irradiation with x rays or cyclotron neutrons: response of the EMT-6/UW tumor system treated under various growth conditions in vitro and in vivo  

SciTech Connect

Postirradiation potentially lethal damage (PLD) repair was examined in the EMT-6/UW tumor system under a variety of in vitro and in vivo growth conditions. Following x irradiation, surviving fraction increased in fed and unfed plateau cultures if subculture and plating were delayed; in exponentially growing cultures if they were covered with depleted medium for the first 6 h postirradiation; and in tumors in vivo if excision for preparation of a cell suspension was delayed. Following irradiation with 21.5 meV (d/sup +/ ..-->.. Be) neutrons, PLD repair was measurable only in unfed plateau cultures when subculture was delayed and in exponentially growing cells exposed to depleted culture medium immediately after irradiation. In x-irradiated EMT-6/UW cells, the greatest repair capacity and the highest surviving fraction ratios were measured in unfed plateau cultures; the least repair was observed in exponentially growing cells exposed to depleted medium. Thus post-neutron repair was not limited to situations where the amount of repair of photon PLD is large. The demonstration of PLD repair in tumors irradiated in vivo with X rays and the absence of such repair after neutrons could have important implications in radiotherapy if this is a general phenomenon.

Rasey, J.S.; Nelson, N.J.

1981-01-01

144

Alterations in peptidoglycan chemical composition associated with rod-to-sphere transition in a conditional mutant of Klebsiella pneumoniae.  

PubMed Central

Klebsiella pneumoniae Mir M7 is a spontaneous parentless morphology mutant which grows as cocci at pH 7 and as rods at pH 5.8. This strain has been characterized as defective in lateral wall formation (at pH7). Data suggest that the cell wall is mainly made up of poles of the rods (G. Satta, R. Fontana, P. Canepari, and G. Botta, J. Bacteriol. 137:727--734, 1979). In this work the isolation and the biochemical properties of the peptidoglycan of both Mir M7 rods and cocci and a nonconditional rod-shaped Mir M7 revertant (strain Mir A12) are described. The peptidoglycan of Mir M7 (both rods and cocci) and Mir A12 strains carried covalently bound proteins which could be easily removed by pronase treatment in Mir M7 rods and Mir A12 cells, but not in Mir M7 round cells. However, when the sodium dodecyl sulfate-insoluble residues of Mir M7 cocci were pretreated with ethylenediaminetetraacetic acid (EDTA), pronase digestion removed the covalently bound proteins, and pure peptidoglycan was obtained. EDTA treatment of the rigid layer of Mir M7 cocci removed amounts of Mg2+ and Ca2+, which were 10- and 50-fold higher, respectively, than the amount liberated from the rigid layer of Mir M7 rods and Mir A12 cells. Amino acid composition was qualitatively similar in both strains, but Mir M7 cocci contained a higher amount of alanine and glucosamine. Mir M7 cocci contained approximately 50% less peptidoglycan than rods. Under electron microscopy, the rigid layer of the Mir M7 rods and Mir A12 cells appeared to be rod-shaped and their shape remained unchanged after EDTA and pronase treatment. On the contrary, the Mir M7 cocci rigid layer appeared to be round, and after EDTA treatment it collapsed and lost any definite morphology. In spite of these alterations, the peptidoglycan of Mir M7 cocci still appeared able to determine the shape of the cell and protect it from osmotic shock and mechanical damages. The accumluation of divalent cations appeared necessary for the peptidoglycan to acquire sufficient rigidity for shape determination and cell protection. We concluded that the coccal shape in Mir M7 cells is not due to loss of cell wall rigidity but is a consequence of the formation of a round peptidoglycan molecule. The possibility that the alterations found in the Mir M7 cocci rigid layer may reflect natural differences in the biochemical composition of the septa and lateral wall of normally shaped bacteria is discussed. Images

Fontana, R; Canepari, P; Satta, G

1979-01-01

145

Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1  

Microsoft Academic Search

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA process- ing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to tem- perature. Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with

L. F. Revers; J. M. Cardone; D. Bonatto; J. Saffi; M. Grey; H. Feldmann; M. Brendel; J. A. P. Henriques

2002-01-01

146

Serum Amyloid A Protects Murine Macrophages from Lethal Toxin-Mediated Death1  

PubMed Central

Lethal toxin, a key virulence factor produced by Bacillus anthracis, induces cell death, in part by disrupting numerous signaling pathways, in mouse macrophages. However, exposure to sublethal doses of lethal toxin allows some cells to survive. Because these pro-survival signaling events occur within a few hours after exposure to sublethal doses, we hypothesized that acute phase proteins might influence macrophage survival. Our data show that serum amyloid A (SAA) is produced in response to lethal toxin treatment. Moreover, pre-treatment of macrophages with exogenous SAA protected macrophages from lethal toxin-mediated death. Exogenous SAA activated the p38 mitogen activated protein kinase (MAP) kinase pathway, while lethal toxin mutants incapable of p38 activation were incapable of causing cell death. Chemical inhibition of the p38 activation pathway abrogated the protective effects of SAA. These data show that SAA affords protection against lethal toxin in mouse macrophages and link this response to the p38 pathway.

Rose, Kira; Long, Paul; Shankar, Malini; Ballard, Jimmy D.; Webb, Carol F.

2011-01-01

147

Extinction conditions for Y-linked mutant-alleles through two-sex branching processes with blind-mating structure.  

PubMed

A new two-sex bidimensional branching process is introduced to model the evolution of the number of carriers of an allele and its mutations of a Y-linked gene. A population is assumed in which females and males coexist and mate without the gene influencing the mating process. It is deduced from the model that the key determining conditions for the extinction or survival of the allele are given by the probability that an offspring is female, the rate of mutation, and the mean number of offspring per mating unit. It is also proved that the destiny of the allele's mutations in the population also depends on the survival or extinction of the original allele. PMID:22588021

González, M; Gutiérrez, C; Martínez, R

2012-05-12

148

Less lethal technology: medical issues  

Microsoft Academic Search

Purpose – Less lethal weapons have become a critical tool for law enforcement when confronting dangerous, combative individuals in the field. The purpose of this paper is to review the medical aspects and implications of three different types of less lethal weapons. Design\\/methodology\\/approach – The paper conducted a comprehensive medical literature review on blunt projectiles, irritant sprays including oleoresin capsicum

Gary M. Vilke; Theodore C. Chan

2007-01-01

149

Conditional cannabinoid receptor type 1 mutants reveal neuron subpopulation-specific effects on behavioral and neuroendocrine stress responses.  

PubMed

The complete genetic loss or pharmacological blockade of cannabinoid receptor type 1 (CB1) in mice results in both altered behavioral performance and increased stress hormone secretion in response to stressful encounters such as forced swim test (FST) exposure. CB1 is expressed on nerve terminals belonging to different neurotransmitter systems, including the glutamatergic and GABAergic system, where it is able to suppress excitatory and inhibitory neurotransmission, respectively. In the current study, we used the conditional mutagenesis approach in mice to investigate the neurotransmitter systems involved in these behavioral and neuroendocrine phenotypes in regard to CB1 signaling. Mice lacking CB1 in cortical glutamatergic neurons (Glu-CB1(-/-)) showed decreased passive stress coping (decreased immobility) in the FST, whereas mice lacking CB1 in principal forebrain neurons (CaMK-CB1(-/-)) and GABAergic neurons (GABA-CB1(-/-)), respectively, behaved as littermate controls. However, we found increased FST-induced corticosterone secretion only in CaMK-CB1(-/-) mice, whereas Glu-CB1(-/-) and GABA-CB1(-/-) mice exhibited normal corticosterone release as compared to controls. Thus, behavioral and neuroendocrine acute stress coping in response to the FST is mainly influenced by CB1 signaling on different glutamatergic neuronal subpopulations, but not by CB1 on GABAergic neurons. PMID:18653287

Steiner, Michel A; Marsicano, Giovanni; Wotjak, Carsten T; Lutz, Beat

2008-07-23

150

Insights into the role of bcl6 in follicular th cells using a new conditional mutant mouse model.  

PubMed

The transcriptional repressor Bcl6 controls development of the follicular Th cell (TFH) lineage, but the precise mechanisms by which Bcl6 regulates this process are unclear. A model has been proposed whereby Bcl6 represses the differentiation of T cells into alternative effector lineages, thus favoring TFH cell differentiation. Analysis of T cell differentiation using Bcl6-deficient mice has been complicated by the strong proinflammatory phenotype of Bcl6-deficient myeloid cells. In this study, we report data from a novel mouse model where Bcl6 is conditionally deleted in T cells (Bcl6(fl/fl)Cre(CD4) mice). After immunization, programmed death -1 (PD-1)(high) TFH cells in Bcl6(fl/fl)Cre(CD4) mice are decreased >90% compared with control mice, and Ag-specific IgG is sharply reduced. Residual PD-1(high)CXCR5(+) TFH cells in Bcl6(fl/fl)Cre(CD4) mice show a significantly higher rate of apoptosis than do PD-1(high)CXCR5(+) TFH cells in control mice. Immunization of Bcl6(fl/fl)Cre(CD4) mice did not reveal enhanced differentiation into Th1, Th2, or Th17 lineages, although IL-10 expression by CD4 T cells was markedly elevated. Thus, T cell-extrinsic factors appear to promote the increased Th1, Th2, and Th17 responses in germline Bcl6-deficient mice. Furthermore, IL-10 may be a key target gene for Bcl6 in CD4 T cells, which enables Bcl6 to promote the TFH cell phenotype. Finally, our data reveal a novel mechanism for the role of Bcl6 in promoting TFH cell survival. PMID:23980208

Hollister, Kristin; Kusam, Saritha; Wu, Hao; Clegg, Ninah; Mondal, Arpita; Sawant, Deepali V; Dent, Alexander L

2013-08-26

151

Investigations on the organization of genetic loci in Drosophila melanogaster : lethal mutations affecting 6-phosphogluconate dehydrogenase and their suppression  

Microsoft Academic Search

The molecular nature of lethal and semilethal mutations in the Pgd locus of D. melanogaster coding for 6-phosphogluconate dehydrogenase (6PGD) was studied. All the 11 mutations affect the structural gene of the Pgd locus: 3 semilethal mutations resulted in altered 6PGD molecules with decreased catalytic activities; the rest 8 lethals were “null” alleles characterized by mutant polypeptides capable of reacting

V. A. Gvozdev; T. I. Gerasimova; G. L. Kogan; J. M. Rosovsky

1977-01-01

152

Lethal mitochondrial genotypes in Podospora anserina : A model for senescence  

Microsoft Academic Search

Crosses between spg1 and spg2, two mitochondrial mutants of Podospora anserina, yield a new type of strain, called pseudo wild-type (PSW), in addition to wild-type recombinants. PSW strains are characterized by a variable phenotype for germination of ascospores and a variable longevity. By autofecondation, PSW strains yield early lethal strains (which die soon after the germination of the spores and

Léon Belcour; Odile Begel

1978-01-01

153

Mutant Cockroaches  

NSDL National Science Digital Library

Images of mutant cockroaches, provided by the Genetic Stock Center for the German Cockroach at Virginia Tech. Useful as a visual aid for explaining the effects of mutation. A linkage map is also available.

0002-11-30

154

Meningitis Mutants  

MedlinePLUS

... Favorites Delicious Digg Facebook Google Bookmarks Twitter Case file: Meningitis Mutants Real name: viral meningitis and Bacterial ... down the bacterial trio. Immune Platoon Disease Database File Formats Help: How do I view different file ...

155

Anthrax lethal toxin induces endothelial barrier dysfunction.  

PubMed

Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both of which are involved in barrier function regulation. Endothelial monolayers cultured on porous membrane inserts were treated with the LT components lethal factor (LF) and protective antigen (PA) individually, or in combination. LT induced a concentration- and time-dependent decrease in transendothelial electrical resistance that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution as revealed by immunofluorescence microscopy and cell surface enzyme-linked immunosorbent assay. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection. PMID:15920171

Warfel, Jason M; Steele, Amber D; D'Agnillo, Felice

2005-06-01

156

Deletion of integrin-linked kinase from neural crest cells in mice results in aortic aneurysms and embryonic lethality.  

PubMed

Neural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Integrin-linked kinase (ILK) is a serine/threonine kinase and a major regulator of integrin signaling. It links integrins to the actin cytoskeleton and recruits other adaptor molecules into a large complex to regulate actin dynamics and integrin function. Using the Cre-lox system, we deleted Ilk from NCCs of mice to investigate its role in NCC morphogenesis. The resulting mutants developed a severe aneurysmal arterial trunk that resulted in embryonic lethality during late gestation. Ilk mutants showed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and the outflow tract. Within the conotruncal cushions, Ilk-deficient NCCs exhibited disorganization of F-actin stress fibers and a significantly rounder morphology, with shorter cellular projections. Additionally, absence of ILK resulted in reduced in vivo phosphorylation of Smad3 in NCCs, which correlated with reduced ?SMA levels. Our findings resemble those seen in Pinch1 and ?1 integrin conditional mutant mice, and therefore support that, in neural crest-derived cells, ILK and Pinch1 act as cytoplasmic effectors of ?1 integrin in a pathway that protects against aneurysms. In addition, our conditional Ilk mutant mice might prove useful as a model to study aortic aneurysms caused by reduced Smad3 signaling, as occurs in the newly described aneurysms-osteoarthritis syndrome, for example. PMID:23744273

Arnold, Thomas D; Zang, Keling; Vallejo-Illarramendi, Ainara

2013-06-05

157

A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.  

PubMed

Increased nuclear accumulation of ?-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/?-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the ?-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the ?-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the ?-catenin destruction complex, followed by increased degradation of ?-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of ?-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/?-catenin signaling through inhibiting the PARP domain of TNKS1/2. PMID:22440753

Waaler, Jo; Machon, Ondrej; Tumova, Lucie; Dinh, Huyen; Korinek, Vladimir; Wilson, Steven Ray; Paulsen, Jan Erik; Pedersen, Nina Marie; Eide, Tor J; Machonova, Olga; Gradl, Dietmar; Voronkov, Andrey; von Kries, Jens Peter; Krauss, Stefan

2012-03-22

158

Naturally acquired Pneumocystis carinii pneumonia in gene disruption mutant mice: roles of distinct T-cell populations in infection.  

PubMed Central

When kept under strict specific-pathogen-free conditions, H-21-Abeta (Abeta(-/-),T-cell receptor beta (TCRbeta(-/-)), and recombinase-activating gene 1 (RAG-1(-/-) gene disruption mutant mice, deficient in conventional CD4+ T cells, TCRalphabeta cells, and all peripheral T and B lymphocytes, respectively, consistently developed lethal Pneumocystis carinii pneumonia through natural infection. The most severe symptoms appeared in RAG-1(-/-) mutants. In contrast, TCRdelta(-/-) and beta2-microglobulin(-/-)(beta2m-/-) mutants, deficient in TCRgammadelta cells and conventional CD8alphabeta+ TCRalphabeta cells, respectively, were fully resistant to infection. Our data indicate not only the insufficiency but also the dispensability of CD8 alphabeta+TCRalphabeta cells and of TCRgammadelta lymphocytes in resistance to P. carinii infection. Under disease conditions, large numbers of unusual single-positive CD4+ and CD8alphabeta+ as well as double-negative TCRgammadelta subpopulations of cells accumulated in lungs of TCRbeta(-/-) mutants. This accumulation was consistently accompanied by a drastic increase in the pulmonary B-cell population. In contrast, CD8alphabeta+ TCR alpha beta cells, but no B cells, appeared in lungs of parasitized Abeta (-/-) mutants. Since lung damage and parasite numbers were less prominent in morbid TCRbeta(-/-) and Abeta(-/-) mutants than in diseased RAG-1(-/-) mice, the remaining lymphocytes accumulating in lungs of the former two mutants seem to perform residual resistance functions.

Hanano, R; Reifenberg, K; Kaufmann, S H

1996-01-01

159

Members of a Novel Family of Mammalian Protein Kinases Complement the DNA-Negative Phenotype of a Vaccinia Virus ts Mutant Defective in the B1 Kinase  

Microsoft Academic Search

Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking

Kathleen A. Boyle; Paula Traktman

2004-01-01

160

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain  

Microsoft Academic Search

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are

Ka-Wah Chu; Shirley K. W. Chan; King L. Chow

2005-01-01

161

Potentially lethal complications of tracheostomy: autopsy considerations.  

PubMed

Tracheostomy is widely used to facilitate respiration by protecting the airways. It may be performed to relieve upper airway obstruction from congenital stenoses or from acquired conditions such as foreign body impaction, swelling from neck trauma or anaphylaxis, benign or malignant tumors, and infection. Tracheostomy may also be performed in individuals with respiratory impairment who require suctioning for accumulated mucoid secretions and in those with obstructive sleep apnea. Review of autopsy files and the literature was undertaken to demonstrate the range of lethal circumstances that may involve tracheostomy. Unexpected death may result from incorrect positioning of an endotracheal tube with failure of oxygenation, tracheal perforation with pneumothorax, mucus plugging, accidental extubation, and hemorrhage from tracheovascular fistulas. Lethal tracheovascular fistulas usually involve the innominate artery and result from mural perforation by the tip of a tracheostomy tube, mural necrosis from a high-pressure cuff, prolonged intubation, radiotherapy, and low tracheal incisions. Increased movement of tubes in patients with impaired consciousness and excessive head movements may also increase the chances of hemorrhage, as may infiltrating tumors. Given the wide range of potential fatal mechanisms that may be found in such cases, careful autopsy evaluation and dissection will be required to demonstrate the exact nature and site of the lethal lesion in individuals who underwent tracheostomy and die unexpectedly. PMID:21817868

Byard, Roger W; Gilbert, John D

2011-12-01

162

Lethal electric currents  

Microsoft Academic Search

Commercial-frequency currents of a few milliamperes flowing through the body will cause muscular contractions, resulting in the inability of the victim to release his grasp on a live conductor. Values of ``let-go'' current are very important criteria in the establishment of safe-current requirements. Since ventricular fibrillation, a condition in which circulation is arrested, is probably the most common cause of

Charles F. Dalziel; W. R. Lee

1969-01-01

163

Drosophila embryonic cell-cycle mutants.  

PubMed

Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified, because larval development occurs without a requirement for cell division. Only cells in the nervous system and the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus, most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell-cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of Cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null Cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system. PMID:23979936

Unhavaithaya, Yingdee; Park, Eugenia A; Royzman, Irena; Orr-Weaver, Terry L

2013-10-03

164

Drosophila Embryonic Cell-Cycle Mutants  

PubMed Central

Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified, because larval development occurs without a requirement for cell division. Only cells in the nervous system and the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus, most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell-cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of Cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null Cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system.

Unhavaithaya, Yingdee; Park, Eugenia A.; Royzman, Irena; Orr-Weaver, Terry L.

2013-01-01

165

Characterization of Saccharomyces cerevisiae dna2 Mutants Suggests a Role for the Helicase Late in S Phase  

PubMed Central

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like “lipid kinase” domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.

Fiorentino, David F.; Crabtree, Gerald R.

1997-01-01

166

Histidinol Dehydrogenase (hisD) Mutants of Salmonella typhimurium1  

PubMed Central

A multidisciplinary analysis has been applied to over 150 hisD mutants of Salmonella typhimurium in a study of gene-enzyme relationship. The mutants were examined for production of immunologically cross-reacting material by using antibody to purified histidinol dehydrogenase, and for genetic complementation by using a set of F? factors bearing Escherichia coli hisD complementing mutants. Classifications as to missense, nonsense, frameshift, or deletion mutant are proposed on the basis of mutagenesis and suppression tests. For the suppression tests the mutants were examined both by a simultaneous suppression technique and by testing for response to E. coli F? factors bearing a recessive lethal amber and a recessive lethal ochre suppressor. The data are interpreted in relation to the position of the mutations in the recombination and complementation maps and in relation to the known composition of histidinol dehydrogenase. The gene hisD appears to be single cistron for the production of a single biosynthetic polypeptide. Images

Greeb, J.; Atkins, J. F.; Loper, J. C.

1971-01-01

167

Residue Histidine 669 Is Essential for the Catalytic Activity of Bacillus anthracis Lethal Factor?  

PubMed Central

The lethal factor (LF) of Bacillus anthracis is a Zn2+-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.

Cao, Sha; Guo, Aizhen; Wu, Gaobing; Liu, Ziduo; Chen, Wei; Feng, Chunfang; Zhang, Cheng-Cai; Chen, Huanchun

2010-01-01

168

Expression of galactose permease and pyruvate carboxylase in Escherichia coli ptsG mutant increases the growth rate and succinate yield under anaerobic conditions.  

PubMed

In Escherichia coli, disruption of ptsG, which encodes the glucose-specific permease of the phosphotransferase transport system (PTS) protein EIICB(Glc), is crucial for high succinate production. This mutation can, however, cause very slow growth and low glucose consumption rates. The ptsG mutant (TUQ2), from wild type E. coli W1485, and E. coli galP (encoding galactose permease) and glk (encoding glucose kinase) gene expression plasmids were constructed. TUQ2 increased the generation time to approximately 4 h and gave a higher final cell density of 0.5 g/l by expression of galP. However, glk expression had no effect on the mutant. After expression of pyruvate carboxylase (PYC) and galactose permease, the ptsG mutant showed higher succinate yield (1.2 mol/mol glucose) and the specific rate of glucose consumption from 0.33 to 0.6 g/1 h. PMID:16369691

Wang, Qingzhao; Wu, Chanyuan; Chen, Tao; Chen, Xun; Zhao, Xueming

2006-01-01

169

Characterization of dominant lethal mutations in the yeast plasma membrane H +ATPase gene  

Microsoft Academic Search

Site-directed mutants of yeast ATPase were studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed and the expression of the wild-type chromosomal ATPase gene was turned off. One objection to this constitutive expression system was made apparent recently, as dominant lethal mutations are lost by gene conversion with the wild-type allele during the

Francisco Portillo

1997-01-01

170

Generating conditional knockout mice.  

PubMed

Gene targeting in ES cells is extensively used to generate designed mouse mutants and to study gene function in vivo. Knockout mice that harbor a null allele in their germline provide appropriate genetic models of inherited diseases and often exhibit embryonic or early postnatal lethality. To study gene function in adult mice and in selected cell types, a refined strategy for conditional gene inactivation has been developed that relies on the DNA recombinase Cre and its recognition (loxP) sites. For conditional mutagenesis, a target gene is modified by the insertion of two loxP sites that enable to excise the flanked (floxed) gene segment through Cre-mediated recombination. Conditional mutant mice are obtained by crossing the floxed strain with a Cre transgenic line such that the target gene becomes inactivated in vivo within the expression domain of Cre. A large collection of Cre transgenic lines has been generated over time and can be used in a combinatorial manner to achieve gene inactivation in many different cell types. A growing number of CreER(T2) transgenic mice further allows for inducible inactivation of floxed alleles in adult mice upon administration of tamoxifen. This chapter covers the design and construction of loxP flanked alleles and refers to the vectors, ES cells, and mice generated by the European conditional mouse mutagenesis (EUCOMM) project. We further describe the design and use of Cre and CreER(T2) transgenic mice and a convenient breeding strategy to raise conditional mutants and controls for phenotype analysis. PMID:21080282

Friedel, Roland H; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

2011-01-01

171

Synthetic Lethality of Cohesins with PARPs and Replication Fork Mediators  

PubMed Central

Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work demonstrates that large-scale genetic interaction screening in yeast can identify clinically relevant genetic interactions and suggests that PARP inhibitors, which are currently undergoing clinical trials as a treatment of homologous recombination-deficient cancers, may be effective in treating cancers that harbor cohesin mutations.

Barrett, Irene; Ferree, Elizabeth; van Pel, Derek M.; Ushey, Kevin; Sipahimalani, Payal; Bryan, Jennifer; Rose, Ann M.; Hieter, Philip

2012-01-01

172

A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide.  

PubMed Central

A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells. A mutation was identified in the msbB gene of E. coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A. Unlike all previously reported lipid A mutants, the msbB mutant was not conditionally lethal for growth. Viable cells or purified LPS from an msbB mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF alpha production by adherent monocytes. The cloned msbB gene was able to functionally complement the msbB mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells. Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain. These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism. In addition, the msbB mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells.

Somerville, J E; Cassiano, L; Bainbridge, B; Cunningham, M D; Darveau, R P

1996-01-01

173

Lethal Chemical Warfare: Option or Myth.  

National Technical Information Service (NTIS)

The events which led to the US obtaining a lethal chemical offensive capability are examined, as are intelligence indicators of Soviet policy concerning lethal chemical warfare. US public opinion is assessed to the extent of determining the general trends...

R. J. Baird

1974-01-01

174

A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.  

PubMed Central

The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes. Images

Costigan, C; Gehrung, S; Snyder, M

1992-01-01

175

Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.  

PubMed Central

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

Budziszewski, G J; Lewis, S P; Glover, L W; Reineke, J; Jones, G; Ziemnik, L S; Lonowski, J; Nyfeler, B; Aux, G; Zhou, Q; McElver, J; Patton, D A; Martienssen, R; Grossniklaus, U; Ma, H; Law, M; Levin, J Z

2001-01-01

176

A photosystem 1 psaFJ-null mutant of the cyanobacterium Synechocystis PCC 6803 expresses the isiAB operon under iron replete conditions.  

PubMed

A psaFJ-null mutant of Synechocystis sp. strain PCC 6803 was characterised. As opposed to similar mutants in chloroplasts of green algae, electron transfer from plastocyanin to photosystem 1 was not affected. Instead, a restraint in full chain photosynthetic electron transfer was correlated to malfunction of photosystem 1 at its stromal side. Our hypothesis is that absence of PsaF causes oxidative stress, which triggers the induction of the 'iron stress inducible' operon isiAB. Products are the IsiA chlorophyll-binding protein (CP43') and the isiB gene product flavodoxin. Supporting evidence was obtained by similar isiAB induction in wild type cells artificially exposed to oxidative stress. PMID:12914924

Jeanjean, Robert; Zuther, Ellen; Yeremenko, Nataliya; Havaux, Michel; Matthijs, Hans C P; Hagemann, Martin

2003-08-14

177

Electroshock weapons can be lethal!  

Microsoft Academic Search

Electroshock weapons (EWs)-stun guns, tasers, riot shields-are electroconductive devices designed to safely incapacitate healthy men neuromuscularly, so they are called nonlethal or less-lethal. EW firms seeking large nonmilitary markets targeted law enforcement and corrections personnel, who began using EWs in prisons\\/jails and on public patrol in 1980 in the USA. This shifted the EW-shocked population from healthy soldiers to a

Marjorie Lundquist

2008-01-01

178

Introduction to Lethal School Violence  

Microsoft Academic Search

\\u000a In this chapter, we offer an introduction to the topic of the book, lethal school violence (LSV). We begin with an introduction\\u000a to and definition of LSV, and then highlight five different situations that often result in fatalities (i.e., suicide, rampage\\u000a shootings, gang-related deaths, domestic murder\\/suicide that occurs on campus, and barricaded captive events). We then turn\\u000a our attention to

Jeffrey A. Daniels; Mary C. Bradley

179

Enhancement of Diacetyl Production by a Diacetyl-Resistant Mutant of Citrate-Positive Lactococcus lactis ssp. lactis 3022 and by Aerobic Conditions of Growth  

Microsoft Academic Search

Addition of Cu2+ and Fe3+ inhibited citrate uptake by citrate-positive Lacto- coccus lacris ssp. lads 3022 and stimu- lated the production of diacetyl, probably because citrate uptake activity of the cells is lowered by diacetyl. A diacetyl-resis- tant mutant, HK-7, derived from citrate- positive L. lactis ssp. lactis 3022, in which diacetyl production was deregulat- ed, produced a larger amount

Tsutomu Kaneko; Yukari Watanabe; Hideki Suzuki

1990-01-01

180

Suppression Analysis of esa1 Mutants in Saccharomyces cerevisiae Links NAB3 to Transcriptional Silencing and Nucleolar Functions  

PubMed Central

The acetyltransferase Esa1 is essential in the yeast Saccharomyces cerevisiae and plays a critical role in multiple cellular processes. The most well-defined targets for Esa1 are lysine residues on histones. However, an increasing number of nonhistone proteins have recently been identified as substrates of Esa1. In this study, four genes (LYS20, LEU2, VAP1, and NAB3) were identified in a genetic screen as high-copy suppressors of the conditional temperature-sensitive lethality of an esa1 mutant. When expressed from a high-copy plasmid, each of these suppressors rescued the temperature-sensitivity of an esa1 mutant. Only NAB3 overexpression also rescued the rDNA-silencing defects of an esa1 mutant. Strengthening the connections between NAB3 and ESA1, mutants of nab3 displayed several phenotypes similar to those of esa1 mutants, including increased sensitivity to the topoisomerase I inhibitor camptothecin and defects in rDNA silencing and cell-cycle progression. In addition, nuclear localization of Nab3 was altered in the esa1 mutant. Finally, posttranslational acetylation of Nab3 was detected in vivo and found to be influenced by ESA1.

Chang, Christie S.; Clarke, Astrid; Pillus, Lorraine

2012-01-01

181

Identification of lethal cluster of genes in the yeast transcription network  

NASA Astrophysics Data System (ADS)

Identification of essential or lethal genes would be one of the ultimate goals in drug designs. Here we introduce an in silico method to select the cluster with a high population of lethal genes, called lethal cluster, through microarray assay. We construct a gene transcription network based on the microarray expression level. Links are added one by one in the descending order of the Pearson correlation coefficients between two genes. As the link density p increases, two meaningful link densities p and p are observed. At p, which is smaller than the percolation threshold, the number of disconnected clusters is maximum, and the lethal genes are highly concentrated in a certain cluster that needs to be identified. Thus the deletion of all genes in that cluster could efficiently lead to a lethal inviable mutant. This lethal cluster can be identified by an in silico method. As p increases further beyond the percolation threshold, the power law behavior in the degree distribution of a giant cluster appears at p. We measure the degree of each gene at p. With the information pertaining to the degrees of each gene at p, we return to the point p and calculate the mean degree of genes of each cluster. We find that the lethal cluster has the largest mean degree.

Rho, K.; Jeong, H.; Kahng, B.

2006-05-01

182

Virulence of a Salmonella typhimurium OmpD Mutant  

Microsoft Academic Search

An ompD mutation caused by a Tn10 insertion was transduced into Salmonella typhimurium SL1344 and UK-1. The adherence and invasion capabilities of the resultant ompD mutants were examined by tissue culture analysis. The virulence of the S. typhimurium ompD mutants was ascertained by a 50% lethal dose (LD50) study and by determining colonization ability with BALB\\/c mice. We found no

PAUL N. MEYER; MARY R. WILMES-RIESENBERG; CHRISTOS STATHOPOULOS; ROY CURTISS III

183

Abdominal tuberculosis: still a potentially lethal disease.  

PubMed

The findings in a 4-yr survey of 82 patients with abdominal tuberculosis are described and compared with those encountered in previous surveys. Fourteen cases of intestinal, 11 of mesenteric-lymphnodal, and 57 of peritoneal tuberculosis were identified. The disease occurred essentially in patients living under worsening socioeconomic conditions, and 51 of them had associated pulmonary tuberculosis. Symptoms and clinical findings were again nonspecific, but newer imaging, endoscopic, and other invasive procedures were helpful in establishing a definite diagnosis. In addition, adenosine deaminase determination showed great promise as a noninvasive diagnostic procedure in patients with tuberculous ascites. The six hospital deaths in the series highlight the hazard of potentially lethal delays in early diagnosis and treatment, even in centers with a high awareness of the disease. PMID:8480741

Lingenfelser, T; Zak, J; Marks, I N; Steyn, E; Halkett, J; Price, S K

1993-05-01

184

Escherichia coli mutants with defects in the biosynthesis of 5-methylaminomethyl-2-thio-uridine or 1-methylguanosine in their tRNA.  

PubMed Central

Two tRNA methyltransferase mutants, isolated as described in the accompanying paper (G.R. Björk and K. Kjellin-Stråby, J. Bacteriol. 133:499-207, 1978), are biochemicaaly and genetically characterized. tRNA from mutant IB13 lacks 5-methylaminomethyl-2-thio-uridine in vivo due to a permanently nonfunctional methyltransferase. Thus tRNA from this mutant is a specific substrate for the corresponding tRNA methyltransferase in vitro. In spite of this defect in tRNA, such a mutant is viable. Mutant IB11 is conditionally defective in the biosynthesis of 1-methylguanosine in tRNA due to a temperature-sensitive tRNA (1-methyl-guanosine) methyltransferase. In mutant cells grown at a high temperature, the level of 1-methylguanosine in bulk tRNA is 20% of that of the wild type, demonstrating that in this mutant an 80% deficiency of 1-methylguanosine in tRNA is not lethal. Genetically these two distinct lesions, trmC2, causing 5=methylaminomethyl-2-thio-uridine deficiency, and trmD1, giving a temperature-sensitive tRNA (1-methylguanosine)methyltransferase, are both located between 50 and 61 min on the Escherichia coli chromosome. Images

Bjork, G R; Kjellin-Straby, K

1978-01-01

185

Early events of lethal action by tobramycin in Pseudomonas aeruginosa  

SciTech Connect

The immediate activities of the aminoglycoside antibiotic, tobramycin, were investigated in Pseudomonas aeruginosa PAO1. The influence of carbon growth substate and the antibiotic exposure environment in the magnitude of activity were examined. Lethality by 8 {mu}g/ml tobramycin occurred rapidly (1 to 3 minutes). The release of specific cellular components into the supernatant was associated with lethality. This material was initially detected as an increase in UV-absorbance. Magnesium in the reaction mixture provided protection against lethality and leakage, but did not reverse lethal damage after a 3 minute tobramycin treatment. Also, uptake of {sup 3}H-tobramycin was reduced in the presence of magnesium. Cells grown with glucose as a carbon source were more susceptible than organic acid grown cells as was the rapidity and amount of cell damage. Analyses of the leakage material revealed a 2-fold increase of protein in the supernatant after a 1-3 minute treatment which paralleled lethality. A prominent 29 kDa protein was observed by SDS-PAGE in the released material, which has been identified as the periplasmic enzyme, {beta}-lactamase. The immediate activities of tobramycin did not involve (i) release of overall cell protein, (ii) massive loss of total pool amino acids, (iii) cell lysis, (iv) inhibition of proline uptake, (v) release of lipopolysaccharide, or (vi) leakage of ATP. Electron microscopy showed no apparent damage after a 3 minute exposure. 40% inhibition of protein synthesis had occurred by 3 minutes of exposure, while release of UV-absorbing material and lethality were detectable after only 1 minute. Resistant cystic fibrosis isolates of P. aeruginosa did not leak under the same experimental conditions, but one of two susceptible strains examined did show increased UV-absorbance following treatment.

Raulston, J.E.

1988-01-01

186

Conditional Expression of Parkinson disease-related Mutant ?-synuclein in the Midbrain Dopaminergic Neurons causes Progressive Neurodegeneration and Degradation of Transcription Factor Nuclear Receptor Related 1  

PubMed Central

?-synuclein(?-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on ?-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of ?-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the ?-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related ?-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the ?-syn-dependent degeneration of mDA neurons, we found that over-expression of ?-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the ?-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the ?-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.

Lin, Xian; Parisiadou, Loukia; Sgobio, Carmelo; Liu, Guoxiang; Yu, Jia; Sun, Lixin; Shim, Hoon; Gu, Xing-Long; Luo, Jing; Long, Cai-Xia; Ding, Jinhui; Mateo, Yolanda; Sullivan, Patricia H.; Wu, Ling-Gang; Goldstein, David S.; Lovinger, David; Cai, Huaibin

2012-01-01

187

D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function.  

PubMed

Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knock-in (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP(+)/NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1? (Hif1?) and up-regulated Hif1? target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects. PMID:22925884

Sasaki, Masato; Knobbe, Christiane B; Itsumi, Momoe; Elia, Andrew J; Harris, Isaac S; Chio, Iok In Christine; Cairns, Rob A; McCracken, Susan; Wakeham, Andrew; Haight, Jillian; Ten, Annick You; Snow, Bryan; Ueda, Takeshi; Inoue, Satoshi; Yamamoto, Kazuo; Ko, Myunggon; Rao, Anjana; Yen, Katharine E; Su, Shinsan M; Mak, Tak Wah

2012-08-27

188

Cross-talk between Chk1 and Chk2 in double-mutant thymocytes  

PubMed Central

Chk1 is a checkpoint kinase and an important regulator of mammalian cell division. Because null mutation of Chk1 in mice is embryonic lethal, we used the Cre-loxP system and the Lck promoter to generate conditional mutant mice in which Chk1 was deleted only in the T lineage. In the absence of Chk1, the transition of CD4?CD8? double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) cells was blocked due to an increase in apoptosis at the DN2 and DN3 stages. Strikingly, loss of Chk1 activated the checkpoint kinase Chk2 as well as the tumor suppressor p53 in these thymocytes. However, the developmental defects caused by Chk1 deletion were not rescued by p53 inactivation. Significantly, even though Chk1 deletion is highly lethal in proliferating tissues, we succeeded in using in vivo methods to generate Chk1/Chk2 double-knockout T cells. Analysis of these T cells revealed an interesting interaction between Chk1 and Chk2 functions that partially rescued the apoptosis of the double-mutant cells. Thus, Chk1 is both critical for the survival of proliferating cells and engages in cross-talk with the Chk2 checkpoint kinase pathway. These factors have implications for the targeting of Chk1 as an anticancer therapy.

Zaugg, Kathrin; Su, Yu-Wen; Reilly, Patrick T.; Moolani, Yasmin; Cheung, Carol C.; Hakem, Razquallah; Hirao, Atsushi; Liu, Quinghua; Elledge, Stephen J.; Mak, Tak W.

2007-01-01

189

Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis  

Microsoft Academic Search

A Burkholderia pseudomallei mutant which was attenuated in a mouse model of melioidosis was identified by a signature tagged mutagenesis approach. The transposon was shown to be inserted into a gene within the capsular biosynthetic operon. Compared with the wild-type bacteria this mutant demonstrated a 105-fold increase in the median lethal dose in a mouse model and it did not

TIMOTHY ATKINS; RICHARD PRIOR; KERRI MACK; PAUL RUSSELL; MICHELLE NELSON; JILL ELLIS; PETRA C. F. OYSTON; GORDON DOUGAN; RICHARD W. TITBALL

2002-01-01

190

The Maternally Expressed WRKY Transcription Factor TTG2 Controls Lethality in Interploidy Crosses of Arabidopsis  

PubMed Central

The molecular mechanisms underlying lethality of F1 hybrids between diverged parents are one target of speciation research. Crosses between diploid and tetraploid individuals of the same genotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a role in polyploid speciation. We have identified variation in F1 lethality in interploidy crosses of Arabidopsis thaliana and determined the genetic architecture of the maternally expressed variation via QTL mapping. A single large-effect QTL, DR. STRANGELOVE 1 (DSL1), was identified as well as two QTL with epistatic relationships to DSL1. DSL1 affects the rate of postzygotic lethality via expression in the maternal sporophyte. Fine mapping placed DSL1 in an interval encoding the maternal effect transcription factor TTG2. Maternal parents carrying loss-of-function mutations in TTG2 suppressed the F1 lethality caused by paternal excess interploidy crosses. The frequency of cellularization in the endosperm was similarly affected by both natural variation and ttg2 loss-of-function mutants. The simple genetic basis of the natural variation and effects of single-gene mutations suggests that F1 lethality in polyploids could evolve rapidly. Furthermore, the role of the sporophytically active TTG2 gene in interploidy crosses indicates that the developmental programming of the mother regulates the viability of interploidy hybrid offspring.

Dilkes, Brian P; Spielman, Melissa; Weizbauer, Renate; Watson, Brian; Burkart-Waco, Diana; Scott, Rod J; Comai, Luca

2008-01-01

191

Nonchemotactic Mutants of Escherichia coli  

PubMed Central

We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged. Images

Armstrong, John B.; Adler, Julius; Dahl, Margaret M.

1967-01-01

192

Extinction of hepatitis C virus by ribavirin in hepatoma cells involves lethal mutagenesis.  

PubMed

Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. PMID:23976977

Ortega-Prieto, Ana M; Sheldon, Julie; Grande-Pérez, Ana; Tejero, Héctor; Gregori, Josep; Quer, Josep; Esteban, Juan I; Domingo, Esteban; Perales, Celia

2013-08-16

193

conditions  

Microsoft Academic Search

Real-time scheduling is both based on a broad theoretical background and available through a multitude of tools and infrastructures. The central input parameters to this discipline are the demand for execution time and the real- time conditions given as deadlines or periods. The former has attracted a lot of research efforts, mainly in the scope of worst case execution time

Dieter Z

194

Conditions  

Microsoft Academic Search

A shallow, RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)- contaminated aquifer at Naval Submarine Base Bangor has been characterized as predominantly manganese-reducing, anoxic with local pockets of oxic conditions. The potential contribution of microbial RDX degradation to localized decreases observed in aquifer RDX concentrations was assessed in sediment microcosms amended with (U- 14 C) RDX. Greater than 85% mineralization of 14 C-RDX to 14 CO2

Paul M. Bradley; Richard S. Dinicola

195

Mutant mice lacking ryanodine receptor type 3 exhibit deficits of contextual fear conditioning and activation of calcium\\/calmodulin-dependent protein kinase II in the hippocampus  

Microsoft Academic Search

As it is known that ryanodine receptor type 3 is expressed in the hippocampus, we examined the contribution of this receptor to contextual fear conditioning behavior and to the activation of Ca2+\\/calmodulin-dependent protein kinase II using mice lacking the receptor. Ryanodine receptor type 3-deficient mice exhibited impairments of performance in the contextual fear conditioning test, passive avoidance test, and Y-maze

Yasuko Kouzu; Takahiro Moriya; Hiroshi Takeshima; Tohru Yoshioka; Shigenobu Shibata

2000-01-01

196

Alcohol Consumption and Nearly Lethal Suicide Attempts.  

ERIC Educational Resources Information Center

|Presents a case-control study of the association between nearly lethal suicide attempts and facets of alcohol consumption; namely, drinking frequency, drinking quantity, binge drinking, alcoholism, drinking within 3 hours of suicide attempt, and age began drinking. In bivariate analyses, all measures were associated with nearly lethal suicide…

Powell, Kenneth E.; Kresnow, Marcie-jo; Mercy, James A.; Potter, Lloyd B.; Swann, Alan C.; Frankowski, Ralph F.; Lee, Roberta K.; Bayer, Timothy L.

2002-01-01

197

Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin.  

PubMed Central

The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.

Little, S F; Leppla, S H; Friedlander, A M

1990-01-01

198

The Yeast Med1 Mutant Undergoes Both Meiotic Homolog Nondisjunction and Precocious Separation of Sister Chromatids  

PubMed Central

A mutant at the yeast MED1 locus was isolated in a screen for sporulation-proficient, meiotic-lethal mutants. Synaptonemal complex formation in the med1 mutant is apparently normal and med1 strains undergo meiotic crossing over at approximately 50% of the wild-type level. The med1 mutant undergoes homolog nondisjunction at meiosis I, presumably as a consequence of the decrease in crossing over. In addition, the mutant undergoes precocious separation of sister chromatids, resulting in chromosome missegregation at both meiotic divisions. We suggest that the med1 mutation perturbs chromosome structure, leading to a reduction in recombination and a defect in sister chromatid cohesion.

Rockmill, B.; Roeder, G. S.

1994-01-01

199

Properties of r Mutants of Bacteriophage T4 Photodynamically Induced in the Presence of Thiopyronin and Psoralen  

PubMed Central

About 4 × 10?4r mutants were induced per lethal hit, a frequency characteristic of weak mutagens. Collections of mutants produced in the presence of either dye were indistinguishable in most of their properties. The rII mutants differed sharply from spontaneous mutants in their mutational spectra (fine scale map distribution) and their reversion responses to specific mutagens. Few or none of the induced mutants were induced to revert with proflavine (sign mutants; reading frame shift mutants). A majority were induced to revert with base analogues (base pair substitution mutants), and about half of these also responded to the hydroxymethylcytosine-specific agent hydroxylamine. A large minority of the mutants reverted spontaneously but failed to respond either to proflavine or to base analogues. We believe these mutants, as well as some of the mutants which did respond to base analogues, to be transversions (base pair substitutions which reverse the purine-pyrimidine orientation).

Drake, John W.; McGuire, Janice

1967-01-01

200

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER  

PubMed Central

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile. All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells. The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.

Garcia-Bellido, Antonio; Robbins, Leonard G.

1983-01-01

201

Construction of Listeria monocytogenes mutants with in-frame Deletions in the phosphotransferase transport system (PTS) and analysis of their growth under stress conditions  

Technology Transfer Automated Retrieval System (TEKTRAN)

Background: Listeria monocytogenes is a food-borne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress ...

202

Hypopigmentation and Maternal-Zygotic Embryonic Lethality Caused by a Hypomorphic Mbtps1 Mutation in Mice  

PubMed Central

The site 1 protease, encoded by Mbtps1, mediates the initial cleavage of site 2 protease substrates, including sterol regulatory element binding proteins and CREB/ATF transcription factors. We demonstrate that a hypomorphic mutation of Mbtps1 called woodrat (wrt) caused hypocholesterolemia, as well as progressive hypopigmentation of the coat, that appears to be mechanistically unrelated. Hypopigmentation was rescued by transgenic expression of wild-type Mbtps1, and reciprocal grafting studies showed that normal pigmentation depended upon both cell-intrinsic or paracrine factors, as well as factors that act systemically, both of which are lacking in wrt homozygotes. Mbtps1 exhibited a maternal-zygotic effect characterized by fully penetrant embryonic lethality of maternal-zygotic wrt mutant offspring and partial embryonic lethality (~40%) of zygotic wrt mutant offspring. Mbtps1 is one of two maternal-zygotic effect genes identified in mammals to date. It functions nonredundantly in pigmentation and embryogenesis.

Rutschmann, Sophie; Crozat, Karine; Li, Xiaohong; Du, Xin; Hanselman, Jeffrey C.; Shigeoka, Alana A.; Brandl, Katharina; Popkin, Daniel L.; McKay, Dianne B.; Xia, Yu; Moresco, Eva Marie Y.; Beutler, Bruce

2012-01-01

203

Hypopigmentation and maternal-zygotic embryonic lethality caused by a hypomorphic mbtps1 mutation in mice.  

PubMed

The site 1 protease, encoded by Mbtps1, mediates the initial cleavage of site 2 protease substrates, including sterol regulatory element binding proteins and CREB/ATF transcription factors. We demonstrate that a hypomorphic mutation of Mbtps1 called woodrat (wrt) caused hypocholesterolemia, as well as progressive hypopigmentation of the coat, that appears to be mechanistically unrelated. Hypopigmentation was rescued by transgenic expression of wild-type Mbtps1, and reciprocal grafting studies showed that normal pigmentation depended upon both cell-intrinsic or paracrine factors, as well as factors that act systemically, both of which are lacking in wrt homozygotes. Mbtps1 exhibited a maternal-zygotic effect characterized by fully penetrant embryonic lethality of maternal-zygotic wrt mutant offspring and partial embryonic lethality (~40%) of zygotic wrt mutant offspring. Mbtps1 is one of two maternal-zygotic effect genes identified in mammals to date. It functions nonredundantly in pigmentation and embryogenesis. PMID:22540041

Rutschmann, Sophie; Crozat, Karine; Li, Xiaohong; Du, Xin; Hanselman, Jeffrey C; Shigeoka, Alana A; Brandl, Katharina; Popkin, Daniel L; McKay, Dianne B; Xia, Yu; Moresco, Eva Marie Y; Beutler, Bruce

2012-04-01

204

Lethal photosensitization of Helicobacter species  

NASA Astrophysics Data System (ADS)

Helicobacter pylori (H. pylori) is associated with a large number of gastroduodenal disorders. Clearance of the bacteria has been shown to benefit patients with duodenal ulcers, gastric ulcers, and certain rare types of gastric tumors. Broad-spectrum antibiotics are the mainstay of current treatment strategies but side-effects, poor compliance, and drug resistance limit their usefulness. We sensitized H. pylori with toluidine blue, haematoporphyrin derivative, aluminum disulphonated phthalocyanine, methylene blue or protoporphyrin IX prior to exposure to low-power laser light from either a gallium aluminum arsenide laser or a helium neon gas laser. All 5 sensitizers caused reductions of greater than 1000-fold in the number of viable bacteria. Light alone had no effect and only HpD caused a significant decrease in bacterial numbers without laser light. Next, we sensitized H. mustelae on explanted ferret gastric mucosa (ex vivo) with the same sensitizers and exposed them to light from a copper vapor pumped dye laser tuned appropriately. MB caused significant reductions in bacterial counts. Successful lethal photosensitization of Helicobacter pylori both in vitro and ex vivo raises the possibility of a local method for eradicating the bacteria, especially as the bacteria are only found in those parts of the upper gastrointestinal tract that are accessible to the endoscope.

Millson, Charles E.; Wilson, Michael; MacRobert, Alexander J.; Thurrell, Wendy; Mlkvy, Peter; Davies, Claire; Bown, S. G.

1995-01-01

205

Analysis of hybrid lethality in F 1 wheat-rye hybrid embryos  

Microsoft Academic Search

The effect of the Embryo lethality mutant (Eml) of rye was studied in crosses between hexaploid wheat and corresponding inbred rye line (L2). Histological analysis of hybrid\\u000a embryos revealed morphological differences 16 days after pollination. Eml was found to arrest the formation of shoot meristem but had no influence on root meristem formation. The effect of Eml cannot be overcome by

N. Tikhenko; T. Rutten; A. Voylokov; A. Houben

2008-01-01

206

Mutant E. Coli Strain with Increased Succinic Acid Production.  

National Technical Information Service (NTIS)

The invention relates to a mutant strain of bacteria, which either lacks or contains mutant genes for several key metabolic enzymes, and which produces high amounts of succinic acid under anaerobic conditions.

A. Sanchez G. N. Bennett S. K. Yiu

2005-01-01

207

Dominant lethal phenotype of a mutation in the -35 recognition region of Escherichia coli sigma 70.  

PubMed Central

A dominant lethal mutation in the Escherichia coli rpoD gene, which encodes sigma 70, the promoter recognition subunit of RNA polymerase, was isolated after random mutagenesis. The lethal gene was maintained under control of the lac repressor on a low copy plasmid. An amount of lethal sigma 70 that was nearly equimolar with the chromosomally encoded sigma 70 was sufficient to cause cessation of growth. RNA synthesis per unit cell mass was unaffected, but protein synthesis was inhibited by the mutant sigma 70. The amino acid change (Glu-585 to Gln) was in a region of sigma 70 thought to bind the -35 hexamer of the promoter, and the mutant sigma 70 caused increased expression from promoters with nonconsensus bases in the third position of the -35 hexamer. A null mutation of the fis gene could partially suppress the mutant phenotype. These properties are consistent with those expected of a sigma 70 insensitive to growth rate control of rRNA and tRNA promoters. Images Fig. 2

Keener, J; Nomura, M

1993-01-01

208

Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+)-ATPase.  

PubMed Central

Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca(2+)-mediated signaling in many cells. Yeast cells lacking functional calcineurin (cna1 cna2 or cnb1 mutants) display growth defects under specific environmental conditions, for example, in the presence of high concentrations of Na+, Li+, Mn2+, or OH- but are indistinguishable from wild-type cells under standard culture conditions. To characterize regulatory pathways that may overlap with calcineurin, we performed a synthetic lethal screen to identify mutants that require calcineurin on standard growth media. The characterization of one such mutant, cnd1-8, is presented. The CND1 gene was cloned, and sequence analysis predicts that it encodes a novel protein 1,876 amino acids in length with multiple membrane-spanning domains. CND1 is identical to the gene identified previously as FKS1, ETG1, and CWH53, cnd1 mutants are sensitive to FK506 and cyclosporin A and exhibit slow growth that is improved by the addition of osmotic stabilizing agents. This osmotic agent-remedial growth defect and microscopic evidence of spontaneous cell lysis in cnd1 cultures suggest that cell integrity is compromised in these mutants. Mutations in the genes for yeast protein kinase C (pkc1) and a MAP kinase (mpk1/slt2) disrupt a Ca(2+)-dependent signaling pathway required to maintain a normal cell wall and cell integrity. We show that pkc1 and mpk1/slt2 growth defects are more severe in the absence of calcineurin function and less severe in the presence of a constitutively active form of calcineurin. These observations suggest that calcineurin and protein kinase C perform independent but physiologically related functions in yeast cells. We show that several mutants that lack a functional vacuolar H(+)-ATPase (vma) require calcineurin for vegetative growth. We discuss possible roles for calcineurin in regulating intracellular ion homeostasis and in maintaining cell integrity.

Garrett-Engele, P; Moilanen, B; Cyert, M S

1995-01-01

209

Suppression of lethal toxicity of endotoxin by biscoclaurine alkaloid cepharanthin.  

PubMed

Suppressive effects of Cepharanthin (CE) on lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFalpha) production followed by liver injury were investigated. Pretreatment with CE reduced limulus activity of LPS. Intraperitoneal treatment with CE 10 min before an i.v. challenge of LPS resulted in protection from LPS lethality in D-galactosamine (GalN)-sensitized BALB/c but not in C57BL/6 and C57BL/10ScSn mice. Treatment with CE before the LPS challenge significantly reduced serum TNF levels in a dose-dependent manner. The suppression was most effective when CE was administered 10 min before the LPS challenge. Increased levels of enzymes released from hepatocytes into the circulation, as a result of LPS-induced liver injury, were reduced by CE administration. Histological evaluation demonstrated that massive cell infiltration after severe injury developed in liver of mice injected with LPS plus D-GalN unless they were pretreated with CE. Apoptotic cells decreased by treatment with CE. Treatment with CE retarded lethal shock induced by an infection with 10(8) CFU Salmonella typhimurium deltaaroA mutant. These results suggest that action of CE is initiated through suppression of LPS-induced TNF production. PMID:10670847

Maruyama, H; Kikuchi, S; Kawaguchi, K; Hasunuma, R; Ono, M; Ohbu, M; Kumazawa, Y

2000-02-01

210

Yeast mutants auxotrophic for choline or ethanolamine.  

PubMed Central

Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (cho1) on chromosome V. The lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally. However, mitochondrial abnormalities were evident even when ethanolamine and choline were supplied. Diploids homozygous for the cho1 mutation were defective in sporulation. Growth on nonfermentable carbon sources was slow, and a high proportion of respiratory-deficient (petite) cells were generated in cho1 cultures.

Atkinson, K D; Jensen, B; Kolat, A I; Storm, E M; Henry, S A; Fogel, S

1980-01-01

211

Directed Energy Non-lethal Weapons.  

National Technical Information Service (NTIS)

This basic research initiative has been an ongoing interdisciplinary effort to lay the foundation for developing, novel effective and safe non- lethal technologies that alter skeletal muscle contraction and/or neural functioning (i.e., neurosecretion) via...

G. L. Craviso I. Chatterjee

2010-01-01

212

Reaming experiments for the lethality test system  

SciTech Connect

Various reaming techniques were tried for use on the barrel of the Lethality Test System railgun. This report covers the successes and failures of the reamers and the techniques that were tried. 5 figs.

Hooten, D.; Stanley, P.

1988-01-01

213

Gibberella fujikuroi mutants obtained with UV radiation and N-methyl-N'-nitro-nitrosoguanidine  

SciTech Connect

N-methyl-N'-nitrosoguanidine and to a lesser extent UV radiation are very mutagenic for Gibberella microconidia. The recommended nitrosoguanidine doses lead to much higher frequencies of mutants than are found in other microorganisms. The frequency of mutants among the survivors increases linearly with the nitrosoguanidine dose (molar concentration x time); the absolute number of viable mutants in a given population reaches a maximum for a dose of ca. 0.7 M x s. The microconidia are uninucleate. The onset of germination brings about increased lethality of nitrosoguanidine, but it does not modify the action of UV radiation. Mycelia are more resistant than spores to both agents. Visible illumination effectively prevents lethality when given immediately after UV irradiation. Auxotrophs and color mutants are very easily obtained. Pink adenine auxotrophs and several classes of color mutants are affected in the biosynthesis of the carotenoid pigment, neurosporaxanthin.

Avalos, J.; Casadesus, J.; Cerda-Olmedo, E.

1985-01-01

214

Dominant lethal mutations in male mice  

Microsoft Academic Search

Dominant lethal mutations are due to chromosome aberrations as demonstrated by analysis of first cleavage. With a sample size of 40–45 mice per dose the induction of dominant lethal mutations by 10 mg\\/kg of methyl methanesulfonat (MMS) can be detected in spermatids in the mating interval 9–12 days posttreatment (6–11%). In the same mating interval a dose of 150 mg\\/kg

U. H. Ehling

1977-01-01

215

Deletion of the chloroplast-localized AtTerC gene product in Arabidopsis thaliana leads to loss of the thylakoid membrane and to seedling lethality.  

PubMed

Early seedling development in plants depends on the biogenesis of chloroplasts from proplastids, accompanied by the formation of thylakoid membranes. An Arabidopsis thaliana gene, AtTerC, whose gene product shares sequence similarity with bacterial tellurite resistance C (TerC), is shown to be involved in a critical step required for the normal organization of prothylakoids and transition into mature thylakoid stacks. The AtTerC gene encodes an integral membrane protein, which contains eight putative transmembrane helices, localized in the thylakoid of the chloroplast, as shown by localization of an AtTerC-GFP fusion product in protoplasts and by immunoblot analysis of subfractions of chloroplasts. T-DNA insertional mutation of AtTerC resulted in a pigment-deficient and seedling-lethal phenotype under normal light conditions. Transmission electron microscopic analysis revealed that mutant etioplasts had normal prolamellar bodies (PLBs), although the prothylakoids had ring-like shapes surrounding the PLBs. In addition, the ultrastructures of mutant chloroplasts lacked thylakoids, did not have grana stacks, and showed numerous globular structures of varying sizes. Also, the accumulation of thylakoid membrane proteins was severely defective in this mutant. These results suggest that the AtTerC protein plays a crucial role in prothylakoid membrane biogenesis and thylakoid formation in early chloroplast development. PMID:18429937

Kwon, Kwang-Chul; Cho, Myeon Haeng

2008-04-17

216

Selection for intragenic suppressors of lethal 23S rRNA mutations in Escherichia coli identifies residues important for ribosome assembly and function  

Microsoft Academic Search

Mutations in several functionally important regions of the 23S rRNA of E. coli increase the levels of frameshifting and readthrough of stop codons. These mutations include U2555A, U2555G, ?A1916 and U2493C.\\u000a The mutant rRNAs are lethal when expressed at high levels from a plasmid, in strains also expressing wild type rRNA from chromosomal\\u000a rrn operons. The lethal phenotype can be

Michael O’Connor

2007-01-01

217

The parkin Mutant Phenotype in the Fly Is Largely Rescued by Metal-Responsive Transcription Factor (MTF-1) ? †  

PubMed Central

The gene for Parkin, an E3 ubiquitin ligase, is mutated in some familial forms of Parkinson's disease, a severe neurodegenerative disorder. A homozygous mutant of the Drosophila ortholog of human parkin is viable but results in severe motoric impairment including an inability to fly, female and male sterility, and a decreased life span. We show here that a double mutant of the genes for Parkin and the metal-responsive transcription factor 1 (MTF-1) is not viable. MTF-1, which is conserved from insects to mammals, is a key regulator of heavy metal homeostasis and detoxification and plays additional roles in other stress conditions, notably oxidative stress. In contrast to the synthetic lethality of the double mutant, elevated expression of MTF-1 dramatically ameliorates the parkin mutant phenotype, as evidenced by a prolonged life span, motoric improvement including short flight episodes, and female fertility. At the cellular level, muscle and mitochondrial structures are substantially improved. A beneficial effect is also seen with a transgene encoding human MTF-1. We propose that Parkin and MTF-1 provide complementary functions in metal homeostasis, oxidative stress and other cellular stress responses. Our findings also raise the possibility that MTF-1 gene polymorphisms in humans could affect the severity of Parkinson's disease.

Saini, Nidhi; Georgiev, Oleg; Schaffner, Walter

2011-01-01

218

Differential response to intracellular stress in the skin from osteogenesis imperfecta Brtl mice with lethal and non lethal phenotype: a proteomic approach.  

PubMed

Phenotypic variability in the presence of an identical molecular defect is a recurrent feature in heritable disorders and it was also reported in osteogenesis imperfecta (OI). OI is a prototype for skeletal dysplasias mainly caused by mutations in the two genes coding for type I collagen. No definitive cure is available for this disorder, but the understanding of molecular basis in OI phenotypic modulation will have a pivotal role in identifying possible targets to develop novel drug therapy. We used a functional proteomic approach to address the study of phenotypic variability using the skin of the OI murine model Brtl. Brtl mice reproduce the molecular defect, dominant transmission and phenotypic variability of human OI patients. In the presence of a Gly349Cys substitution in ?1(I)-collagen Brtl mice can have a lethal or a moderately severe outcome. Differential expression of chaperones, proteasomal subunits, metabolic enzymes, and proteins related to cellular fate demonstrated that a different ability to adapt to cellular stress distinguished mutant from wild-type mice and mutant lethal from surviving mutant animals. Interestingly, class discovery analysis identified clusters of differentially expressed proteins associated with a specific outcome, and functional analysis contributed to a deeper investigation into biochemical and cellular pathways affected by the disease. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22846432

Bianchi, Laura; Gagliardi, Assunta; Gioia, Roberta; Besio, Roberta; Tani, Chiara; Landi, Claudia; Cipriano, Maria; Gimigliano, Anna; Rossi, Antonio; Marini, Joan C; Forlino, Antonella; Bini, Luca

2012-02-18

219

Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells.  

PubMed

An alternative to therapeutic targeting of oncogenes is to perform "synthetic lethality" screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with "undruggable" genetic alterations. PMID:19490892

Scholl, Claudia; Fröhling, Stefan; Dunn, Ian F; Schinzel, Anna C; Barbie, David A; Kim, So Young; Silver, Serena J; Tamayo, Pablo; Wadlow, Raymond C; Ramaswamy, Sridhar; Döhner, Konstanze; Bullinger, Lars; Sandy, Peter; Boehm, Jesse S; Root, David E; Jacks, Tyler; Hahn, William C; Gilliland, D Gary

2009-05-29

220

Crystallographic studies of the Anthrax lethal toxin. Annual report  

SciTech Connect

The lethal form of Anthrax results from the inhalation of anthrax spores. Death is primarily due to the effects of the lethal toxin (Protective Antigen (PA) + Lethal Factor) from the causative agent, Bacillus anthracis. All the Anthrax vaccines currently in use or under development contain or produce PA, the major antigenic component of anthrax toxin, and there is a clear need for an improved vaccine for human use. In the previous report we described the first atomic resolution structure of PA, revealing that the molecule is composed largely of beta-sheets organized into four domains. This information can be used in the design. of recombinant PA vaccines. In this report we describe additional features of the full-length PA molecule derived from further crystallographic refinement and careful examination of the structure. We compare two crystal forms of PA grown at different pH values and discuss the functional implications. A complete definition of the function of each domain must await the crystal structure of the PA63 heptamer. We have grown crystals of the heptamer under both detergent and detergent-free conditions, and made substantial progress towards the crystal structure. The mechanism of anthrax intoxication in the light of our results is reviewed.

Frederick, C.A.

1996-07-01

221

The MAP Kinase Slt2 Is Involved in Vacuolar Function and Actin Remodeling in Saccharomyces cerevisiae Mutants Affected by Endogenous Oxidative Stress.  

PubMed

Oxidative stress causes transient actin cytoskeleton depolarization and also provokes vacuole fragmentation in wild-type cells. Under conditions of oxidative stress induced by hydrogen peroxide, the Slt2 protein is required to repolarize the actin cytoskeleton and to promote vacuole fusion. In this study, we show that grx3 grx4 and grx5 mutants are cellular models of endogenous oxidative stress. This stress is the result of alterations in iron homeostasis that lead to impairment of vacuolar function and also to disorganization of the actin cytoskeleton. Slt2 overexpression suppresses defects in vacuolar function and actin cytoskeleton organization in the grx3 grx4 mutant. Slt2 exerts this effect independently of the intracellular levels of reactive oxygen species (ROS) and of iron homeostasis. The deletion of SLT2 in the grx3 grx4 mutant results in synthetic lethality related to vacuolar function with substantial vacuole fragmentation. The observation that both Vps4 and Vps73 (two proteins related to vacuole sorting) suppress vacuole fragmentation and actin depolarization in the grx3 grx4 slt2 triple mutant strengthens the hypothesis that Slt2 plays a role in vacuole homeostasis related to actin dynamics. Here we show that in sod1, grx5, and grx3 grx4 slt2 mutants, all of which are affected by chronic oxidative stress, the overexpression of Slt2 favors vacuole fusion through a mechanism dependent on an active actin cytoskeleton. PMID:23956390

Pujol-Carrion, Nuria; Petkova, Mima I; Serrano, Luis; de la Torre-Ruiz, Maria Angeles

2013-08-16

222

Development of ELISA based detection system for lethal toxin of Clostridium sordellii  

PubMed Central

Background & objectives: Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii. Methods: The catalytic domain (1.6kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund's complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin. Results: The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody. Interpretation & conclusions: An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples.

Arya, Preetika; Ponmariappan, S.; Singh, Lokendra; Kumar, Om

2013-01-01

223

Developing a Combined Lethal and Non-Lethal Capability for the Individual Marine.  

National Technical Information Service (NTIS)

The United States Marine Corps should research, develop, and field a new weapon that provides both a lethal and non-lethal capability for the individual Marine. Discussion: The future of military conflict will include a cluttered battlefield mixed with bo...

G. T. Polland

2010-01-01

224

Lethality and synthetic lethality in the genome-wide metabolic network of Escherichia coli  

Microsoft Academic Search

Recent genomic analyses on the cellular metabolic network show that reaction flux across enzymes are diverse and exhibit power-law behavior in its distribution. While intuition might suggest that the reactions with larger fluxes are more likely to be lethal under the blockade of its catalysing gene products or gene knockouts, we find, by in silico flux analysis, that the lethality

Cheol-Min Ghim; Kwang-Il Goh; Byungnam Kahng

2005-01-01

225

Lethality and synthetic lethality in the genome-wide metabolic network of Escherichia coli  

Microsoft Academic Search

Recent genomic analyses on the cellular metabolic network show that reaction flux across enzymes are diverse and exhibit power-law behavior in its distribution. While one may guess that the reactions with larger fluxes are more likely to be lethal under the blockade of its catalyzing gene products or gene knockouts, we find, by in silico flux analysis, that the lethality

C.-M. Ghim; K.-I. Goh; B. Kahng

2004-01-01

226

Lethal and mutagenic effects of elevated temperature on haploid yeast  

Microsoft Academic Search

When after heat treatment at 52°, yeast cells are held in water at 28° with aeration for 24 hours to 5 days before plating, their survival is greatly enhanced in comparison to what is obtained on immediate plating. Moreover, the frequency of induced nuclear (canavanine-sensitivity to canavanine-resistance) and cytoplasmic (?+ to ?-) mutants decreases in these conditions of liquid holding.

A. Schenberg-Frascino

1972-01-01

227

Dominant lethal testing of theobromine in rats.  

PubMed

Theobromine (TBR) 3,7-dimethylxanthine, the major purine alkaloid present in chocolate and cocoa products, was evaluated for dominant lethality in male Sprague-Dawley rats after administration of subacute oral doses of 0, 50, 150 and 450 mg/kg. Dominant lethal mutations were assessed weekly for a 10-week period by killing the females 13 days after the midweek of presumptive mating with treated males and counting total implants (living and dead), corpora lutea and number of pregnant females. Dead implants per total implants and preimplantation loss per total corpora lutea were evaluated statistically. No significant dose-dependent effects were observed in the % dead implants of preimplantation loss throughout the study; pregnancy rate averaged 94%. These results indicated no induction of dominant lethal mutations or adverse effects on pregnancy rate after TBR doses equivalent to 25-225 times the maximum human consumption level. PMID:6701919

Shively, C A; White, D M; Blauch, J L; Tarka, S M

1984-03-01

228

Lethal and mutagenic effects of ion beams and ?-rays in Aspergillus oryzae.  

PubMed

Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was (12)C(5+) ion beams with an LET of 121keV/?m. The (12)C(5+) ion beams had a 3.6-times higher lethal effect than low-LET (0.2keV/?m) ?-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. (12)C(6+) ion beams with an LET of 86keV/?m were the most effective in inducing selenate resistance. The mutant frequency following exposure to (12)C(6+) ion beams increased with an increase in dose and reached 3.47×10(-3) at 700Gy. In the dose range from 0 to 700Gy, (12)C(5+) ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67×10(-3)) at 400Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between (12)C(5+) ion beams and ?-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions >2bp was generally low, deletions >20bp were characteristic for (12)C(5+) ion beams. ?-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation. PMID:23280012

Toyoshima, Yoshiyuki; Takahashi, Akemi; Tanaka, Hisaki; Watanabe, Jun; Mogi, Yoshinobu; Yamazaki, Tatsuo; Hamada, Ryoko; Iwashita, Kazuhiro; Satoh, Katsuya; Narumi, Issay

2012-12-29

229

Appearance of recessive lethal mutations in derivatives of an unstable X{sup Z} chromosome of Drosophila melanogaster  

SciTech Connect

An X{sup Z} chromosome isolated from a natural population of Drosophila melanogaster is characterized by spontaneous mutability of the genes yellow, white, and singed and the appearance of chromosomal rearrangements. In mutant lines derived from the line carrying the X{sup Z} chromosome that had one, two, or three unstable vision mutations (markers), the rate of appearance of sex-linked lethal mutations was analyzed. This rate was shown to increase with an increase in the number of markers in a line. This phenomenon, termed {open_quotes}marker induction,{close_quotes} might explain the phenotypic homogeneity of natural Drosophila populations. Spontaneous lethal mutations were mapped, and their nonrandom distribution along the X{sup Z} chromosome was shown. Along with common {open_quotes}hot spot{close_quotes} sites of lethal mutations, the derivatives of the X{sup Z} chromosomes had their own specific sites for lethal mutations. In some cases, the appearance of lethal mutations was accompanied by the formation of inversions in the X{sup Z} chromosome. The lethal destabilization of the X{sup Z} derivatives, caused by selection for accumulation of visible mutations, is associated with an increase in the number of hot spots for nuclear mutations. Presumably, these hot spots are hot sites for the transposition of mobile genetic elements. 18 refs., 4 figs., 3 tabs.

Yurchenko, N.N.; Koryakov, D.E.; Zakharov, I.K. [Institute of Cytology and Genetics, Novosibirsk (Russian Federation)

1995-09-01

230

Targeted mutation of Ncam to produce a secreted molecule results in a dominant embryonic lethality.  

PubMed Central

The neural cell adhesion molecule (NCAM) is a membrane-associated member of the immunoglobulin superfamily capable of both homophilic and heterophilic binding. To investigate the significance of this binding, a gene targeting strategy in embryonic stem (ES) cells was used to replace the membrane-associated forms of NCAM with a soluble, secreted form of its extracellular domain. Although the heterozygous mutant ES cells were able to generate low coat color chimeric mice, only the wild-type allele was transmitted, suggesting the possibility of dominant lethality. Analysis of chimeric embryos with high level of ES cell contribution revealed severe growth retardation and morphological defects by E8.5-E9.5. The second allele was also targeted, and embryos derived almost entirely from the homozygous mutant ES cells exhibited the same lethal phenotype as observed with heterozygous chimeras. Together, these results indicate that dominant lethality associated with the secreted NCAM does not require the presence of membrane-associated NCAM. Furthermore, the data indicate that potent bioactive cues or signals can be generated by NCAM. Images Fig. 1 Fig. 2 Fig. 3

Rabinowitz, J E; Rutishauser, U; Magnuson, T

1996-01-01

231

Metformin is synthetically lethal with glucose withdrawal in cancer cells.  

PubMed

Glucose deprivation is a distinctive feature of the tumor microecosystem caused by the imbalance between poor supply and an extraordinarily high consumption rate. The metabolic reprogramming from mitochondrial respiration to aerobic glycolysis in cancer cells (the "Warburg effect") is linked to oncogenic transformation in a manner that frequently implies the inactivation of metabolic checkpoints such as the energy rheostat AMP-activated protein kinase (AMPK). Because the concept of synthetic lethality in oncology can be applied not only to genetic and epigenetic intrinsic differences between normal and cancer cells but also to extrinsic ones such as altered microenvironment, we recently hypothesized that stress-energy mimickers such as the AMPK agonist metformin should produce metabolic synthetic lethality in a glucose-starved cell culture milieu imitating the adverse tumor growth conditions in vivo. Under standard high-glucose conditions, metformin supplementation mostly caused cell cycle arrest without signs of apoptotic cell death. Under glucose withdrawal stress, metformin supplementation circumvented the ability of oncogenes (e.g., HER2) to protect breast cancer cells from glucose-deprivation apoptosis. Significantly, representative cell models of breast cancer heterogeneity underwent massive apoptosis (by >90% in some cases) when glucose-starved cell cultures were supplemented with metformin. Our current findings may uncover crucial issues regarding the cell-autonomous metformin's anti-cancer actions: (1) The offently claimed clinically irrelevant, non-physiological concentrations needed to observe the metformin's anti-cancer effects in vitro merely underlie the artifactual interference of erroneous glucose-rich experimental conditions that poorly reflect glucose-starved in vivo conditions; (2) the preferential killing of cancer stem cells (CSC) by metformin may simply expose the best-case scenario for its synthetically lethal activity because an increased dependency on Warburg-like aerobic glycolysis (hyperglycolytic phenotype) is critical to sustain CSC stemness and immortality; (3) the microenvironment-mediated contextual synthetic lethality of metformin should be expected to enormously potentiate the anti-cancer effect of anti-angiogenesis agents that promote severe oxygen and glucose deprivation in certain areas of the tumor tissues. PMID:22809961

Menendez, Javier A; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Corominas-Faja, Bruna; Joven, Jorge; Martin-Castillo, Begoña; Vazquez-Martin, Alejandro

2012-08-01

232

Phanerochaete mutants with enhanced ligninolytic activity  

SciTech Connect

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.

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

1993-06-01

233

Random mutagenesis of proximal mouse chromosome 5 uncovers predominantly embryonic lethal mutations  

PubMed Central

A region-specific ENU mutagenesis screen was conducted to elucidate the functional content of proximal mouse Chr 5. We used the visibly marked, recessive, lethal inversion Rump White (Rw) as a balancer in a three-generation breeding scheme to identify recessive mutations within the ?50 megabases spanned by Rw. A total of 1003 pedigrees were produced, representing the largest inversion screen performed in mice. Test-class animals, homozygous for the ENU-mutagenized proximal Chr 5 and visibly distinguishable from nonhomozygous littermates, were screened for fertility, hearing, vestibular function, DNA repair, behavior, and dysmorphology. Lethals were identifiable by failure to derive test-class animals within a pedigree. Embryonic lethal mutations (total of 34) were overwhelmingly the largest class of mutants recovered. We characterized them with respect to the time of embryonic death, revealing that most act at midgestation (8.5–10.5) or sooner. To position the mutations within the Rw region and to guide allelism tests, we performed complementation analyses with a set of new and existing chromosomal deletions, as well as standard recombinational mapping on a subset of the mutations. By pooling the data from this and other region-specific mutagenesis projects, we calculate that the mouse genome contains ?3479–4825 embryonic lethal genes, or about 13.7%–19% of all genes.

Wilson, Lawriston; Ching, Yung-Hao; Farias, Michael; Hartford, Suzanne A.; Howell, Gareth; Shao, Hongguang; Bucan, Maja; Schimenti, John C.

2005-01-01

234

Could Antidepressant Combat Lethal Lung Cancer?  

MedlinePLUS

... page, please enable JavaScript. Could Antidepressant Combat Lethal Lung Cancer? Little-used depression drug shows early promise in ... Friday, September 27, 2013 Related MedlinePlus Pages Antidepressants Lung Cancer FRIDAY, Sept. 27 (HealthDay News) -- An older and ...

235

Pharmacology: Screening inhibitors of anthrax lethal factor  

Microsoft Academic Search

The disease anthrax is caused by lethal factor, an enzyme component of the toxin produced by the spore-forming bacterium Bacillus anthracis. Here we describe substrate molecules for this factor that offer a means for high-throughput screening of potential inhibitors for use in anthrax treatment. Our assay should help to answer the urgent call for new and specific therapies to combat

Fiorella Tonello; Michela Seveso; Oriano Marin; Michèle Mock; Cesare Montecucco

2002-01-01

236

Deadly Lessons: Understanding Lethal School Violence.  

ERIC Educational Resources Information Center

This collection of papers is the outcome of the National Academies' effort to glean information from six different case studies of student-perpetrated school shootings. Part 1, "Case Studies of Lethal School Violence," includes: "The Copycat Factor: Mental Illness, Guns, and the Shooting Incident at Heritage High School, Rockdale County, Georgia"…

Moore, Mark H., Ed.; Petrie, Carol V., Ed.; Braga, Anthony A., Ed.; McLaughlin, Brenda L., Ed.

237

Deadly Lessons: Understanding Lethal School Violence.  

ERIC Educational Resources Information Center

|This collection of papers is the outcome of the National Academies' effort to glean information from six different case studies of student-perpetrated school shootings. Part 1, "Case Studies of Lethal School Violence," includes: "The Copycat Factor: Mental Illness, Guns, and the Shooting Incident at Heritage High School, Rockdale County, Georgia"…

Moore, Mark H., Ed.; Petrie, Carol V., Ed.; Braga, Anthony A., Ed.; McLaughlin, Brenda L., Ed.

238

Sarcocystis Species Lethal for Domestic Pigeons  

PubMed Central

A large number of Sarcocystis spp. infect birds as intermediate hosts, but pigeons are rarely affected. We identified a novel Sarcocystis sp. that causes lethal neurologic disease in domestic pigeons in Germany. Experimental infections indicated transmission by northern goshawks, and sequence analyses indicated transnational distribution. Worldwide spread is possible.

Gruber, Achim D.; Kohls, Andrea; Hafez, Hafez M.; Heydorn, Alfred Otto; Mehlhorn, Heinz; Lierz, Michael

2010-01-01

239

The evolution of lethal intergroup violence  

PubMed Central

Recent findings and analyses in evolutionary biology, archaeology, and ethnology provide a favorable conjuncture for examining the evolution of lethal intergroup violence among hominids during the 2.9-million-year Paleolithic time span. Here, I seek to identify and investigate the main turning points in this evolutionary trajectory and to delineate the periodization that follows from this inquiry.

Kelly, Raymond C.

2005-01-01

240

Hypervelocity impact and lethality: Experiment, theory, computation  

SciTech Connect

A study directed at experimental, theoretical and computational issues in hypervelocity impact and lethality phenomena is described. The work is in progress. A series of lead-on-lead impact experiments has provided the focus for testing theoretical and computational capabilities. 9 refs., 6 figs.

Grady, D.E.; Trucano, T.G.; McGlaun, J.M.

1988-01-01

241

Lethal Malaria: Marchiafava and Bignami Were Right  

PubMed Central

One hundred and twenty years ago, the Italian malariologists Marchiafava and Bignami proposed that the fundamental pathological process underlying lethal falciparum malaria was microvascular obstruction. Since then, several alternative hypotheses have been proposed. These formed the basis for adjunctive interventions, which have either been ineffective or harmful. Recent evidence strongly suggests that Marchiafava and Bignami were right.

White, Nicholas J.; Turner, Gareth D. H.; Day, Nicholas P. J.; Dondorp, Arjen M.

2013-01-01

242

A novel RNA helicase gene tightly linked to the Triplo-lethal locus of Drosophila.  

PubMed Central

The Triplo-lethal (Tpl) locus of Drosophila is the only known locus which is lethal when present in three copies rather than the normal two. After recovering a hybrid-dysgenesis-induced mutation of Tpl we used a rapid combination of transposon tagging, chromosome microdissection and PCR to clone the P element that had transposed into the Tpl region. That P element is located within the gene for a new and unique member of the RNA helicase family. This new helicase differs from all others known by having glycine-rich repeats at both the amino and carboxyl termini. Curiously, genetic analysis shows that the P element inserted into this gene is not responsible for the Tpl mutant phenotype. We present possible explanations for these findings. Images

Dorer, D R; Christensen, A C; Johnson, D H

1990-01-01

243

Unexpected double lethal oleander poisoning.  

PubMed

Nerium oleander is a very popular urban ornamental plant in Europe, but it is also extremely dangerous because it contains several types of glycosides, accidental ingestion of which can cause cardiac arrhythmias and even deaths. The rarity of such cases makes it difficult to think of oleander poisoning without evidences that suggest this possibility as the cause of the unexpected death. This report concerns the discovery of the bodies of 2 young people, a man and a woman, in a forest in conditions of extreme malnutrition. Medicolegal investigations showed neither pathologic nor traumatic causes of death, but the presence of vegetal remains in the stomach was noticed. A common toxicological analysis resulted negative, but the implementation of more detailed investigations showed the presence of digoxin in the blood of both cadavers, excluding the possibility of a pharmaceutical provenience of digoxin, this laboratory result was interpreted as evidence of ingestion of oleander, which contains oleandrine, the cross reaction of which with digoxin is widely described in the literature. Identification of the 2 subjects, which occurred after 4 years, strengthened the hypothesis of accidental poisoning by oleander because it was ascertained that the 2 young people were vegans--extreme vegetarians who reject the ingestion of foods of animal origin and live by eating only what they find in nature. PMID:21926903

Papi, Luigi; Luciani, Alessandro Bassi; Forni, David; Giusiani, Mario

2012-03-01

244

Evaluating mutant mice: anatomic pathology.  

PubMed

As the human and mouse genome projects approach their goals, initiatives in functional genomics are advancing. When the nucleotide sequences are available, identification of gene functions will assume even greater importance. Determination of gene products and their proximal biochemical functions provide a part of the picture, but determination of their functions in the context of the whole organism is the ultimate goal. The manipulated mouse genome has become accepted as a model for understanding the genetic basis of human conditions and diseases. Consequently, biomedical research institutions have seen significant increases in the use of mice since the early 1980s, and these increases are largely attributable to the use of genetically modified mice. The role of comparative pathology in research on mutant mouse models of disease is increasing in response to these trends. Evaluation and phenotypic characterization of mutant mice, via clinical and anatomic pathology techniques, will be an important component of functional genomics initiatives. PMID:11199155

Brayton, C; Justice, M; Montgomery, C A

2001-01-01

245

A genetically engineered attenuated coxsackievirus B3 strain protects mice against lethal infection.  

PubMed

Coxsackievirus B3 (CVB3) is a common human pathogen that is endemic throughout the world. There is currently no vaccine available, although the virus is known to be highly lethal to newborns and has been associated with heart disease and pancreatitis in older children and adults. Previously, we showed that the virulence of CVB3 is reduced by a lysine-to-arginine substitution in the capsid protein VP2 (K2168R) or a glutamic acid-to-glycine substitution in VP3 (E3060G). In this report, we show that the double mutant virus CVB3(KR/EG) displays additional attenuation, particularly for the pancreas, in A/J mice. In addition, two other attenuating mutations have been identified in the capsid protein VP1. When either the aspartic acid residue D1155 was replaced with glutamic acid or the proline residue P1126 was replaced with methionine, the resulting mutant also possessed an attenuated phenotype. Moreover, when either of these mutations was incorporated into CVB3(KR/EG), the resulting triple mutant viruses, CVB3(KR/EG/DE) and CVB3(KR/EG/PM), were completely noncardiovirulent and caused only small foci of damage to the pancreas, even at a high dose. Both triple mutants were found to be immunogenic, and a single injection of young A/J mice with either was found to protect them from a subsequent lethal challenge with wild-type CVB3. These findings indicate that the triple mutants could be exploited for the development of a live attenuated vaccine against CVB3. PMID:15994822

Dan, M; Chantler, J K

2005-07-01

246

Carbon monoxide and lethal arrhythmias  

SciTech Connect

The effect of acute exposure to carbon monoxide on ventricular arrhythmias was studied in a previously described chronically maintained animal model of sudden cardiac death. In 60 percent of dogs with a healed anterior myocardial infarction, the combination of mild exercise and acute myocardial ischemia induces ventricular fibrillation. The events in this model are highly reproducible, thus allowing study by internal control analysis. Dogs that develop ventricular fibrillation during the test of exercise and acute myocardial ischemia are considered at high risk for sudden death and are defined as 'susceptible'; dogs that survive the test without a fatal arrhythmia are considered at low risk for sudden death and are defined as 'resistant.' In the current study, the effects of carboxyhemoglobin levels ranging from 5 to 15 percent were tested in resistant and susceptible dogs. A trend toward higher heart rates was observed at all levels of carboxyhemoglobin, although significant differences were observed only with 15 percent carboxyhemoglobin. This trend was observed at rest and during exercise in both resistant and susceptible dogs. In resistant animals, in which acute myocardial ischemia is typically associated with bradycardia even under the control condition, this reflex response occurred earlier and was augmented after exposure to carbon monoxide. This effect may depend on the increased hypoxic challenge caused by carbon monoxide, and thus on an augmentation of the neural reflex activation or a sensitization of the sinus node to acetylcholine induced by hypoxia. In both resistant and susceptible dogs, carbon monoxide exposure induced a worsening of ventricular arrhythmias in a minority of cases. This worsening was not reproducible in subsequent trials. These data indicate that acute exposure to carbon monoxide is seldom arrhythmogenic in dogs that have survived myocardial infarction. (Abstract Truncated)

Farber, J.P.; Schwartz, P.J.; Vanoli, E.; Stramba-Badiale, M.; De Ferrari, G.M. (Univ. of Oklahoma Health Sciences Center, Oklahoma City (USA))

1990-12-01

247

Disruption of the rice plastid ribosomal protein s20 leads to chloroplast developmental defects and seedling lethality.  

PubMed

Plastid ribosomal proteins (PRPs) are essential for ribosome biogenesis, plastid protein biosynthesis, chloroplast differentiation, and early chloroplast development. This study identifies the first rice PRP mutant, asl1 (albino seedling lethality1), which exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered chlorophyll (Chl) content and chloroplast development. Map-based cloning revealed that ASL1 encodes PRP S20 (PRPS20), which localizes to the chloroplast. ASL1 showed tissue-specific expression, as it was highly expressed in plumule and young seedlings but expressed at much lower levels in other tissues. In addition, ASL1 expression was regulated by light. The transcript levels of nuclear genes for Chl biosynthesis and chloroplast development were strongly affected in asl1 mutants; transcripts of some plastid genes for photosynthesis were undetectable. Our findings indicate that nuclear-encoded PRPS20 plays an important role in chloroplast development in rice. PMID:23979931

Gong, Xiaodi; Jiang, Quan; Xu, Jianlong; Zhang, Jianhui; Teng, Sheng; Lin, Dongzhi; Dong, Yanjun

2013-10-03

248

Disruption of the Rice Plastid Ribosomal Protein S20 Leads to Chloroplast Developmental Defects and Seedling Lethality  

PubMed Central

Plastid ribosomal proteins (PRPs) are essential for ribosome biogenesis, plastid protein biosynthesis, chloroplast differentiation, and early chloroplast development. This study identifies the first rice PRP mutant, asl1 (albino seedling lethality1), which exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered chlorophyll (Chl) content and chloroplast development. Map-based cloning revealed that ASL1 encodes PRP S20 (PRPS20), which localizes to the chloroplast. ASL1 showed tissue-specific expression, as it was highly expressed in plumule and young seedlings but expressed at much lower levels in other tissues. In addition, ASL1 expression was regulated by light. The transcript levels of nuclear genes for Chl biosynthesis and chloroplast development were strongly affected in asl1 mutants; transcripts of some plastid genes for photosynthesis were undetectable. Our findings indicate that nuclear-encoded PRPS20 plays an important role in chloroplast development in rice.

Gong, Xiaodi; Jiang, Quan; Xu, Jianlong; Zhang, Jianhui; Teng, Sheng; Lin, Dongzhi; Dong, Yanjun

2013-01-01

249

Lethal Time of Centruroides Sculpturatus Venom in Rats.  

National Technical Information Service (NTIS)

The effects of sex, weight, and dosage on the lethal time of scorpion Centruroides sculpturatus venom was studied. Sex and weight of the rats did not significantly alter lethal time whereas increased dosages of venom did.

H. L. Stahnke

1967-01-01

250

40 CFR 798.5450 - Rodent dominant lethal assay.  

Code of Federal Regulations, 2013 CFR

...ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT (CONTINUED) HEALTH EFFECTS TESTING GUIDELINES Genetic Toxicity § 798.5450 Rodent dominant lethal assay. (a) Purpose. Dominant lethal (DL) effects cause...

2013-07-01

251

Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1  

PubMed Central

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene’s influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT–PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2?, rlf2? and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2? pso4-1 and rlf2? pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.

Revers, L. F.; Cardone, J. M.; Bonatto, D.; Saffi, J.; Grey, M.; Feldmann, H.; Brendel, M.; Henriques, J. A. P.

2002-01-01

252

The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization  

Microsoft Academic Search

A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen

Kathleen L. Pfaff; Christian T. Straub; Ken Chiang; Daniel M. Bear; Yi Zhou; Leonard I. Zon

2007-01-01

253

A Saccharomyces cerevisiae Genome-Wide Mutant Screen for Altered Sensitivity to K1 Killer Toxin  

Microsoft Academic Search

Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall -glucan receptor and subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186

Nicolas Page ´; Manon Gerard-Vincent; Patrice Menard; Maude Beaulieu; Masayuki Azuma; Gerrit J. P. Dijkgraaf; Thuy Nguyen; Tim Dowse; Anne-Marie Sdicu; Howard Bussey

254

Androstenediol regulates systemic resistance against lethal infections in mice  

Microsoft Academic Search

Summary We previously reported that subcutaneous injection of DHEA (5-androsten-3 ß-ol-17-one, dehydroepiandrosterone) protected mice from lethal infection. This included both a lethal herpes virus type 2 encephalitis and a lethal systemic coxsackievirus B 4 (CB 4) infection. Androstenediol (5-andros-ten-3 ß-17 ß-diol, AED), a metabolic product of DHEA is up to 100×more effective in regulating systemic resistance against lethal infection with

R. M. Loria; D. A. Padgett

1992-01-01

255

Lethal pulmonary infection with Francisella novicida is associated with severe sepsis  

PubMed Central

The bacterial or host determinants of lethality associated with respiratory Francisella infections are currently unknown. No exo- or endotoxins that contribute to the severity of this disease have been identified. However, a deregulated host immune response upon infection is characterized by an initial 36- to 48-h delay followed by a rapid and excessive inflammatory response prior to death at 72–120 h. Here, we extend these findings by comparing host immune responses between sublethal and lethal respiratory infections of mice with an attenuated transposon mutant (Mut) of F. novicida (F.n.) strain U112 (sublethal) versus the wild-type (WT) strain (lethal). Infection with WT bacteria, but not the Mut, was characterized by sustained bacteremia and systemic dissemination of the pathogen with temporal increases in bacterial burdens in liver and spleen. Severe pathology with large foci of infiltrates associated with extensive tissue damage was evident in WT-infected lungs, and Mut-infected mice displayed much reduced pathology with intact lung architecture. Similar to other experimental models of severe sepsis, WT- but not the Mut-infected mice exhibited a robust increase in numbers of Gr1+ and CD11b+ cells, while displaying a significant depletion of ?? T cells. Further, a dramatic up-regulation of multiple cytokines and chemokines was observed only in lethal WT infection. In addition, an earlier and larger increased expression of S100A9, a known mediator of sepsis, was observed in WT-infected mice. Taken together, these results show that a hyperinflammatory host immune response, culminating in severe sepsis, is responsible for the lethal outcome of respiratory tularemia.

Sharma, Jyotika; Li, Qun; Mishra, Bibhuti B.; Pena, Christopher; Teale, Judy M.

2009-01-01

256

Sex-lethal Facilitates the Transition From Germline Stem Cell to Committed Daughter Cell in the Drosophila Ovary  

Microsoft Academic Search

In Drosophila, the female-specific SEX-LETHAL (SXL) protein is required for oogenesis, but how Sxl interfaceswiththegeneticcircuitrycontrollingoogenesisremainsunknown.Hereweuseanalleleofsansfille (snf) that specifically eliminates SXL protein in germ cells to carry out a detailed genetic and cell biological analysis of the resulting ovarian tumor phenotype.Wefindthattumor growth requiresbothCyclinBandzero population growth, demonstrating that these mutant cells retain at least some of the essential growth-control mechanisms used by

Johnnie Chau; Laura Shapiro Kulnane; Helen K. Salz

2009-01-01

257

Small Arms Mortality: Access to Firearms and Lethal Violence  

Microsoft Academic Search

There is an intuitive appeal to the notion that the more lethal the weaponry the more lethal the violence. We explore one aspect of lethal weaponry, firearm accessibility. Using nation-level (N = 168) data from the Small Arms Survey and the World Health Organization's measures of mortality we examine whether rates of small arm ownership have a positive effect on rates of

Mark Konty; Brian Schaefer

2012-01-01

258

Induction of dominant lethal mutations in male mice by fosfestrol  

Microsoft Academic Search

Using the dominant lethal assay, the ability of fosfestrol, a diethylstilboestrol derivate, to induce mutations in male mice was tested and confirmed. Up to 300 mg\\/kg of fosfestrol the induction of mutations occurs exclusively in spermatozoa. A dose of 600 mg\\/kg of fosfestrol induces dominant lethal mutations up to 10 days posttreatment. The majority of induced dominant lethal mutations in

U. H. Ehling; Ingolstiidter Landstrasse

1978-01-01

259

Mutant yeast on drugs  

Microsoft Academic Search

Analyzing drug-treated and mutant yeast cells with the new tools of genomics enables the identification of drug targets and should improve the odds of developing useful therapeutics (pages 1293–1301).

David J. Lockhart

1998-01-01

260

Generation and characterization of a conditional deletion allele for Lmna in mice.  

PubMed

Extensive efforts have been devoted to study A-type lamins because mutations in their gene, LMNA in humans, are associated with a number of diseases. The mouse germline mutations in the A-type lamins (encoded by Lmna) exhibit postnatal lethality at either 4-8 postnatal (P) weeks or P16-18days, depending on the deletion alleles. These mice exhibit defects in several tissues including hearts and skeletal muscles. Despite numerous studies, how the germline mutation of Lmna, which is expressed in many postnatal tissues, affects only selected tissues remains poorly understood. Addressing the tissue specific functions of Lmna requires the generation and careful characterization of conditional Lmna null alleles. Here we report the creation of a conditional Lmna knockout allele in mice by introducing loxP sites flanking the second exon of Lmna. The Lmna(flox/flox) mice are phenotypically normal and fertile. We show that Lmna homozygous mutants (Lmna(?/?)) generated by germline Cre expression display postnatal lethality at P16-18days with defects similar to a recently reported germline Lmna knockout mouse that exhibits the earliest lethality compared to other germline knockout alleles. This conditional knockout mouse strain should serve as an important genetic tool to study the tissue specific roles of Lmna, which would contribute toward the understanding of various human diseases associated with A-type lamins. PMID:23998933

Kim, Youngjo; Zheng, Yixian

2013-08-30

261

Plant hormone mutants  

Microsoft Academic Search

The techniques used for the production and identification of plant hormone mutants are described. The properties used to classify\\u000a these mutants into the broad synthesis and response categories are discussed, and the genetic considerations needed to allow\\u000a their effective use in plant hormone research examined. A brief outline of significant work on gibberellin (GA), abscisic\\u000a acid (ABA), auxin, ethylene, cytokinin

James B. Reid

1993-01-01

262

Lethality in Desbuquois dysplasia: three new cases  

Microsoft Academic Search

Three new cases of Desbuquois syndrome in two brothers and a sporadic male, all of whom died in early infancy, are presented\\u000a to emphasize the high rate (33 %) of lethality in this variable, but serious skeletal dysplasia. Including the three presented\\u000a patients and 10 of the 36 cases in the literature who died, most did so between birth and

Bryan D. Hall

2001-01-01

263

Bullying and Lethal Acts of School Violence  

Microsoft Academic Search

\\u000a In Chap. 3, we address a topic that has received considerable research attention over the past decade and has been implicated as a causal\\u000a factor in LSV. It has been reported that many lethal school shootings were in part in retaliation for being bullied. Within\\u000a this chapter, we address prevalence of bullying in the USA and then discuss different forms

Jeffrey A. Daniels; Mary C. Bradley

264

Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-toxin.  

PubMed

Clostridium sordellii lethal toxin and Clostridium novyi ?-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (?-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP(6)) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP(6) (<1 ?M), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi ?-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and ?-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of ?-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and ?-toxin, autocatalytic cleavage and InsP(6) binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi ?-toxin are InsP(6)-dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP(6) and subsequent processing of the toxin. PMID:21385871

Guttenberg, Gregor; Papatheodorou, Panagiotis; Genisyuerek, Selda; Lü, Wei; Jank, Thomas; Einsle, Oliver; Aktories, Klaus

2011-03-08

265

Variegation mutants and mechanisms of chloroplast biogenesis.  

PubMed

Variegated plants typically have green- and white-sectored leaves. Cells in the green sectors contain normal-appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development. PMID:17263779

Yu, Fei; Fu, Aigen; Aluru, Maneesha; Park, Sungsoon; Xu, Yang; Liu, Huiying; Liu, Xiayan; Foudree, Andrew; Nambogga, Milly; Rodermel, Steven

2007-03-01

266

Uncoupler-resistant mutants of bacteria.  

PubMed Central

The chemiosmotic model of energy transduction offers a satisfying and widely confirmed understanding of the action of uncouplers on such processes as oxidative phosphorylation; the uncoupler, by facilitating the transmembrane movement of protons or other compensatory ions, reduces the electrochemical proton gradient that is posited as the energy intermediate for many kinds of bioenergetic work. In connection with this formulation, uncoupler-resistant mutants of bacteria that neither exclude nor inactivate these agents represent a bioenergetic puzzle. Uncoupler-resistant mutants of aerobic Bacillus species are, in fact, membrane lipid mutants with bioenergetic properties that are indeed challenging in connection with the chemiosmotic model. By contrast, uncoupler-resistant mutants of Escherichia coli probably exclude uncouplers, sometimes only under rather specific conditions. Related phenomena in eucaryotic and procaryotic systems, as well as various observations on uncouplers, decouplers, and certain other membrane-active agents, are also briefly considered. Images

Krulwich, T A; Quirk, P G; Guffanti, A A

1990-01-01

267

Tissue-specific Expression of Dominant Negative Mutant Drosophila HSC70 Causes Developmental Defects and Lethality  

Microsoft Academic Search

The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding\\/hydrolysis activi- ties that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and

Felice Elefant; Karen B. Palter

1999-01-01

268

Temperature-Sensitive Lethal Pseudorevertants of ste Mutations in Saccharomyces cerevisiae  

PubMed Central

A procedure was devised to isolate mutations that could restore conjugational competence to temperature sensitive ste mutants and simultaneously confer temperature-sensitive lethal growth phenotypes. Three such mutations, falling into two complementation groups, were identified on the basis of suppression of ste5 alleles. These same mutations were later shown to be capable of suppressing ste4 and ste7 alleles. Five mutations in a single complementation group were isolated as suppressors of ste2 alleles. None of the mutations described in this study conferred a homogeneous cell cycle arrest phenotype, and all were shown to define complementation groups distinct from those previously identified in studies of cell division cycle (cdc) mutations. In no instance did pseudoreversion appear to be achieved by mutational G1 arrest of ste mutant cells. Instead, it is proposed that the mutations restore conjugation by reestablishing the normal pheromone response.

Katz, Margaret E.; Ferguson, Jill; Reed, Steven I.

1987-01-01

269

Detection of low numbers of healthy and sub-lethally injured Salmonella enterica in chocolate.  

PubMed

The capacity to detect low levels of healthy and sub-lethally injured Salmonella enterica cells in chocolate by two alternative rapid detection methods iQ-Check(TM)Salmonella II real-time PCR (Bio-Rad) and VIDAS® Easy SLM (BioMérieux) was assessed and compared with ISO 6579:2005. Chocolate, a low moisture food known to support the survival of Salmonella, was challenged as food matrix. Buffered peptone water (BPW) did not support the recovery of low levels of sub-lethally injured S. enterica independent of the detection method, while BPW supplemented with milk powder enabled detection by the three examined methods. However, inhibition of real-time PCR was observed since for one out of three repetitions of chocolate inoculated with a low number of sub-lethally injured S. enterica cells, no PCR signal was obtained. Therefore, attention should be paid to the enrichment step to avoid false negative results due to the presence of especially sub-lethally injured Salmonella cells in chocolate. An appropriate sample preparation (such as enrichment media and conditions for incubation) remains the key factor for reliable detection including sub-lethally injured cells and should be evaluated, if necessary optimized, for each detection assay. PMID:21320732

Jasson, Vicky; Baert, Leen; Uyttendaele, Mieke

2011-01-31

270

Importance of repair of potentially lethal damage in assays of cytotoxic agents  

SciTech Connect

Most in vitro assays expose growing cells to cytotoxic agents for a fixed period of time. However, these assays fail to account for repair of potentially lethal damage, the enhancement in survival that occurs when cells are maintained under nongrowth conditions after treatment with cytotoxic agents. This study extends previous observations of the repair of potentially lethal damage in human melanoma cells to parallel in vitro and in vivo experiments with the F10 subline of B16 melanoma. An in vivo (in C57BL/6 mice) and in vitro (in plastic plates) radiation dose-response relationship for the F10 subline of B16 melanoma was determined (200 to 1100 rads). Time delay until explant, allowing repair of potentially lethal damage, resulted in a significant and progressive enhancement of survival, five- to sixfold, both in vivo and in vitro. Survival with 700 rads and 24-hour delay was equivalent to that after treatment with 300 rads and no delay. Repair of potentially lethal damage, demonstrated in human cells in vitro and in animal preparations in vitro and in vivo, may account for the lack of clinical efficacy of some cytotoxic agents. Our results suggest that repair of potentially lethal damage should be taken into consideration in the design of in vitro chemotherapy and radiation therapy assays.

Richie, J.P.; Weichselbaum, R.R.; Little, J.B.

1982-08-01

271

Characterization of Lethal Zygosis Associated with Conjugation in Escherichia coli K-12  

PubMed Central

When F? cells are mixed with an excess of Hfr cells there is a lethal event which results in a decrease in the number of F? survivors. We have described and discussed the parameters affecting this phenomenon of lethal zygosis, and these include the cultural conditions of both donor and recipient cells prior to mixing and the use of aeration throughout the period of the experiment. The absence of lethal zygosis with filtrates and supernatant fluids from donors suggests a dependence on direct cell-cell contact as found in conjugation. The phenomenon, which is normally observed in liquid media, also occurs on solid media, and use of these two methods has allowed examination of strains of different mating types. Whereas most Hfr strains capable of producing normal yields of recombinants showed killing activity, no F+ and only one F? donor produced lethal zygosis. Only F? strains were sensitive to this phenomenon. The relationship between lethal zygosis and the various stages of conjugation is discussed. Images

Skurray, Ronald A.; Reeves, Peter

1973-01-01

272

Lethal infection thresholds of Paenibacillus larvae for honeybee drone and worker larvae (Apis mellifera).  

PubMed

We compared the mortality of honeybee (Apis mellifera) drone and worker larvae from a single queen under controlled in vitro conditions following infection with Paenibacillus larvae, a bacterium causing the brood disease American Foulbrood (AFB). We also determined absolute P. larvae cell numbers and lethal titres in deceased individuals of both sexes up to 8 days post infection using quantitative real-time PCR (qPCR). Our results show that in drones the onset of infection induced mortality is delayed by 1 day, the cumulative mortality is reduced by 10% and P. larvae cell numbers are higher than in worker larvae. Since differences in bacterial cell titres between sexes can be explained by differences in body size, larval size appears to be a key parameter for a lethal threshold in AFB tolerance. Both means and variances for lethal thresholds are similar for drone and worker larvae suggesting that drone resistance phenotypes resemble those of related workers. PMID:20545737

Behrens, Dieter; Forsgren, Eva; Fries, Ingemar; Moritz, Robin F A

2010-07-07

273

Combination of opium smoking and hypercholesterolemia augments susceptibility for lethal cardiac arrhythmia and atherogenesis in rabbit.  

PubMed

Opium consumption is increasing in some eastern societies, where it is grown. We investigated the effect of opium smoking on plasma atherogenic index and incidence of lethal cardiac arrhythmia, i.e. ventricular tachycardia (VT) and ventricular fibrillation (VF) in rabbits. Animals were divided into two-, normo- and hyper-cholesterolemic main groups fed with normal or high cholesterol diet prior and during short-term and long-term exposure to opium smoke. Then, isoproterenol (3mg/kg, i.p.) was injected to induce cardiac ischemia and animals were followed for 3h for counting of lethal arrhythmia incidence. Long-term opium smoking significantly increased the plasma atherogenic index. In ischemic hearts, opium smoking along with hypercholesterolemia significantly enhanced the incidence of fatal arrhythmia. This vulnerability was not mediated by changes in QT interval. These data suggest that opium smoking, especially in hypercholesterolemic conditions, can be a predisposing factor for atherogenesis and lethal arrhythmia. PMID:22522425

Najafipour, Hamid; Joukar, Siyavash

2012-03-28

274

Identification of new dominant-negative mutants of anthrax protective antigen using directed evolution.  

PubMed

The anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema toxin (EF). The PA moiety carries EF and LF into the cytosol of mammalian cells via a mechanism that depends on the oligomerization of PA and transmembrane pore formation by the PA oligomer. Certain mutants of PA, termed dominant-negative (DN) mutants, can co-oligomerize with wild-type PA and disrupt the translocation ability of the pore. Here, we constructed a PA mutant library by introducing random mutations into domain II of PA and screened three new DN mutants of PA: V377E, T380S, and I432C. All the mutants inhibited the anthrax toxin action against sensitive cells. V377E had the strongest inhibitory effect and was further confirmed to be able to protect mice against a challenge with anthrax lethal toxin. Furthermore, we functionally characterized these mutants. The result showed that these mutations did not impair proteolytic activation or oligomer formation of PA, but impeded the prepore-pore conversion of the oligomer. These DN mutants of PA identified in our study may provide valuable information for elucidating the structure-function relationship of PA and for designing therapeutics for anthrax treatment. PMID:22948605

Wu, Gaobing; Feng, Chunfang; Cao, Sha; Guo, Aizhen; Liu, Ziduo

2012-09-05

275

Deciphering the Mechanisms of Developmental Disorders (DMDD): a new programme for phenotyping embryonic lethal mice  

PubMed Central

Summary International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The ‘Deciphering the Mechanisms of Developmental Disorders’ (DMDD) programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013)] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes.

Mohun, Timothy; Adams, David J.; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J.; Hemberger, Myriam; Houart, Corinne; Hurles, Matt E.; Robertson, Elizabeth; Smith, James C.; Weaver, Tom; Weninger, Wolfgang

2013-01-01

276

Reduced LPS phosphorylation in Escherichia coli lowers the elevated ori/ter ratio in seqA mutants  

PubMed Central

Summary The seqA defect in E. coli increases the ori/ter ratio and causes chromosomal fragmentation, making seqA mutants dependent on recombinational repair (the seqA recA co-lethality). To understand the nature of this chromosomal fragmentation, we characterized ?seqA mutants and isolated suppressors of the ?seqA recA lethality. We demonstrate that our ?seqA alleles have normal function of the downstream pgm gene and normal ratios of the major phospholipids in the membranes, but increased surface lipopolysaccharide (LPS) phosphorylation. The predominant class of ?seqA recA suppressors disrupts the rfaQGP genes, reducing phosphorylation of the inner core region of LPS. The rfaQGP suppressors also reduce the elevated ori/ter ratio of the ?seqA mutants, but, unexpectedly, the suppressed mutants still exhibit the high levels of chromosomal fragmentation and SOS induction, characteristic of the ?seqA mutants. We also found that co-lethality of rfaP with defects in the production of acidic phospholipids is suppressed by alternative initiation of chromosomal replication, suggesting that LPS phosphorylation stimulates replication initiation. The rfaQGP suppression of the seqA recA lethality provides genetic support for the surprising physical evidence that the oriC DNA forms complexes with the outer membrane.

Rotman, Ella; Bratcher, Preston; Kuzminov, Andrei

2009-01-01

277

Genome-Wide P-Element Screen for Drosophila Synaptogenesis Mutants  

PubMed Central

A molecular understanding of synaptogenesis is a critical step toward the goal of understanding how brains “wire themselves up,” and then “rewire” during development and experience. Recent genomic and molecular advances have made it possible to study synaptogenesis on a genomic scale. Here, we describe the results of a screen for genes involved in formation and development of the glutamatergic Drosophila neuromuscular junction (NMJ). We screened 2185 P-element transposon mutants representing insertions in ?16% of the entire Drosophila genome. We first identified recessive lethal mutants, based on the hypothesis that mutations causing severe disruptions in synaptogenesis are likely to be lethal. Two hundred twenty (10%) of all insertions were homozygous lethal. Two hundred five (93%) of these lethal mutants developed at least through late embryogenesis and formed neuromusculature. We examined embryonic/larval NMJs in 202 of these homozygous mutants using immunocytochemistry and confocal microscopy. We identified and classified 88 mutants with altered NMJ morphology. Insertion loci in these mutants encode several different types of proteins, including ATP- and GTPases, cytoskeletal regulators, cell adhesion molecules, kinases, phosphatases, RNA regulators, regulators of protein formation, transcription factors, and transporters. Thirteen percent of insertions are in genes that encode proteins of novel or unknown function. Complementation tests and RT-PCR assays suggest that approximately 51% of the insertion lines carry background mutations. Our results reveal that synaptogenesis requires the coordinated action of many different types of proteins—perhaps as much as 44% of the entire genome—and that transposon mutageneses carry important caveats that must be respected when interpreting results generated using this method.

Liebl, Faith L.W.; Werner, Kristen M.; Sheng, Qi; Karr, Julie E.; McCabe, Brian D.; Featherstone, David E.

2006-01-01

278

Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation?  

PubMed Central

Synthetic lethality is inviability of a double-mutant combination of two fully viable single mutants, commonly interpreted as redundancy at an essential metabolic step. The dut-1 defect in Escherichia coli inactivates dUTPase, causing increased uracil incorporation in DNA and known synthetic lethalities [SL(dut) mutations]. According to the redundancy logic, most of these SL(dut) mutations should affect nucleotide metabolism. After a systematic search for SL(dut) mutants, we did identify a single defect in the DNA precursor metabolism, inactivating thymidine kinase (tdk), that confirmed the redundancy explanation of synthetic lethality. However, we found that the bulk of mutations interacting genetically with dut are in DNA repair, revealing layers of damage of increasing complexity that uracil-DNA incorporation sends through the chromosomal metabolism. Thus, we isolated mutants in functions involved in (i) uracil-DNA excision (ung, polA, and xthA); (ii) double-strand DNA break repair (recA, recBC, and ruvABC); and (iii) chromosomal-dimer resolution (xerC, xerD, and ftsK). These mutants in various DNA repair transactions cannot be redundant with dUTPase and instead reveal “defect-damage-repair” cycles linking unrelated metabolic pathways. In addition, two SL(dut) inserts (phoU and degP) identify functions that could act to support the weakened activity of the Dut-1 mutant enzyme, suggesting the “compensation” explanation for this synthetic lethality. We conclude that genetic interactions with dut can be explained by redundancy, by defect-damage-repair cycles, or as compensation.

Ting, Helen; Kouzminova, Elena A.; Kuzminov, Andrei

2008-01-01

279

Dominant lethal mutagenicity study on hair dyes  

Microsoft Academic Search

A dominant lethal mutagenicity study was performed in rats with the following chemicals that may be used to dye hair: 2?nitro?p?phenylenediamine, 4?nitro?o?phenylenediamine, m?phenylenediamine, o?phenylenediamine, p?phenylenediamine, p?toluenediamine, 2,4?diaminoanisole, 2,5?diaminoanlsole, 2?amino?4?nitrophenol, 2?amino?5?nitrophenol, and 4?amino?2?nitrophenol. The compounds were administered intraperitoneally three times weekly for 8 weeks to groups of 20 sexually mature Charles River CD male rats at a dose of 20 mg\\/kg.

C. Burnett; R. Loehr; J. Corbett

1977-01-01

280

Gdnf Haploinsufficiency Causes Hirschsprung-Like Intestinal Obstruction and Early-Onset Lethality in Mice  

PubMed Central

Hirschsprung disease (HSCR) is a common congenital disorder that results in intestinal obstruction and lethality, as a result of defective innervation of the gastrointestinal (GI) tract. Despite its congenital origin, the molecular etiology of HSCR remains elusive for >70% of patients. Although mutations in the c-RET receptor gene are frequently detected in patients with HSCR, mutations in the gene encoding its ligand (glial cell line–derived neurotrophic factor [GDNF]), are rarely found. In an effort to establish a possible link between human HSCR and mutations affecting the Gdnf locus, we studied a large population of mice heterozygous for a Gdnf null mutation. This Gdnf+/? mutant cohort recapitulates complex features characteristic of HSCR, including dominant inheritance, incomplete penetrance, and variable severity of symptoms. The lack of one functioning Gdnf allele causes a spectrum of defects in gastrointestinal motility and predisposes the mutant mice to HSCR-like phenotypes. As many as one in five Gdnf+/? mutant mice die shortly after birth. Using a transgenic marking strategy, we identified hypoganglionosis of the gastrointestinal tract as a developmental defect that renders the mutant mice susceptible to clinical symptoms of HSCR. Our findings offer a plausible way to link an array of seemingly disparate features characteristic of a complex disease to a much more narrowly defined genetic cause. These findings may have general implications for the genetic analysis of cause and effect in complex human diseases.

Shen, Liya; Pichel, Jose G.; Mayeli, Thomas; Sariola, Hannu; Lu, Bai; Westphal, Heiner

2002-01-01

281

Lethal Mutagenesis of Foot-and-Mouth Disease Virus Involves Shifts in Sequence Space?†  

PubMed Central

Lethal mutagenesis or virus transition into error catastrophe is an antiviral strategy that aims at extinguishing a virus by increasing the viral mutation rates during replication. The molecular basis of lethal mutagenesis is largely unknown. Previous studies showed that a critical substitution in the foot-and-mouth disease virus (FMDV) polymerase was sufficient to allow the virus to escape extinction through modulation of the transition types induced by the purine nucleoside analogue ribavirin. This substitution was not detected in mutant spectra of FMDV populations that had not replicated in the presence of ribavirin, using standard molecular cloning and nucleotide sequencing. Here we selectively amplify and analyze low-melting-temperature cDNA duplexes copied from FMDV genome populations passaged in the absence or presence of ribovirin Hypermutated genomes with high frequencies of A and U were present in both ribavirin -treated and untreated populations, but the major effect of ribavirin mutagenesis was to accelerate the occurrence of AU-rich mutant clouds during the early replication rounds of the virus. The standard FMDV quasispecies passaged in the absence of ribavirin included the salient transition-modulating, ribavirin resistance mutation, whose frequency increased in populations treated with ribavirin. Thus, even nonmutagenized FMDV quasispecies include a deep, mutationally biased portion of sequence space, in support of the view that the virus replicates close to the error threshold for maintenance of genetic information.

Perales, Celia; Henry, Michel; Domingo, Esteban; Wain-Hobson, Simon; Vartanian, Jean-Pierre

2011-01-01

282

Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations  

PubMed Central

By taking advantage of a lethal phenotype characteristic of Caenorhabditis elegans embryos that fail to move, we have identified 13 genes required for muscle assembly and function and discovered a new lethal class of alleles for three previously known muscle-affecting genes. By staining mutant embryos for myosin and actin we have recognized five distinct classes of genes: mutations in four genes disrupt the assembly of thick and thin filaments into the myofilament lattice as well as the polarized location of these components to the sarcolemma. Mutations in another three genes also disrupt thick and thin filament assembly, but allow proper polarization of lattice components based on the myosin heavy chain isoform that we analyzed. Another two classes of genes are defined by mutations with principal effects on thick or thin filament assembly into the lattice, but not both. The final class includes three genes in which mutations cause relatively minor defects in lattice assembly. Failure of certain mutants to stain with antibodies to specific muscle cell antigens suggest that two genes associated with severe disruptions of myofilament lattice assembly may code for components of the basement membrane and the sarcolemma that are concentrated where dense bodies (Z- line analogs) and M-lines attach to the cell membrane. Similar evidence suggests that one of the genes associated with mild effects on lattice assembly may code for tropomyosin. Many of the newly identified genes are likely to play critical roles in muscle development and function.

1994-01-01

283

Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function  

PubMed Central

The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.

Langer, Marybeth; Duggan, Elizabeth Stewart; Booth, John Leland; Patel, Vineet Indrajit; Zander, Ryan A.; Silasi-Mansat, Robert; Ramani, Vijay; Veres, Tibor Zoltan; Prenzler, Frauke; Sewald, Katherina; Williams, Daniel M.; Coggeshall, Kenneth Mark; Awasthi, Shanjana; Lupu, Florea; Burian, Dennis; Ballard, Jimmy Dale; Braun, Armin

2012-01-01

284

Issues surrounding lethal injection as a means of capital punishment.  

PubMed

Lethal injection as a method of state-sanctioned capital punishment was initially proposed in the United States in 1977 and used for the first time in 1982. Most lethal injection protocols use a sequential drug combination of sodium thiopental, pancuronium bromide, and potassium chloride. Lethal injection was originally introduced as a more humane form of execution compared with existing mechanical methods such as electrocution, toxic gassing, hanging, or firing squad. Lethal injection has not, however, been without controversy. Several states are considering whether lethal injection meets constitutional scrutiny forbidding cruel and unusual punishment. Recently in the case of Ralph Baze and Thomas C. Bowling, Petitioners, v John D. Rees, Commissioner, Kentucky Department of Corrections et al, the United States Supreme Court upheld the constitutionality of the lethal injection protocol as carried out in the Commonwealth of Kentucky. Most of the debate has surrounded the dosing and procedures used in lethal injection and whether the drug combinations and measures for administering the drugs truly produce a timely, pain-free, and fail-safe death. Many have also raised issues regarding the "medicalization" of execution and the ethics of health care professionals' participation in any part of the lethal injection process. As a result of all these issues, the future of lethal injection as a means of execution in the United States is under significant scrutiny. Outcomes of ongoing legislative and judicial reviews might result in cessation of lethal injection in totality or in alterations involving specific drug combinations or administration procedures. PMID:19025423

Romanelli, Frank; Whisman, Tyler; Fink, Joseph L

2008-12-01

285

Analysis of the Albino-Locus Region of the Mouse. III. Time of Death of Prenatal Lethals  

PubMed Central

The stage at which homozygotes die was determined for 28 mutations (general symbol c*) at the albino (c) locus, of which 26 had earlier been found to be probably prenatally lethal. Within each of the mutant stocks, the uterine contents of c*/cch females, made pregnant either by c*/cch males ("Ex" series) or by cch/cch males ("Co" series), were examined between 13 and 17 days postconception. Altogether, 743 females were dissected and 7197 corpora lutea (representing ovulations) counted. In selected stocks, an additional 40 and 13 females were dissected on days seven or nine, respectively.—In each of the 26 presumed prenatally lethal mutants, there was a deficiency of living fetuses in the Ex, as compared with the Co, group. Overall, this deficiency was 23.6% (expectation, 25% c*/c*). All meaningful excesses were in numbers either of moles (death shortly before, during, or just after implantation), or of early preimplantation losses. Homozygotes in none of the mutant stocks die between days nine and 19 postconception. Of 24 c-locus mutants known to be deficiencies since they lack the closely linked Mod-2, 13 clearly kill before implantation, ten around implantation, and one neonatally. The c and Mod-2 loci and the region between them are not needed for intrauterine survival.—There are indications that the distinction between early-preimplantation death and implantation death may, in a general way, be related to length of the deficiency.

Russell, Liane B.; Raymer, G. D.

1979-01-01

286

Suicide Intent and Accurate Expectations of Lethality: Predictors of Medical Lethality of Suicide Attempts  

ERIC Educational Resources Information Center

|The degree of intent to commit suicide and the severity of self-injury were examined in individuals (N = 180) who had recently attempted suicide. Although a minimal association was found between the degree of suicide intent and the degree of lethality of the attempt, the accuracy of expectations about the likelihood of dying was found to moderate…

Brown, Gregory K.; Henriques, Gregg R.; Sosdjan, Daniella; Beck, Aaron T.

2004-01-01

287

Gonadosomatic mosaicism for lethal mutations in Drosophila lethal mutations disturbing larval development  

SciTech Connect

Phenogenetic analysis of autonomous lethal mutations obtained by the method of gonadosomatic mosaicism which manifested during larval stages, established that the nuclei of hypodermal cells, salivary glands suprapharyngeal ganglion, pharynx, esophagus, gizzard, and hindgut are the derivatives of the same nucleus (from the first two nuclei of cleavage) as the nuclei of the cells of the imaginal-somatic tissues.

Ivanov, A.I.; Sakharova, N.Yu.

1988-11-01

288

Role of macrophage oxidative burst in the action of anthrax lethal toxin.  

PubMed Central

BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.

Hanna, P. C.; Kruskal, B. A.; Ezekowitz, R. A.; Bloom, B. R.; Collier, R. J.

1994-01-01

289

A Reduction of Tropomyosin Limits Development of Sarcomeric Structures in Cardiac Mutant Hearts of the Mexican Axolotl  

Microsoft Academic Search

The cardiac lethal mutation in Mexican axolotl (Ambystoma mexicanum) results in a lack of contractions in the ventricle of mutant embryos. Previous studies have demonstrated that tropomyosin,\\u000a a component of thin filaments, is greatly reduced in mutant hearts lacking myofibril organization. Confocal microscopy was\\u000a used to examine the structure and comparative amount of tropomyosin at heartbeat initiation and at a

Robert W. Zajdel; Harold Thurston; Sastry Prayaga; Syamalima Dube; Bernard J. Poiesz; Dipak K. Dube

2007-01-01

290

The deoxyhypusine synthase mutant dys1-1 reveals the association of eIF5A and Asc1 with cell wall integrity.  

PubMed

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1? and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1? strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1? mutants in comparison with the pkc1? mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. PMID:23573236

Galvão, Fabio Carrilho; Rossi, Danuza; Silveira, Wagner da Silva; Valentini, Sandro Roberto; Zanelli, Cleslei Fernando

2013-04-01

291

Virulence, persistence, and immunogenicity of Yersinia enterocolitica O:8 aroA mutants.  

PubMed Central

The virulent Yersinia enterocolitica strain 8081 killed BALB/c mice within 5 days of oral infection with a 50% lethal dose of log10 7.1, whereas an aroA mutant of 8081, YAM.1, and the plasmidless variant 8081c failed to kill mice. Unlike 8081, YAM.1 and 8081c did not persist or grow in the Peyer's patches, mesenteric lymph nodes, livers, or spleens of mice. Mice immunized orally with single doses of live YAM.1 were poorly protected against a lethal 8081 challenge, whereas mice immunized with three doses of YAM.1 were moderately well protected.

Bowe, F; O'Gaora, P; Maskell, D; Cafferkey, M; Dougan, G

1989-01-01

292

Riot Control Agents, Tasers, and Other Less Lethal Weapons  

Microsoft Academic Search

Less lethal weapons have become increasingly popular for law enforcement use when confronting dangerous, combative individuals\\u000a in the field. On the use-of-force continuum, these technologies occupy an intermediate level between verbal and physical control\\u000a methods and lethal force such as actual firearms. Less lethal weapons include riot control agents, electric stun devices such\\u000a as tasers, and other blunt projectile weapons.

Christian Sloane; Gary M. Vilke

293

The lethal giant larvae tumour suppressor mutation requires dMyc oncoprotein to promote clonal malignancy  

PubMed Central

Background Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues. Results We observed that lgl mutant cells growing in wild-type imaginal wing discs show poor viability and are eliminated by Jun N-terminal Kinase (JNK)-dependent cell death. Furthermore, they express very low levels of dMyc oncoprotein compared with those found in the surrounding normal tissue. Evidence that this is a cause of lgl mutant cells elimination was obtained by increasing dMyc levels in lgl mutant clones: their overgrowth potential was indeed re-established, with mutant cells overwhelming the neighbouring tissue and forming tumourous masses displaying several cancer hallmarks. Moreover, when lgl mutant clones were induced in backgrounds of slow-dividing cells, they upregulated dMyc, lost apical-basal cell polarity and were able to overgrow. Those phenotypes were abolished by reducing dMyc levels in the mutant clones, thereby confirming its key role in lgl-induced tumourigenesis. Furthermore, we show that the eiger-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating lgl mutant cells in the wing pouch; lgl-/- clonal death in this region is instead driven mainly by dMyc-induced Cell Competition. Conclusions Our results provide the first evidence that dMyc oncoprotein is required in lgl tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside versus outside the mutant clones plays a key role in driving neoplastic overgrowth.

2010-01-01

294

Growth and development of maize that contains mutant tubulin genes  

SciTech Connect

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.

Susan M. Wick

2004-07-23

295

Physiological characterization of 'stay green' mutants in durum wheat.  

PubMed

Four mutants with delayed leaf senescence were selected from seed of durum wheat mutagenized with ethylmethane sulphonate. Changes in net photosynthetic rate, efficiency of photosystem II and chlorophyll concentration during the maturation and senescence of the flag leaves of both mutant and parental plants were determined under glasshouse conditions. The four mutant lines maintained photosynthetic competence for longer than the parental line and are therefore functionally 'stay green'. The mutant lines also had higher seed weights and grain yields per plant than the parental line. PMID:12709488

Spano, G; Di Fonzo, N; Perrotta, C; Platani, C; Ronga, G; Lawlor, D W; Napier, J A; Shewry, P R

2003-05-01

296

A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae.  

PubMed Central

Saccharomyces cerevisiae whi2Delta cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Delta. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Delta, siw14Delta, fen2Delta, and thr4Delta mutants. Furthermore, whi2Delta and siw14Delta mutants produce large actin clumps in stationary phase similar to those seen in prk1Delta ark1Delta mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Delta strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Delta, sla1Delta, sla2Delta, vrp1Delta, ypt51Delta, ypt52Delta, and end3Delta mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Delta mutants.

Care, Alison; Vousden, Katherine A; Binley, Katie M; Radcliffe, Pippa; Trevethick, Janet; Mannazzu, Ilaria; Sudbery, Peter E

2004-01-01

297

Replacement of Lipopolysaccharide with Free Lipid A Molecules in Escherichia coli Mutants Lacking All Core Sugars  

PubMed Central

Escherichia coli mutants deficient in 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by over-expression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetra-acylated precursor lipid IVA replacing lipopolysaccharide (Meredith, T. C. et al. ACS Chem. Biol. 1, 33–42, 2006). Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IVA reaches the outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexa-acylated lipid A, we over-expressed the lauroyl or the myristoyl transferase of lipid A biosynthesis, encoded by lpxL and lpxM respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo-disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IVA. Over-expression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 °C without the need for MsbA over-production. These strains accumulated penta- and hexa-acylated free lipid A containing a secondary laurate chain, or a laurate and a myristate chain, respectively. Deletion of kdtA in strains over-expressing LpxM accumulated penta-acylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 °C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexa-acylated lipid A, which is optimal for the MsbA flippase.

Reynolds, C. Michael; Raetz, Christian R. H.

2009-01-01

298

Neurobehavioral Mutants Identified in an ENU Mutagenesis Project  

SciTech Connect

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.

Cook, Melloni N. [University of Memphis; Dunning, Jonathan P [University of Memphis; Wiley, Ronald G [Vanderbilt University and Veterans Administration, Nashville, TN; Chesler, Elissa J [ORNL; Johnson, Dabney K [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

2007-01-01

299

Six post-implantation lethal knockouts of genes for lipophilic MAPK pathway proteins are expressed in preimplantation mouse embryos and trophoblast stem cells.  

PubMed

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations. PMID:15736129

Xie, Yufen; Wang, Yingchun; Sun, Tong; Wang, Fangfei; Trostinskaia, Anna; Puscheck, Elizabeth; Rappolee, Daniel A

2005-05-01

300

Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels.  

PubMed

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function. PMID:23558439

Lübkemeier, Indra; Requardt, Robert Pascal; Lin, Xianming; Sasse, Philipp; Andrié, René; Schrickel, Jan Wilko; Chkourko, Halina; Bukauskas, Feliksas F; Kim, Jung-Sun; Frank, Marina; Malan, Daniela; Zhang, Jiong; Wirth, Angela; Dobrowolski, Radoslaw; Mohler, Peter J; Offermanns, Stefan; Fleischmann, Bernd K; Delmar, Mario; Willecke, Klaus

2013-04-05

301

Lethal fish hook attachment - An unusual occurrence.  

PubMed

A 39-year-old fisherman is reported who was dragged into the water from a boat after he became entangled in fishing line. His death was attributed to salt water drowning. At autopsy the cause of death was confirmed and the mechanism of the lethal event elucidated. Specifically, a large fish hook attached to line was embedded in his right wrist. The hook had passed beneath flexor tendons and had firmly attached him to fishing line that was being dropped from the vessel. There were no other significant injuries or underlying diseases present. This case demonstrates another rare situation in the commercial fishing industry that may result in a victim being dragged from a boat and drowned. PMID:23357398

Byard, Roger W

2012-06-17

302

Isolation of protein glycosylation mutants in the fission yeast Schizosaccharomyces pombe.  

PubMed Central

We have isolated mutants in the fission yeast Schizosaccharomyces pombe that are defective in protein glycosylation. A collection of osmotically sensitive mutants was prepared and screened for glycosylation defects using lectin staining as an assay. Mutants singly defective in four glycoprotein synthesis genes (gps1-4) were isolated, all of which bind less galactose-specific lectin. Acid phosphatase and other glycoproteins from the gps mutants have increased electrophoretic mobility, suggesting that these mutants make glycans of reduced size. N-linked glycan analysis revealed that terminal oligosaccharide modification is defective in the gps1 and gps2 mutants. Both mutants synthesize the Man9GlcNAc2 core glycan but have reduced amounts of larger structures. Modified core glycans from gps1 cells have normal amounts of galactose (Gal) residues, but reduced amounts of Man, consistent with a defect in a Golgi mannosyltransferase in this mutant. In contrast, N-linked oligosaccharides from gps2 mutants have much less Gal than wild type, because of reduced levels of the Gal donor, UDP-Gal. This reduction is caused by decreased activity of UDP-glucose 4-epimerase, which synthesizes UDP-Gal. Neither the gps1 or gps2 mutations are lethal, although the cells grow at reduced rates. These findings suggest that S. pombe cells can survive with incompletely glycosylated cell wall glycoproteins. In particular, these results suggest that Gal, which comprises approximately 30% by weight of cell wall glycoprotein glycans, is not crucial for cell growth or survival. Images

Huang, K M; Snider, M D

1995-01-01

303

Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis.  

PubMed

cpSRP54 (for chloroplast SIGNAL RECOGNITION PARTICLE54) is involved in cotranslational and posttranslational sorting of thylakoid proteins. The Arabidopsis (Arabidopsis thaliana) cpSRP54 null mutant, ffc1-2, is pale green with delayed development. Western-blot analysis of individual leaves showed that the SRP sorting pathway, but not the SecY/E translocon, was strongly down-regulated with progressive leaf development in both wild-type and ffc1-2 plants. To further understand the impact of cpSRP54 deletion, a quantitative comparison of ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves using stable isotope labeling (isobaric stable isotope labeling and isotope-coded affinity tags) and two-dimensional gels. This showed that cpSRP54 deletion led to a change in light-harvesting complex composition, an increase of PsbS, and a decreased photosystem I/II ratio. Moreover, the cpSRP54 deletion led in young leaves to up-regulation of thylakoid proteases and stromal chaperones, including ClpC. In contrast, the stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. A differential response between young and mature leaves was also found in carbon metabolism, with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves but not in old leaves. The Calvin cycle was down-regulated in mature leaves to adjust to the reduced capacity of the light reaction, while reactive oxygen species defense proteins were up-regulated. The significance of ClpC up-regulation was confirmed through the generation of an ffc2-1 clpc1 double mutant. This mutant was seedling lethal under autotrophic conditions but could be partially rescued under heterotrophic conditions. PMID:18633119

Rutschow, Heidi; Ytterberg, A Jimmy; Friso, Giulia; Nilsson, Robert; van Wijk, Klaas J

2008-07-16

304

Chronic Exposure of Corals to Fine Sediments: Lethal and Sub-Lethal Impacts  

PubMed Central

Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l?1 TSS (25 mg cm?2 day?1) for M. aequituberculata and 100 mg l?1 TSS (83 mg cm?2 day?1) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue.

Flores, Florita; Hoogenboom, Mia O.; Smith, Luke D.; Cooper, Timothy F.; Abrego, David; Negri, Andrew P.

2012-01-01

305

Chronic exposure of corals to fine sediments: lethal and sub-lethal impacts.  

PubMed

Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l(-1) TSS (25 mg cm(-2) day(-1)) for M. aequituberculata and 100 mg l(-1) TSS (83 mg cm(-2) day(-1)) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue. PMID:22662225

Flores, Florita; Hoogenboom, Mia O; Smith, Luke D; Cooper, Timothy F; Abrego, David; Negri, Andrew P

2012-05-25

306

Non-Lethal Weaponry: Applications to AC-130 Gunships.  

National Technical Information Service (NTIS)

The theory and application of non-lethal weapons is not new and has been in use by ground combat troops and civil authorities for some time, in situations requiring the application of less than lethal force, With the increasing involvement of US military ...

J. L. Bobb

2002-01-01

307

Non?lethal weapons for UN military operations  

Microsoft Academic Search

Rapid advances in Non?Lethal Weapons (NLWs) technology are offering UN military peacekeepers and peace enforcers a wider range of options before having to resort to lethal force. In this article we define and review the operational history of NLWs; discuss the issues and controversies, such as the legal and ethical implications, which surround the development and deployment of NLWs; and

Nick Lewer; Steven Schofield

1997-01-01

308

Preparing Entomocidal Products with Oligosporogenic Mutants of 'Bacillus thuringiensis'.  

National Technical Information Service (NTIS)

Six oligosporogenic mutant strains obtained from Bacillus thuringiensis subsp. kurstaki yield parasporal products substantially free of interfering spores when cultivated under normal sporulation conditions. These parasporal products have utility in the b...

D. E. Johnson

1980-01-01

309

Improved method of selection for mutants of Pseudomonas putida.  

PubMed Central

Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis with an improved D-cycloserine selection.

Carhart, G; Hegeman, G

1975-01-01

310

Improved method of selection for mutants of Pseudomonas putida.  

PubMed

Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis with an improved D-cycloserine selection. PMID:1108792

Carhart, G; Hegeman, G

1975-12-01

311

Deletion of Candida albicans SPT6 Is Not Lethal but Results in Defective Hyphal Growth  

PubMed Central

As a means to study surface proteins involved in the yeast to hypha transition, human monoclonal antibody fragments (single chain variable fragments, scFv) have been generated that bind to antigens expressed on the surface of Candida albicans yeast and/or hyphae. A cDNA expression library was constructed from hyphae, and screened for immunoreactivity with scFv5 as a means to identify its cognate antigen. A reactive clone contained the 3? end of the C. albicans gene, orf 19.7136, designated SPT6 based on homology to S. cerevisiae, where its product functions as a transcription elongation factor. A mutant containing a homozygous deletion of SPT6 was isolated, demonstrating that unlike S. cerevisiae, deletion of this gene in C. albicans is not lethal. Growth of this strain was severely impaired, however, as was its capacity to undergo filamentous growth. Reactivity with scFv5 was not detected in the mutant strain, although its impaired growth complicates the interpretation of this finding. To assess C. albicans SPT6 function, expression of the C. albicans gene was induced in a defined S. cerevisiae spt6 mutant. Partial complementation was seen, confirming that the C. albicans and S. cerevisiae genes are functionally related in these species.

Al-Rawi, Nada; Laforce-Nesbitt, Sonia S.; Bliss, Joseph M.

2010-01-01

312

A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis.  

PubMed Central

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila.

Pultz, M A; Zimmerman, K K; Alto, N M; Kaeberlein, M; Lange, S K; Pitt, J N; Reeves, N L; Zehrung, D L

2000-01-01

313

Membrane fusion mutants of Semliki Forest virus  

PubMed Central

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild- type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.

1984-01-01

314

Non-lethal heat shock protects gnotobiotic Artemia franciscana larvae against virulent Vibrios  

Microsoft Academic Search

Brine shrimp Artemia were exposed under gnotobiotic conditions to a non-lethal heat shock (NLHS) from 28 to 32, 37 and 40°C. Different recovery periods (2, 6, 12 and 24h) and different heat-exposure times (15, 30, 45 and 60min) were tested. After these NLHS, Artemia was subsequently challenged with Vibrio. Challenge tests were performed in stressed and unstressed nauplii at concentrations

Yeong Yik Sung; Els J. M. Van Damme; Patrick Sorgeloos; Peter Bossier

2007-01-01

315

Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim  

Microsoft Academic Search

We performed stem cell rescue and allogeneic skin transplantation on a lethally neutron-irradiated nuclear accident victim. HLA-DRB1 mismatched unrelated umbilical cord blood cells (2.08 × 107\\/kg recipient body weight) were transplanted to an 8–10 Gy equivalent neutron-irradiated patient because of a lack of a suitable bone marrow or peripheral blood donor. Pre-transplant conditioning consisted of anti-thymocyte ?-globulin alone, and GVHD

H Nagayama; K Misawa; H Tanaka; J Ooi; T Iseki; A Tojo; K Tani; Y Yamada; H Kodo; TA Takahashi; N Yamashita; S Shimazaki; S Asano

2002-01-01

316

Neurodegeneration in Drop-Dead Mutant Drosophila melanogaster Is Associated with the Respiratory System but Not with Hypoxia  

PubMed Central

Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia.

Sansone, Christine Lynn; Blumenthal, Edward M.

2013-01-01

317

Role of a Cytotoxic Enterotoxin in Aeromonas-Mediated Infections: Development of Transposon and Isogenic Mutants  

PubMed Central

Transposon and marker exchange mutagenesis were used to evaluate the role of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections. The transposon mutants were generated by random insertion of Tn5-751 in the chromosomal DNA of a diarrheal isolate SSU of Aeromonas hydrophila. Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild-type Aeromonas when injected intraperitoneally (i.p.). Southern blot data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (act). The transcription of the act gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type Aeromonas by using a suicide vector. In these mutants, the truncated act gene was integrated in place of a functionally active act gene. The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act. These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron microscopy, whereas culture filtrates from wild-type Aeromonas caused complete destruction of the microvilli. The 50% lethal dose of these mutants in mice was 1.0 × 108 when injected i.p., compared to 3.0 × 105 for the wild-type Aeromonas. Reintegration of the native act gene in place of the truncated toxin gene in isogenic mutants resulted in complete restoration of Act’s biological activity and virulence in mice. The animals injected with a sublethal dose of wild-type Aeromonas or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies. Taken together, these studies clearly established a role for Act in the pathogenesis of Aeromonas-mediated infections.

Xu, Xin-J.; Ferguson, M. R.; Popov, V. L.; Houston, C. W.; Peterson, J. W.; Chopra, A. K.

1998-01-01

318

Interaction of metronidazole with DNA repair mutants of Escherichia coli.  

PubMed Central

It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light.

Yeung, T C; Beaulieu, B B; McLafferty, M A; Goldman, P

1984-01-01

319

Systematic Triple Mutant Analysis Uncovers Functional Connectivity Between Pathways Involved in Chromosome Regulation  

PubMed Central

Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed Triple Mutant Analysis (TMA). We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histone chaperones. After subjecting asf1? cac1? to TMA, we found that the Swi/Snf Rdh54 protein, compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1? strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6, cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered using traditional double mutant analyses.

Haber, James E.; Braberg, Hannes; Wu, Qiuqin; Alexander, Richard; Haase, Julian; Ryan, Colm; Lipkin-Moore, Zach; Franks-Skiba, Kathleen E.; Johnson, Tasha; Shales, Michael; Lenstra, Tineke L.; Holstege, Frank C. P.; Johnson, Jeffrey R.; Bloom, Kerry; Krogan, Nevan J.

2013-01-01

320

White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase.  

PubMed

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene. PMID:15579683

McCarthy, Sarah S; Kobayashi, Marilyn C; Niyogi, Krishna K

2004-11-01

321

White Mutants of Chlamydomonas reinhardtii Are Defective in Phytoene Synthase  

PubMed Central

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or “white” mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.

McCarthy, Sarah S.; Kobayashi, Marilyn C.; Niyogi, Krishna K.

2004-01-01

322

Bacillus anthracis cell wall produces injurious inflammation but paradoxically decreases the lethality of anthrax lethal toxin in a rat model  

PubMed Central

Objectives The in vivo inflammatory effects of the Bacillus anthracis cell wall are unknown. We therefore investigated these effects in rats and, for comparison, those of known inflammatory stimulants, Staphylococcus aureus cell wall or lipopolysaccharide (LPS). Method and Results Sprague–Dawley rats (n = 103) were challenged with increasing B. anthracis cell wall doses (10, 20, 40, 80, or 160 mg/kg) or diluent (control) as a bolus or 24-h infusion. The three highest bolus doses were lethal (20–64% lethality rates) as were the two highest infused doses (13% with each). Comparisons among lethal or nonlethal doses on other measured parameters were not significantly different, and these were combined for analysis. Over the 24 h after challenge initiation with lethal bolus or infusion, compared to controls, ten inflammatory cytokines and NO levels were increased and circulating neutrophils and platelets decreased (P ? 0.05). Changes with lethal doses were greater than changes with nonlethal doses (P ? 0.01). Lethal bolus or infusion doses produced hypotension or hypoxemia, respectively (P ? 0.05). The effects with B. anthracis cell wall were similar to those of S. aureus cell wall or LPS. However, paradoxically administration of B. anthracis cell wall or LPS decreased the lethality of concurrently administered B. anthracis lethal toxin (P < 0.0001 and 0.04, respectively). Conclusion B. anthracis cell wall has the potential to produce inflammatory injury during anthrax infection clinically. However, understanding why cell wall or LPS paradoxically reduced lethality with lethal toxin may help understand this toxin’s pathogenic effects.

Cui, Xizhong; Su, Junwu; Li, Yan; Shiloach, Joseph; Solomon, Steven; Kaufman, Jeanne B.; Mani, Haresh; Fitz, Yvonne; Weng, Jia; Altaweel, Laith; Besch, Virginia; Eichacker, Peter Q.

2012-01-01

323

Lethal body burdens of polar narcotics: Chlorophenols  

SciTech Connect

The goal of the present study was to measure in fathead minnow (Pimephales promelas) the lethal body burden (LBB) of three chlorophenols that are known as polar narcotic chemicals. The LBBs of the chlorophenols were compared to LBBs of nonpolar narcotic chemicals to consider if the two classes of narcotic chemicals differ on a body burden level. The LBB of the most acidic chlorophenol was measured at two different levels of pH exposure to determine the influence of the degree of ionization on the magnitude of the LBB. Both n-octanol/water partition coefficients and n-hexane/water partition coefficients of the chlorophenols were determined at different pH levels to consider the influence of ionization on the partition coefficient and to determine the importance of a polar group in the organic phase on the partitioning behavior. Partitioning to n-octanol and n-hexane was used as input in a model to simulate the equilibrium partitioning between hydrophobic and nonhydrophobic and target and nontarget compartments in the fish.

Wezel, A.P. van; Punte, S.S. [Utrecht Univ. (Netherlands). Environmental Chemistry Group; Opperhuizen, A. [Ministry of Transport, Public Works and Water Management, The Hague (Netherlands). National Institute for Coastal and Marine Management

1995-09-01

324

Human cooperation by lethal group competition  

PubMed Central

Why humans are prone to cooperate puzzles biologists, psychologists and economists alike. Between-group conflict has been hypothesized to drive within-group cooperation. However, such conflicts did not have lasting effects in laboratory experiments, because they were about luxury goods, not needed for survival (“looting”). Here, we find within-group cooperation to last when between-group conflict is implemented as “all-out war” (eliminating the weakest groups). Human subjects invested in helping group members to avoid having the lowest collective pay-off, whereas they failed to cooperate in control treatments with random group elimination or with no subdivision in groups. When the game was repeated, experience was found to promote helping. Thus, not within-group interactions alone, not random group elimination, but pay-off-dependent group elimination was found to drive within-group cooperation in our experiment. We suggest that some forms of human cooperation are maintained by multi-level selection: reciprocity within groups and lethal competition among groups acting together.

Egas, Martijn; Kats, Ralph; van der Sar, Xander; Reuben, Ernesto; Sabelis, Maurice W.

2013-01-01

325

Behavioral and Biochemical Defects in Temperature-Sensitive Acetylcholinesterase Mutants of DROSOPHILA MELANOGASTER  

PubMed Central

Temperature-sensitive (ts) mutants of the Ace gene, which codes for acetylcholinesterase (AChE) in Drosophila melanogaster, were analyzed for defects in viability, behavior and function of the enzyme. The use of heat-sensitive and cold-sensitive mutations permited the function of AChE in the nervous system to be analyzed temporally. All ts mutations were lethal, or nearly so, when animals expressing them were subjected to restrictive temperatures during late embryonic and very early larval stages. Heat treatments to Ace-ts mid- and late larvae had little effect on the behavior of these animals or on the viability or behavior of the eventual adults. Heat-sensitive mutants exposed to nonpermissive temperatures as pupae, by contrast, had severe defects in phototaxis and locomotor activity as adults. AChE extracted from adult ts mutants that had developed at a permissive temperature were abnormally heat labile, and they had reduced substrate affinity when assayed at restrictive temperatures. However, enzyme activity did not decline during exposure of heat-sensitive adults to high temperatures even though such treatments caused decrements in phototaxis (29°) and, eventually, cessation of movement (31°). The cold-sensitive mutant also produced readily detectable levels of AChE when exposed to a restrictive temperature during the early developmental stage when this mutation causes almost complete lethality. We suggest that the relationship among the genetic, biochemical and neurobiological defects in these mutants may involve more than merely temperature-sensitive catalytic functions.

Hall, Jeffrey C.; Alahiotis, Stamatis N.; Strumpf, David A.; White, Kristin

1980-01-01

326

Insertion of a Sequence Encoding Light Chain 3 of Microtubule Associated Proteins 1A and 1B in a Pestivirus Genome: Connection with Virus Cytopathogenicity and Induction of Lethal Disease in Cattle  

Microsoft Academic Search

Pestiviruses represent the first RNA viruses for which recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can develop from non- cytopathogenic (noncp) viruses. In the case of bovine viral diarrhea virus (BVDV), the generation of cp mutants is linked to the induction of the lethal syndrome mucosal disease (MD) in cattle.

GREGOR MEYERS; DIETER STOLL; MICHAEL GUNN

1998-01-01

327

28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.  

Code of Federal Regulations, 2013 CFR

...less-than-lethal weapons, including chemical agents. 552.25...less-than-lethal weapons, including chemical agents. (a) The...less-than-lethal weapons, including those containing chemical agents, only...

2013-07-01

328

Dominant-lethal mutation and heritable translocation tests in mice  

SciTech Connect

The spontaneous occurrence of chromosome breakage-related genetic anomalies in humans is estimated to be 0.24%, not including spontaneous abortions. More balanced reciprocal translocations are being discovered among mentally handicapped individuals. Chromosome breakage can result in chromosome loss, which often leads to embryonic lethality and occassionally to viable aneuploids, or to viable reciprocal translocations and inversions. In mice, transmitted chromosome breakage effects may be measured by using dominant-lethal mutations, heritable translocations, heritable inversions, sex-chromosome loss, and chromosome breakage and rearrangement scored cytologically in early embryos. The dominant-lethal mutation and the heritable translocation tests are the most widely used tests. The heritable translocation procedure described here applies only to the induction of translocations in male germ cells. The use of treated females in the dominant-lethal test has its drawback too. When the fertility of treated female is reduced or when embryonic lethality is observed, it is difficult to ascertain whether these effects are attributable to true genetic damage or to physiological imbalances in the treated females. Therefore the dominant-lethal test as it is used in practical testing has almost exclusively employed the treatment of males. This is not to say that treatment of females has no advantages whatsoever. On the contrary, there is a good example of a case whereby dominant-lethal effects of the test compound was unequivocably demonstrated in treated females but not in treated males.

Generoso, W.M.

1980-01-01

329

Transgenics, toxicity and therapeutics in rodent models of mutant SOD1-mediated familial ALS  

Microsoft Academic Search

Gain-of-function mutations in the Cu,Zn-superoxide dismutase (SOD1) gene are implicated in progressive motor neuron death and paralysis in one form of inherited amyotrophic lateral sclerosis (ALS). At present, transgenic expression of 12 human SOD1 mutations driven by the endogenous promoter is disease-causative and uniformly lethal in mice and rats, despite tremendous biochemical and biophysical variation between the mutants tested. This

Bradley J. Turner; Kevin Talbot

2008-01-01

330

Induction and isolation of DNA transformation mutants in the yeast Saccharomyces cerevisiae  

Microsoft Academic Search

The objective of this research was to induce and isolate mutants of the yeast Saccharomyces cerevisiae which have become transformable by purified plasmid DNA. Non-transformable yeast cells were mutagenized by ultraviolet light using a 65% lethal dose (480 ergs\\/mm²). After a period of overnight liquid holding recovery, the irradiated cells were subjected to DNA transformation using our CaClâ protocol with

P. A. Hegerich; C. V. Bruschi

1987-01-01

331

Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax  

PubMed Central

The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis. We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA? mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA? mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

Drysdale, Melissa; Koehler, Theresa M.; Hutt, Julie A.; Lyons, C. Rick

2012-01-01

332

Structural characterization of plancitoxin I, a deoxyribonuclease II-like lethal factor from the crown-of-thorns starfish Acanthaster planci , by expression in Chinese hamster ovary cells  

Microsoft Academic Search

Plancitoxin I, the major lethal factor from the spines of crown-of-thorns starfish Acanthaster planci, is a 37 kDa protein composed of two different subunits, and it has potent hepatotoxicity. It is homologous with mammalian\\u000a deoxyribonuclease II (DNase II) and exhibits DNase activity responsible for the hepatotoxicity. To obtain information on the\\u000a structure–activity relationship of plancitoxin I, various mutants were expressed in

Ai Watanabe; Hiroshi Nagai; Yuji Nagashima; Kazuo Shiomi

2009-01-01

333

A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.  

PubMed Central

To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

Merrill, B J; Holm, C

1999-01-01

334

The Genetics of Catalase in Drosophila Melanogaster: Isolation and Characterization of Acatalasemic Mutants  

PubMed Central

Activated oxygen species have been demonstrated to be the important agents in oxygen toxicity by disrupting the structural and functional integrity of cells through lipid peroxidation events, DNA damage and protein inactivation. The biological consequences of free radical damage have long been hypothesized to be a causal agent in many aging-related diseases. Catalase (H(2)O(2):H(2)O(2) oxidoreductase; EC 1.15.1.1) is one of several enzymes involved in the scavenging of oxygen free radicals and free radical derivatives. The structural gene for catalase in Drosophila melanogaster has been localized to region 75D1-76A on chromosome 3L by dosage responses to segmental aneuploidy. This study reports the isolation of a stable deficiency, Df(3L)Cat(DH104)(75C1-2;75F1), that uncovers the catalase locus and the subsequent isolation of six acatalasemic mutants. All catalase mutants are viable under standard culture conditions and recessive lethal mutations within the 75C1-F1 interval have been shown not to affect catalase activity. Two catalase mutations are amorphic while four are hypomorphic alleles of the Cat(+) locus. The lack of intergenic complementation between the six catalase mutations strongly suggests that there is only one functional gene in Drosophila. One acatalesemic mutation was mapped to position 3-47.0 which resides within the catalase dosage sensitive region. While complete loss of catalase activity confers a severe viability effect, residual levels are sufficient to restore viability to wild type levels. These results suggest a threshold effect for viability and offer an explanation for the general lack of phenotypic effects associated with the known mammalian acatalasemics.

Mackay, W. J.; Bewley, G. C.

1989-01-01

335

Lethal and sub lethal effects of the biocide chlorhexidine on aquatic organisms.  

PubMed

Chlorhexidine is among the most used biocides in Europe, however its toxicity to aquatic organisms is scarcely known. The main objective of this study was to assess the lethal and sub lethal effects of chlorhexidine digluconate (ChD) on four aquatic model organisms: the bacteria Vibrio fischeri, the algae Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the embryos of the fish Danio rerio. ChD was very toxic to algae and crustaceans, with a 72 h-EC50 of 62.2 ?g/l and a 48 h-EC50 of 45.0 ?g/l, respectively. Toxicity to fish embryos and the bacteria was lower, with a 96 h-EC50 of 804.0 ?g/l and a 15 min-EC50 of 1,694.0 ?g/l, respectively. Concerning sub lethal effects on D. magna (feeding inhibition) a 6 h-EC50 of 503.7 ?g/l was obtained. In fish, ChD caused developmental abnormalities, namely alterations in the amniotic fluid (48 h-EC20 of 753.6 ?g/l) and early hatching. Moreover, enzymatic biomarkers on fish embryos showed an induction of cholinesterase activity in all ChD tested concentrations (80-900 ?g/l). The catalase activity was also induced at the highest concentration tested (900 ?g/l) whereas no changes were observed for glutathione-S-transferase and lactate dehydrogenase activities. The toxicity of ChD to the algae and crustacean raises concerns about its potential effects in aquatic food webs, since these organisms are in the base of trophic chains, and highlights the need for further studies on ChD toxicity to aquatic organisms. PMID:24026526

Jesus, Fátima T; Oliveira, Rhaul; Silva, Andreia; Catarino, Ana L; Soares, Amadeu M V M; Nogueira, António J A; Domingues, Inês

2013-09-13

336

Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements  

Microsoft Academic Search

Synthetic lethal interaction is defined as a combination of two mutations that is lethal when present in the same cell; each\\u000a individual mutation is non-lethal. Synthetic lethal interactions attract attention in cancer research fields since the discovery\\u000a of synthetic lethal genes with either oncogenes or tumor suppressor genes (TSGs) provides novel cancer therapeutic targets.\\u000a Due to the selective lethal effect

Shinji Mizuarai; Hidehito Kotani

2010-01-01

337

Neonatal Lethality of LGR5 Null Mice Is Associated with Ankyloglossia and Gastrointestinal Distension  

PubMed Central

The physiological role of an orphan G protein-coupled receptor, LGR5, was investigated by targeted deletion of this seven-transmembrane protein containing a large N-terminal extracellular domain with leucine-rich repeats. LGR5 null mice exhibited 100% neonatal lethality characterized by gastrointestinal tract dilation with air and an absence of milk in the stomach. Gross and histological examination revealed fusion of the tongue to the floor of oral cavity in the mutant newborns and immunostaining of LGR5 expression in the epithelium of the tongue and in the mandible of the wild-type embryos. The observed ankyloglossia phenotype provides a model for understanding the genetic basis of this craniofacial defect in humans and an opportunity to elucidate the physiological role of the LGR5 signaling system during embryonic development.

Morita, Hiroki; Mazerbourg, Sabine; Bouley, Donna M.; Luo, Ching-Wei; Kawamura, Kazuhiro; Kuwabara, Yoshimitsu; Baribault, Helene; Tian, Hui; Hsueh, Aaron J. W.

2004-01-01

338

Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening.  

PubMed

Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data. PMID:23519032

Adams, David; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J; Dickinson, Mary; Greene, Nicholas D E; Henkelman, Mark; Justice, Monica; Mohun, Timothy; Murray, Stephen A; Pauws, Erwin; Raess, Michael; Rossant, Janet; Weaver, Tom; West, David

2013-03-18

339

Mutant Selection Window Hypothesis Updated  

Microsoft Academic Search

The mutant selection window hypothesis postulates that, for each antimicrobial-pathogen combination, an antimicrobial concentration range exists in which selective amplification of single-step, drug-resistant mu- tants occurs. This hypothesis suggests an antimutant dosing strategy that is keyed to the upper boundary of the selection window: the mutant prevention concentration. Correlations are described between the mutant prevention concentration—a static parameter that is

Karl Drlica; Xilin Zhao

2007-01-01

340

Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast.  

PubMed Central

14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR.

Gelperin, Daniel; Horton, Lynn; DeChant, Anne; Hensold, Jack; Lemmon, Sandra K

2002-01-01

341

Germline Transformation Using a Prune Cdna Rescues Prune/Killer of Prune Lethality and the Prune Eye Color Phenotype in Drosophila  

PubMed Central

Null mutations in the prune gene of Drosophila melanogaster result in prune eye color due to reductions in red pigment accumulation. When one copy of the awd(Killer of prune) mutant gene is present in a prune background, the animals die. The cause of prune/Killer of prune lethality remains unknown. The genomic region characterized for the prune locus is transcriptionally active and complex, with multiple and overlapping transcripts. Despite the transcriptional complexity of the genomic region of prune, accumulated evidence suggests that the prune locus is small and consists of a single transcription unit, since every prune allele to date exhibits both prune eye color and prune/Killer of prune lethality. A functional prune product from a single, full-length cDNA was identified in this study that can rescue both the eye phenotype and prune/Killer of prune lethality. The DNA sequences of several mutant prune alleles along with Western blot analysis of mutant proteins provide convincing evidence that prune mutations are nulls, and that the cDNA identified in this study encodes the only product of the prune locus.

Timmons, L.; Shearn, A.

1996-01-01

342

Genetic loci controlling lethal cell death in tomato caused by viral satellite RNA infection.  

PubMed

Cucumber mosaic virus (CMV) associated with D satellite RNA (satRNA) causes lethal systemic necrosis (LSN) in tomato (Solanum lycopersicum), which involves programmed cell death. No resistance to this disease has been found in tomato. We obtained a line of wild tomato, S. habrochaitis, with a homogeneous non-lethal response (NLR) to the infection. This line of S. habrochaitis was crossed with tomato to generate F1 plants that survived the infection with NLR, indicating that NLR is a dominant trait. The NLR trait was successfully passed on to the next generation. The phenotype and genotype segregation was analyzed in the first backcross population. The analyses indicate that the NLR trait is determined by quantitative trait loci (QTL). Major QTL associated with the NLR trait were mapped to chromosomes 5 and 12. Results from Northern blot and in situ hybridization analyses revealed that the F1 and S. habrochaitis plants accumulated minus-strand satRNA more slowly than tomato, and fewer vascular cells were infected. In addition, D satRNA-induced LSN in tomato is correlated with higher accumulation of the minus-strand satRNA compared with the accumulation of the minus strand of a non-necrogenic mutant D satRNA. PMID:22746824

Xu, Ping; Wang, Hua; Coker, Frank; Ma, Jun-Ying; Tang, Yuhong; Taylor, Mark; Roossinck, Marilyn J

2012-08-01

343

EGFR mutant lung cancer.  

PubMed

Thoracic oncologists traditionally have made treatment decisions based upon tumor histology, distinguishing non-small cell lung cancer (NSCLC) from small cell lung cancer (SCLC). However, recent data has revealed that at least one histological subtype of NSCLC, lung adenocarcinoma comprises multiple molecularly distinct diseases. Lung adenocarcinoma subsets now can be defined by specific 'driver' mutations in genes encoding components of the EGFR signaling pathway. Importantly, these mutations have implications regarding targeted therapy. Here, we focus on EGFR mutant NSCLC-a prime example of a clinically relevant molecular subset of lung cancer, with defined mechanisms of drug sensitivity, primary drug resistance, and acquired resistance to EGFR tyrosine kinase inhibitors. Efforts are now being made to overcome mechanisms of acquired resistance. These findings illustrate how knowledge about the genetic drivers of tumors can lead to rational targeted therapy for individual patients. PMID:21866438

Gong, Yixuan; Pao, William

2012-01-01

344

Epsilon-Toxin Is Required for Most Clostridium perfringens Type D Vegetative Culture Supernatants To Cause Lethality in the Mouse Intravenous Injection Model  

PubMed Central

Clostridium perfringens type D enterotoxemias have significant economic impact by causing rapid death of several domestic animal species. Consequently, domestic animals are commonly vaccinated, at varying efficacy, with inactivated type D vegetative supernatants. Improved type D vaccines might become possible if the lethal toxins produced by type D isolates were characterized and the contributions of those toxins to supernatant-induced lethality were established. Therefore, the current study evaluated the presence of lethal toxins in supernatants prepared from late-log-phase vegetative cultures of a large collection of genotype D isolates. Under this growth condition, most genotype D isolates produced variable levels of at least three different lethal toxins, including epsilon-toxin (ETX). To model the rapid lethality of type D enterotoxemias, studies were conducted involving intravenous (i.v.) injection of genotype D vegetative supernatants into mice, which were then observed for neurotoxic distress. Those experiments demonstrated a correlation between ETX (but not alpha-toxin or perfringolysin O) levels in late-log-phase genotype D supernatants and lethality. Consistent with the known proteolytic activation requirement for ETX toxicity, trypsin pretreatment was required for, or substantially increased, the lethality of nearly all of the tested genotype D vegetative supernatants. Finally, the lethality of these trypsin-pretreated genotype D supernatants could be completely neutralized by an ETX-specific monoclonal antibody but not by an alpha-toxin-specific monoclonal antibody. Collectively, these results indicate that, under the experimental conditions used in the present study, ETX is necessary for the lethal properties of most genotype D vegetative supernatants in the mouse i.v. injection model.

Sayeed, Sameera; Fernandez-Miyakawa, M. E.; Fisher, Derek J.; Adams, Vicki; Poon, Rachael; Rood, Julian I.; Uzal, Francisco A.; McClane, Bruce A.

2005-01-01

345

Mitochondrial-mediated suppression of ROS production upon exposure of neurons to lethal stress: Mitochondrial targeted preconditioning  

Microsoft Academic Search

Preconditioning represents the condition where transient exposure of cells to an initiating event leads to protection against subsequent, potentially lethal stimuli. Recent studies have established that mitochondrial-centered mechanisms are important mediators in promoting development of the preconditioning response. However, many details concerning these mechanisms are unclear. The purpose of this review is to describe the initiating and subsequent intracellular events

David W. Busija; Tamas Gaspar; Ferenc Domoki; Prasad V. Katakam; Ferenc Bari

2008-01-01

346

Engineered Repressible Lethality for Controlling the Pink Bollworm, a Lepidopteran Pest of Cotton  

PubMed Central

The sterile insect technique (SIT) is an environmentally friendly method of pest control in which insects are mass-produced, irradiated and released to mate with wild counterparts. SIT has been used to control major pest insects including the pink bollworm (Pectinophora gossypiella Saunders), a global pest of cotton. Transgenic technology has the potential to overcome disadvantages associated with the SIT, such as the damaging effects of radiation on released insects. A method called RIDL (Release of Insects carrying a Dominant Lethal) is designed to circumvent the need to irradiate insects before release. Premature death of insects’ progeny can be engineered to provide an equivalent to sterilisation. Moreover, this trait can be suppressed by the provision of a dietary antidote. In the pink bollworm, we generated transformed strains using different DNA constructs, which showed moderate-to-100% engineered mortality. In permissive conditions, this effect was largely suppressed. Survival data on cotton in field cages indicated that field conditions increase the lethal effect. One strain, called OX3402C, showed highly penetrant and highly repressible lethality, and was tested on host plants where its larvae caused minimal damage before death. These results highlight a potentially valuable insecticide-free tool against pink bollworm, and indicate its potential for development in other lepidopteran pests.

Morrison, Neil I.; Simmons, Gregory S.; Fu, Guoliang; O'Connell, Sinead; Walker, Adam S.; Dafa'alla, Tarig; Walters, Michelle; Claus, John; Tang, Guolei; Jin, Li; Marubbi, Thea; Epton, Matthew J.; Harris, Claire L.; Staten, Robert T.; Miller, Ernest; Miller, Thomas A.; Alphey, Luke

2012-01-01

347

Sirenomelia Phenotype in Bmp7;Shh Compound Mutants: A Novel Experimental Model for Studies of Caudal Body Malformations  

PubMed Central

Sirenomelia is a severe congenital malformation of the lower body characterized by the fusion of the legs into a single lower limb. This striking external phenotype consistently associates severe visceral abnormalities, most commonly of the kidneys, intestine, and genitalia that generally make the condition lethal. Although the causes of sirenomelia remain unknown, clinical studies have yielded two major hypotheses: i) a primary defect in the generation of caudal mesoderm, ii) a primary vascular defect that leaves the caudal part of the embryo hypoperfused. Interestingly, Sirenomelia has been shown to have a genetic basis in mice, and although it has been considered a sporadic condition in humans, recently some possible familial cases have been reported. Here, we report that the removal of one or both functional alleles of Shh from the Bmp7-null background leads to a sirenomelia phenotype that faithfully replicates the constellation of external and internal malformations, typical of the human condition. These mutants represent an invaluable model in which we have analyzed the pathogenesis of sirenomelia. We show that the signaling defect predominantly impacts the morphogenesis of the hindgut and the development of the caudal end of the dorsal aortas. The deficient formation of ventral midline structures, including the interlimb mesoderm caudal to the umbilicus, leads to the approximation and merging of the hindlimb fields. Our study provides new insights for the understanding of the mechanisms resulting in caudal body malformations, including sirenomelia.

Garrido-Allepuz, Carlos; Gonzalez-Lamuno, Domingo; Ros, Maria A.

2012-01-01

348

Internet suicide: communities of affirmation and the lethality of communication.  

PubMed

As a tool of instant information dissemination and social networking, the Internet has made possible the formation and affirmation of public identities based on personality traits that are usually characterized by clinicians as pathological. The wide variety of online communities of affirmation reveals new conditions for permissiveness and inclusiveness in expressions of these socially marginal and clinically pathologized identities. Much the same kind of discourse common to these online communities is evident in some suicide forums. Web sites with suicide as their central raison d'être, taken together, encompass a wide range of ideas and commitments, including many that provide collective affirmation outside of (and often with hostility toward) professional intervention. The paradox of a potentially life-affirming effect of such forums runs counter to a stark dualism between online therapy versus "prochoice" forums and, by extension, to simple models of the influence of ideas on the lethality of suicide. Different forums either intensify or mitigate self-destructive tendencies in ways that are significant for understanding the place of communication in the occurrence of suicide and for therapeutic practice. PMID:23315147

Niezen, Ronald

2013-01-11

349

Precursors of lethal violence: a death row sample.  

PubMed

A qualitative methodology based on the standards of criminal defense investigation was used to analyze the social and family histories of 16 men sentenced to death in California. Using a multisource cross-validation methodology, we assessed patterns of impairment, injury and deficit at each of four ecological levels: family, individual, community and social institutions. Investigation documented consistent and pervasive patterns of serious impairment, injury and deficit across the cases and levels. The men share numerous risk factors and few resiliency factors associated with violence. We found family violence in all 16 cases, including severe physical and/or sexual abuse in 14 cases; individual impairments in 16, including 14 with post-traumatic stress disorder, 13 with severe depression and 12 with histories of traumatic brain injury; community isolation and violence in 12; and institutional failure in 15, including 13 cases of severe physical and/or sexual abuse while in foster care or under state youth authority jurisdiction. Appropriate interventions might have made a difference in reducing lethal violence and its precursor conditions. PMID:10798330

Freedman, D; Hemenway, D

2000-06-01

350

Mice With an Anterior Cleft of the Palate Survive Neonatal Lethality  

PubMed Central

Many genes are known to function in a region-specific manner in the developing secondary palate. We have previously shown that Shox2-deficient embryos die at mid-gestation stage and develop an anterior clefting phenotype. Here, we show that mice carrying a conditional inactivation of Shox2 in the palatal mesenchyme survive the embryonic and neonatal lethality, but develop a wasting syndrome. Phenotypic analyses indicate a delayed closure of the secondary palate at the anterior end, leading to a failed fusion of the primary and secondary palates. Consistent with a role proposed for Shox2 in skeletogenesis, Shox2 inactivation causes a significantly reduced bone formation in the hard palate, probably due to a down-regulation of Runx2 and Osterix. We conclude that the secondary palatal shelves are capable of fusion with each other, but fail to fuse with the primary palate in a developmentally delayed manner. Mice carrying an anterior cleft can survive neonatal lethality.

Gu, Shuping; Wei, Na; Yu, Xueyan; Jiang, Yiping; Fei, Jian; Chen, YiPing

2009-01-01

351

Pleiotropic menaquinone-deficient mutant of Bacillus subtilis.  

PubMed

A multiple aromatic amino acid auxotroph of Bacillus subtilis 168 has been isolated which is unable to synthesize menaquinone-7 (MK-7) unless supplied with shikimic acid (SHK). The mutant, RB163, was isolated by selecting for resistance to low levels (1.5 mug/ml) of kanamycin. Enzymatic and genetic analyses show that the strain is an aroD mutant lacking 5-dehydroshikimate reductase. Under growth conditions in which its MK-7 deficiency is expressed, RB163 is deficient in cytochromes a, b, and c, exhibits low growth yields, and does not sporulate. Genetic analysis indicates that this pleiotropic phenotype is the result of a single genetic event. All phenotypic characteristics are reversible when the mutant is grown under conditions such that MK is synthesized. Comparison of strain RB163 with other aro mutants blocked before SHK ("early-aro" mutants) reveals interesting differences. Most early-aro mutants are cytochrome- and MK-sufficient, sporogenous, and sensitive to kanamycin when grown in the absence of SHK. However, in addition to strain RB163, two other aro mutants were found to show the pleiotropic phenotype. These three mutants have in common, and differ from other early-aro strains in, the inability to synthesize MK. It is suggested that the phenotypically wild-type aro mutants are bradytrophic, allowing enough substrate flow through the common aromatic pathway to satisfy the MK requirement. The pleiotropic mutants are thought to be completely blocked in the common pathway, thus accounting for their inability to synthesize MK. PMID:4199502

Farrand, S K; Taber, H W

1973-09-01

352

Zebrafish Mutants calamity and catastrophe Define Critical Pathways of Gene-Nutrient Interactions in Developmental Copper Metabolism  

PubMed Central

Nutrient availability is an important environmental variable during development that has significant effects on the metabolism, health, and viability of an organism. To understand these interactions for the nutrient copper, we used a chemical genetic screen for zebrafish mutants sensitive to developmental copper deficiency. In this screen, we isolated two mutants that define subtleties of copper metabolism. The first contains a viable hypomorphic allele of atp7a and results in a loss of pigmentation when exposed to mild nutritional copper deficiency. This mutant displays incompletely penetrant skeletal defects affected by developmental copper availability. The second carries an inactivating mutation in the vacuolar ATPase that causes punctate melanocytes and embryonic lethality. This mutant, catastrophe, is sensitive to copper deprivation revealing overlap between ion metabolic pathways. Together, the two mutants illustrate the utility of chemical genetic screens in zebrafish to elucidate the interaction of nutrient availability and genetic polymorphisms in cellular metabolism.

Madsen, Erik C.; Gitlin, Jonathan D.

2008-01-01

353

Less-Than-Lethal Weapons: New Solutions for Law Enforcement.  

National Technical Information Service (NTIS)

This report discusses the search for less-than-lethal (LTL) weapons for United States police organizations. The National Institute of Justice's Science and Technology Division has devised an LTL strategy to develop new technologies that will improve polic...

L. Pilant

1993-01-01

354

40 CFR 798.5450 - Rodent dominant lethal assay.  

Code of Federal Regulations, 2010 CFR

...is lethal to the fertilized egg or developing embryo. ...For example, in a typical experiment, the number of males in...shall be included in each experiment. When acceptable positive...results are available from experiments conducted recently...

2010-07-01

355

40 CFR 798.5450 - Rodent dominant lethal assay.  

Code of Federal Regulations, 2010 CFR

...is lethal to the fertilized egg or developing embryo. ...For example, in a typical experiment, the number of males in...shall be included in each experiment. When acceptable positive...results are available from experiments conducted recently...

2009-07-01

356

Bleomycin: Female-Specific Dominant Lethal Effects in Mice.  

National Technical Information Service (NTIS)

Limited comparative data in mice indicate that chemical mutagens that induce dominant lethal mutations in males are not necessarily effective in females, but those which are effective in females are generally equally or more effective in males. Recently, ...

P. D. Sudman J. C. Rutledge J. B. Bishop W. M. Generoso

1992-01-01

357

Lethal Concentrations of Heavy Metals in Tissue of Earthworms.  

National Technical Information Service (NTIS)

This toxicological research report addresses lethal concentrations of heavy metals in tissue of earthworms. We have presented the work in progress in the first interim report to improve 1) ecotoxicological test, 2) field procedures and 3) standardization ...

A. Rida J. Y. Gal M. B. Bouche P. Brun

1987-01-01

358

Fluorescence resonance energy transfer studies on anthrax lethal toxin  

Microsoft Academic Search

Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA63, a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for

John C. Croney; Kristina M. Cunningham; R. John Collier; David M. Jameson

2003-01-01

359

Dominant-lethal mutations and heritable translocations in mice  

Microsoft Academic Search

Chromosome aberrations are a major component of radiation or chemically induced genetic damage in mammalian germ cells. The types of aberration produced are dependent upon the mutagen used and the germ-cell stage treated. For example, in male meiotic and postmeiotic germ cells certain alkylating chemicals induce both dominant-lethal mutations and heritable translocations while others induce primarily dominant-lethal mutations. Production of

Generoso

1983-01-01

360

Mechanism by Which Caffeine Potentiates Lethality of Nitrogen Mustard  

Microsoft Academic Search

Caffeine is synergistic with many DNA-damaging agents in increasing lethality to mammalian cells. The mechanism is not well understood. Our results show that caffeine potentiates the lethality of the nitrogen mustard 2-chloro-N-(2-chloroethyl)-N-methylethanamine (HN2) by inducing damaged cells to undergo mitosis before properly repairing lesions in their DNA. Treatment with low doses of HN2 (0.5 mu M for 1 hr) caused

Ching C. Lau; Arthur B. Pardee

1982-01-01

361

GPS targeting methods for non-lethal systems  

Microsoft Academic Search

Non-lethal systems consist of devices and methods which can be used to incapacitate an adversary's capability, while minimizing casualties and collateral property or environmental damages. Examples of military non-lethal concepts include wire mesh entanglements to snag tank treads, highly expansive sticky foams to immobilize personnel and material, anti-material agents to degrade supplies, and information warfare tactics such as the use

Gerald Frost; Calvin Shipbaugh

1994-01-01

362

REACTIVATION OF PHAGE FROM LETHAL ULTRAVIOLET DAMAGE IN DNA  

Microsoft Academic Search

Reactivation phenomena can be used to discriminate between different ; types of lethal effects in phage. This report deals with the relationship ; between photorestoration (PhR) and u gene reactivation (uR). The uR is initiated ; by the u gene in T4 phage in which it exists obligatorily, but not in T2. The ; lethal uv lesions reactivable by PhR

Harm

1961-01-01

363

Molecular Foundations of Reproductive Lethality in Arabidopsis thaliana  

Microsoft Academic Search

The SeedGenes database (www.seedgenes.org) contains information on more than 400 genes required for embryo development in Arabidopsis. Many of these EMBRYO-DEFECTIVE (EMB) genes encode proteins with an essential function required throughout the life cycle. This raises a fundamental question. Why does elimination of an essential gene in Arabidopsis often result in embryo lethality rather than gametophyte lethality? In other words,

Rosanna Muralla; Johnny Lloyd; David Meinke

2011-01-01

364

Chaperone-mediated autophagy degrades mutant p53.  

PubMed

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells. PMID:23913924

Vakifahmetoglu-Norberg, Helin; Kim, Minsu; Xia, Hong-Guang; Iwanicki, Marcin P; Ofengeim, Dimitry; Coloff, Jonathan L; Pan, Lifeng; Ince, Tan A; Kroemer, Guido; Brugge, Joan S; Yuan, Junying

2013-08-01

365

Reduction in chlorophyll content without a corresponding reduction in photosynthesis and carbon assimilation enzymes in yellow-green oil yellow mutants of maize  

Microsoft Academic Search

The photosynthetic properties of a yellow lethal mutant, Oy\\/oy, and two yellow-green mutants of maize which are allelic (a homozygous recessive oy\\/oy and a heterozygous dominant Oy\\/+) were examined. Although Oy\\/oy had little or no chlorophyll or capacity for CO2 fixation compared to normal siblings, it had 28% as much ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity, and from 40% to near

Colin L. D. Jenkins; Gerald E. Edwards; John Andrews

1989-01-01

366

Identification and characterization of tomato mutants affected in the Rx-mediated resistance to PVX isolates.  

PubMed

Five tomato mutants affected in the Rx-mediated resistance against Potato virus X (PVX) were identified by screening a mutagenized population derived from a transgenic, Rx1-expressing 'Micro-Tom' line. Contrary to their parental line, they failed to develop lethal systemic necrosis upon infection with the virulent PVX-KH2 isolate. Sequence analysis and quantitative reverse-transcription polymerase chain reaction experiments indicated that the mutants are not affected in the Rx1 transgene or in the Hsp90, RanGap1 and RanGap2, Rar1 and Sgt1 genes. Inoculation with the PVX-CP4 avirulent isolate demonstrated that the Rx1 resistance was still effective in the mutants. In contrast, the virulent PVX-KH2 isolate accumulation was readily detectable in all mutants, which could further be separated in two groups depending on their ability to restrict the accumulation of PVX-RR, a mutant affected at two key positions for Rx1 elicitor activity. Finally, transient expression of the viral capsid protein elicitor indicated that the various mutants have retained the ability to mount an Rx1-mediated hypersensitive response. Taken together, the results obtained are consistent with a modification of the specificity or intensity of the Rx1-mediated response. The five Micro-Tom mutants should provide very valuable resources for the identification of novel tomato genes affecting the functioning of the Rx gene. PMID:22088194

Sturbois, Bénédicte; Dubrana-Ourabah, Marie-Pierre; Gombert, Julie; Lasseur, Bertrand; Macquet, Audrey; Faure, Chantal; Bendahmane, Abdelhafid; Baurès, Isabelle; Candresse, Thierry

2012-03-01

367

Generation and Identification of Arabidopsis EMS Mutants.  

PubMed

EMS mutant analysis is a routine experiment to identify new players in a specific biological process or signaling pathway using forward genetics. It begins with the generation of mutants by treating Arabidopsis seeds with EMS. A mutant with a phenotype of interest (mpi) is obtained by screening plants of the M2 generation under a specific condition. Once the phenotype of the mpi is confirmed in the next generation, map-based cloning is performed to locate the mpi mutation. During the map-based cloning, mpi plants (Arabidopsis Columbia-0 (Col-0) ecotype background) are first crossed with Arabidopsis Landsberg erecta (Ler) ecotype, and the presence or absence of the phenotype in the F1 hybrids indicates whether the mpi is recessive or dominant. F2 plants with phenotypes similar to the mpi, if the mpi is recessive, or those without the phenotype, if the mpi is dominant, are used as the mapping population. As few as 24 such plants are selected for rough mapping. After finding one marker (MA) linked to the mpi locus or mutant phenotype, more markers near MA are tested to identify recombinants. The recombinants indicate the interval in which the mpi is located. Additional recombinants and molecular markers are then required to narrow down the interval. This is an iterative process of narrowing down the mapping interval until no further recombinants or molecular markers are available. The genes in the mapping interval are then sequenced to look for the mutation. In the last step, the wild-type or mutated gene is cloned to generate binary constructs. Complementation or recapitulation provides the most convincing evidence in determining the mutation that causes the phenotype of the mpi. Here, we describe the procedures for generating mutants with EMS and analyzing EMS mutations by map-based cloning. PMID:24057369

Qu, Li-Jia; Qin, Genji

2014-01-01

368

Bioassay Protocol for Lethal and Sub-Lethal Effects of Fungal Pathogens on 'Chrysoperla carnea' (Neuroptera: Chrysopidae).  

National Technical Information Service (NTIS)

The protocol describes procedures for evaluating the lethal and sublethal effects of exposure to fungal pathogens on larvae and adults of the predatory insect Chrysoperla carnea (Stephens). The protocol was developed and tested with the fungal insect path...

K. Donegan B. Lighthart

1991-01-01

369

RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency.  

PubMed

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (?F/?F), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ?F/?F mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (?F/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ?F/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ?F/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ?F/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus. PMID:23212600

Volta, Viviana; Beugnet, Anne; Gallo, Simone; Magri, Laura; Brina, Daniela; Pesce, Elisa; Calamita, Piera; Sanvito, Francesca; Biffo, Stefano

2012-12-02

370

Subcellular location of lincomycin resistance in Nicotiana mutants  

Microsoft Academic Search

Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The

Ágnes Cséplö; Lutz Eigel; Gábor V. Horváth; Péter Medgyesy; Reinhold G. Herrmann; Hans-Ulrich Koop

1993-01-01

371

Mucidin-nonproducing mutants of oudemansiella mucida.  

PubMed

Mutants of Oudemansiella mucida, blocked in the biosynthesis of the antibiotic mucidin, were obtained at a 0.28% frequency after the application of N-methyl-N'-nitro-N-nitrosoguanidine (MNG) to basidiospores under conditions leading to 0.5--5.0% survival rates. Loss of antibiotic activity was in most isolates accompanied by a decrease in mycelium growth rate and a suppression of dikaryotizing and fructification ability. Recombination analysis of two stable mutants revealed that the block in mucidin synthesis is the result of mutation in the same chromosomal gene (muc). In contrast to the action of MNG, UV-irradiation leads neither to the loss of biosynthetic activity nor to any morphological change. PMID:7193624

Semerdzieva, M; Musílek, V

1981-01-01

372

Suppressors of snf2 Mutations Restore Invertase Derepression and Cause Temperature-Sensitive Lethality in Yeast  

PubMed Central

Mutations in the SNF2 gene of Saccharomyces cerevisiae prevent derepression of the SUC2 (invertase) gene, and other glucose-repressible genes, in response to glucose deprivation. We have isolated 25 partial phenotypic revertants of a snf2 mutant that are able to derepress secreted invertase. These revertants all carried suppressor mutations at a single locus, designated SSN20 (suppressor of snf2). Alleles with dominant, partially dominant and recessive suppressor phenotypes were recovered, but all were only partial suppressors of snf2, reversing the defect in invertase synthesis but not other defects. All alleles also caused recessive, temperature-sensitive lethality and a recessive defect in galactose utilization, regardless of the SNF2 genotype. No significant effect on SUC2 expression was detected in a wild-type (SNF2) genetic background. The ssn20 mutations also suppressed the defects in invertase derepression caused by snf5 and snf6 mutations, and selection for invertase-producing revertants of snf5 mutants yielded only additional ssn20 alleles. These findings suggest that the roles of the SNF2, SNF5 and SNF6 genes in regulation of SUC2 are functionally related and that SSN20 plays a role in expression of a variety of yeast genes.

Neigeborn, Lenore; Rubin, Kenneth; Carlson, Marian

1986-01-01

373

Interactions between the crumbs, lethal giant larvae and bazooka pathways in epithelial polarization.  

PubMed

Several protein complexes that are involved in epithelial apicobasal polarity have been identified. However, the mechanism by which these complexes interact to form an integrated polarized cell morphology remains unclear. Crumbs (Crb) and Lethal giant larvae (Lgl) are components of distinct complexes that regulate epithelial polarization in Drosophila melanogaster, but may not interact directly as they localize to the apical and basolateral membrane, respectively. Nevertheless, a genetic screen identifies marked functional interactions between crb and lgl. These interactions extend to other genes within the crb (stardust, sdt) and lgl (discs large, dlg; scribble, scrib) pathways. Our findings suggest that the crb and lgl pathways function competitively to define apical and basolateral surfaces. They also suggest that in the absence of lgl pathway activity, the crb pathway is not required to maintain epithelial polarity. Moreover, we show that crb and lgl cooperate in zonula adherens formation early in development. At later stages, epithelial cells in these mutants acquire normal polarity, indicating the presence of compensatory mechanisms. We find that bazooka (baz) functions redundantly with crb/sdt to support apical polarity at mid- to late-embryogenesis. Despite regaining cell polarity, however, epithelial cells in crb and lgl pathway mutants fail to re-establish normal overall tissue architecture, indicating that the timely acquisition of polarized cell structure is essential for normal tissue organization. PMID:12510193

Tanentzapf, Guy; Tepass, Ulrich

2003-01-01

374

Determination of synthetic lethal interactions in KRAS oncogene-dependent cancer cells reveals novel therapeutic targeting strategies.  

PubMed

Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy. PMID:22613949

Steckel, Michael; Molina-Arcas, Miriam; Weigelt, Britta; Marani, Michaela; Warne, Patricia H; Kuznetsov, Hanna; Kelly, Gavin; Saunders, Becky; Howell, Michael; Downward, Julian; Hancock, David C

2012-05-22

375

Determination of synthetic lethal interactions in KRAS oncogene-dependent cancer cells reveals novel therapeutic targeting strategies  

PubMed Central

Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy.

Steckel, Michael; Molina-Arcas, Miriam; Weigelt, Britta; Marani, Michaela; Warne, Patricia H; Kuznetsov, Hanna; Kelly, Gavin; Saunders, Becky; Howell, Michael; Downward, Julian; Hancock, David C

2012-01-01

376

The population genetics of X-autosome synthetic lethals and steriles.  

PubMed

Epistatic interactions are widespread, and many of these interactions involve combinations of alleles at different loci that are deleterious when present in the same individual. The average genetic environment of sex-linked genes differs from that of autosomal genes, suggesting that the population genetics of interacting X-linked and autosomal alleles may be complex. Using both analytical theory and computer simulations, we analyzed the evolutionary trajectories and mutation-selection balance conditions for X-autosome synthetic lethals and steriles. Allele frequencies follow a set of fundamental trajectories, and incompatible alleles are able to segregate at much higher frequencies than single-locus expectations. Equilibria exist, and they can involve fixation of either autosomal or X-linked alleles. The exact equilibrium depends on whether synthetic alleles are dominant or recessive and whether fitness effects are seen in males, females, or both sexes. When single-locus fitness effects and synthetic incompatibilities are both present, population dynamics depend on the dominance of alleles and historical contingency (i.e., whether X-linked or autosomal mutations occur first). Recessive synthetic lethality can result in high-frequency X-linked alleles, and dominant synthetic lethality can result in high-frequency autosomal alleles. Many X-autosome incompatibilities in natural populations may be cryptic, appearing to be single-locus effects because one locus is fixed. We also discuss the implications of these findings with respect to standing genetic variation and the origins of Haldane's rule. PMID:21900269

Lachance, Joseph; Johnson, Norman A; True, John R

2011-09-06

377

The Population Genetics of X-Autosome Synthetic Lethals and Steriles  

PubMed Central

Epistatic interactions are widespread, and many of these interactions involve combinations of alleles at different loci that are deleterious when present in the same individual. The average genetic environment of sex-linked genes differs from that of autosomal genes, suggesting that the population genetics of interacting X-linked and autosomal alleles may be complex. Using both analytical theory and computer simulations, we analyzed the evolutionary trajectories and mutation–selection balance conditions for X–autosome synthetic lethals and steriles. Allele frequencies follow a set of fundamental trajectories, and incompatible alleles are able to segregate at much higher frequencies than single-locus expectations. Equilibria exist, and they can involve fixation of either autosomal or X-linked alleles. The exact equilibrium depends on whether synthetic alleles are dominant or recessive and whether fitness effects are seen in males, females, or both sexes. When single-locus fitness effects and synthetic incompatibilities are both present, population dynamics depend on the dominance of alleles and historical contingency (i.e., whether X-linked or autosomal mutations occur first). Recessive synthetic lethality can result in high-frequency X-linked alleles, and dominant synthetic lethality can result in high-frequency autosomal alleles. Many X–autosome incompatibilities in natural populations may be cryptic, appearing to be single-locus effects because one locus is fixed. We also discuss the implications of these findings with respect to standing genetic variation and the origins of Haldane’s rule.

Lachance, Joseph; Johnson, Norman A.; True, John R.

2011-01-01

378

Mutagenesis-Mediated Virus Extinction: Virus-Dependent Effect of Viral Load on Sensitivity to Lethal Defection  

PubMed Central

Background Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis. Results The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results. Conclusions (i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.

Moreno, Hector; Tejero, Hector; de la Torre, Juan Carlos; Domingo, Esteban; Martin, Veronica

2012-01-01

379

Selective reconstitution of liver cholesterol biosynthesis promotes lung maturation but does not prevent neonatal lethality in Dhcr7 null mice  

PubMed Central

Background Targeted disruption of the murine 3?-hydroxysterol-?7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver. Results We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice. Conclusion The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice, with the peripheral organs contributing the morbidity.

Yu, Hongwei; Li, Man; Tint, G Stephen; Chen, Jianliang; Xu, Guorong; Patel, Shailendra B

2007-01-01

380

Transposon insertions causing constitutive sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing  

SciTech Connect

Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle {open_quotes}on{close_quotes} vs. {open_quotes}off{close_quotes} reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. {open_quotes}Male-lethal{close_quotes} (Sxl{sup M}) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, Sxl{sup M} haplo-X animals (chromosomal males) die because of improper dosage compensation, and Sxl{sup m} chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Transcript analysis of double-mutant male-viable Sxl{sup M} derivatives in which the Sxl{sup M} insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these Sxl{sup M} alleles is a consequence of an alteration of the structure of the pre-mRNA that allow some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of Sxl{sup M} alleles appears to depend on the mutant alleles` responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce. 47 refs., 10 figs., 4 tabs.

Berstein, M.; Cline, T.W. [Princeton Univ., Princeton, NJ (United States)]|[Yale Univ., New Haven, CT (United States); Lersch, R.A.; Subrahmanyan, L. [Univ. of California, Berkeley, CA (United States)

1995-02-01

381

The Rorschach Suicide Constellation: assessing various degrees of lethality.  

PubMed

In this article we examine the relation between the Rorschach Comprehensive System's Suicide Constellation (S-CON; Exner, 1993; Exner & Wiley, 1977) and lethality of suicide attempts during the course of patients' hospitalization at the Austen Riggs Center (Stockbridge, MA). Patient records were rated as nonsuicidal (n = 37), parasuicidal (n = 37), or near-lethal (n = 30) based on the presence and lethality of self-destructive acts. Diagnostic efficiency statistics utilizing a cutoff score of 7 or more positive indicators successfully predicted which patients would engage in near-lethal suicidal activity relative to parasuicidal patients (overall correct classification rate [OCC] = .79), nonsuicidal inpatients (OCC = .79), and college students (OCC = .89). Although these predictions were influenced by relatively high base rates in the hospital population (14.5%), base rate estimates were calculated for other hypothetical populations revealing different prediction estimates that should be considered when judging the relative efficacy of the S-CON. Logistic regression analysis revealed that an S-CON score of 7 or more was the sole predictor of near-lethal suicide attempts among 9 psychiatric and demographic variables. PMID:11393464

Fowler, J C; Piers, C; Hilsenroth, M J; Holdwick, D J; Padawer, J R

2001-04-01

382

Empirical Complexities in the Genetic Foundations of Lethal Mutagenesis  

PubMed Central

From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias—mutagenesis of virions—but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

Bull, James J.; Joyce, Paul; Gladstone, Eric; Molineux, Ian J.

2013-01-01

383

Construction of Bacillus anthracis mutant strains producing a single toxin component.  

PubMed

The two protein exotoxins secreted by Bacillus anthracis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo. PMID:8254316

Pezard, C; Duflot, E; Mock, M

1993-10-01

384

A Novel Balanced-Lethal Host-Vector System Based on glmS  

PubMed Central

During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS? mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS+p) complemented the phenotype of the GlmS? mutant, and that GlmS+p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS+pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.

Kim, Kwangsoo; Jeong, Jae Ho; Lim, Daejin; Hong, Yeongjin; Yun, Misun; Min, Jung-Joon; Kwak, Sahng-June; Choy, Hyon E.

2013-01-01

385

A novel balanced-lethal host-vector system based on glmS.  

PubMed

During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS(-) mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS(+)p) complemented the phenotype of the GlmS(-) mutant, and that GlmS(+) p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS (+)pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number. PMID:23555984

Kim, Kwangsoo; Jeong, Jae Ho; Lim, Daejin; Hong, Yeongjin; Yun, Misun; Min, Jung-Joon; Kwak, Sahng-June; Choy, Hyon E

2013-03-28

386

Making temperature-sensitive mutants  

PubMed Central

The study of temperature sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter we describe two “shuffling” methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the “plasmid” version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the “chromosomal” version, The Ts allele is integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have the ir strengths and weaknesses, which are discussed here.

Ben-Aroya, Shay; Pan, Xuewen; Boeke, Jef D.; Hieter, Philip

2010-01-01

387

Novel memory mutants in Drosophila: Behavioral characteristics of the mutant nemyP153  

PubMed Central

Background Starting from Benzer's initiative, the approach of forward genetics has been widely used to isolate mutations affecting learning and memory. For this aim, mainly the odor-shock conditioning was employed. We have isolated P insertional mutations affecting memory after courtship conditioning – another form of classical conditioning in Drosophila. Here we report the behavioral characteristics of one of these mutants, which we have called nemy (no extended memory). Results The courtship activity of Drosophila males is reduced when a male has a previous experience of courting a fertilized female. In the wild-type strain C-S (K), this conditioned courtship inhibition lasts for 1–3 h in the test with a virgin female, and at least for 8 h in the test with a subsequent fertilized female. The mutant males nemyP153 display distinct memory deficiency in both tests already 0.5 h after training. The mutant males show an increased level of locomotor activity unrelated to courtship, and spend more time in such an element of courtship as pursuit. This, however, seems to be a pleiotropic effect of the mutation, independent from its influence on the courtship conditioning. The mutation reduces also memory performance after the odor-shock classical conditioning. At the same time, the sensory and motor functions involved in this type of learning seem to be normal. Conclusions Insertion of P-lacW vector into 49B region of the second chromosome (mutation nemyP153) causes an increased level of locomotor activity, memory deficiency after the courtship conditioning and subnormal acquisition after the odor-shock conditioning.

Kamyshev, Nikolai G; Iliadi, Konstantin G; Bragina, Julia V; Kamysheva, Elena A; Tokmatcheva, Elena V; Preat, Thomas; Savvateeva-Popova, Elena V

2002-01-01

388

Molecular characterization of phycobilisome regulatory mutants of Fremyella diplosiphon.  

PubMed Central

Three classes of pigment mutants were generated in Fremyella diplosiphon in the course of electroporation experiments. The red mutant class had high levels of phycoerythrin in both red and green light and no inducible phycocyanin in red light. Thus, this mutant behaved as if it were always in green light, regardless of light conditions. Blue mutants exhibited normal phycoerythrin photoregulation, whereas the inducible phycocyanin was present at high levels in both red- and green-light-grown cells. Furthermore, the absolute amount of allophycocyanin was increased threefold in comparison with our wild-type strain. Green mutants lost the capacity to accumulate phycoerythrin in green light but showed normal photoregulation of phycocyanin. Analyses of transcript abundance in these mutants demonstrated that changes in the levels of the different phycobilisome components correlated with changes in the levels of mRNAs encoding those components. The characterization of these mutants supports hypotheses previously discussed concerning molecular mechanisms involved in the regulation of the phycobiliprotein gene sets during chromatic adaptation. Images

Bruns, B U; Briggs, W R; Grossman, A R

1989-01-01

389

Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.  

PubMed Central

Mutants deficient in the production of bacteriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus. Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions.

Pierson, B K; Keith, L M; Leovy, J G

1984-01-01

390

Mutant Fungus from Space  

NSDL National Science Digital Library

Space expert Yuri Karash of Russia says that he anticipates that the Mir Space Station could bring virulent new strains of fungus to earth when it splashes down later this month. Various types of fungus, whose smell is the first thing visitors to Mir notice, grow behind panels and in air-conditioning units on the spacecraft and have likely mutated. This article from the BBC News online covers the story.

2001-01-01

391

Temperature-sensitive mutants of Actinobacillus pleuropneumoniae induce protection in mice.  

PubMed Central

Temperature-sensitive mutants of Actinobacillus pleuropneumoniae 4074, serotype 1, were isolated after treatment with nitrosoguanidine and enrichment with penicillin and D-cycloserine. Of the four temperature-sensitive mutants evaluated in mice, one (A-1) had a tight phenotype (i.e., it ceased replication immediately after transfer to the nonpermissive temperature [37 degrees C]) and three (1-2, 4-1, and 12-1) were coasters that continued replication for up to three generations after transfer to 37 degrees C. The reversion frequencies ranged from 10(-6) to 10(-9), and cutoff temperatures ranged from 33 to 35 degrees C. No major changes were detected in the biochemical profiles; agglutination reactions; electrophoretic profiles of the lipopolysaccharides, outer membrane proteins, and hemolysin proteins; hemolytic titers; or CAMP factor reactions of the mutants and the wild-type bacteria. Groups of 3- to 5-week-old, female ICR mice were immunized intranasally with three doses of 3.5 x 10(6) CFU of the mutants over 3 weeks and subsequently challenged intranasally with 5 50% lethal doses of the parental wild-type. Protection was induced by both the tight and the coaster mutants, with the 4-1 and 12-1 coasters eliciting greater protection (67 and 82%, respectively) than that induced by the A-1 tight mutant (57%). Intranasal immunization with both phenotypes induced serum antibody responses against the surface antigens and the hemolysin protein.

Byrd, W; Hooke, A M

1997-01-01

392

Characterization and mapping of novel chlorophyll deficient mutant genes in durum wheat.  

PubMed

The yellow-green leaf mutant has a non-lethal chlorophyll-deficient mutation that can be exploited in photosynthesis and plant development research. A novel yellow-green mutant derived from Triticum durum var. Cappelli displays a yellow-green leaf color from the seedling stage to the mature stage. Examination of the mutant chloroplasts with transmission electron microscopy revealed that the shape of chloroplast changed, grana stacks in the stroma were highly variable in size and disorganized. The pigment content, including chlorophyll a, chlorophyll b, total chlorophyll and carotene, was decreased in the mutant. In contrast, the chla/chlb ratio of the mutants was increased in comparison with the normal green leaves. We also found a reduction in the photosynthetic rate, fluorescence kinetic parameters and yield-related agronomic traits of the mutant. A genetic analysis revealed that two nuclear recessive genes controlled the expression of this trait. The genes were designated ygld1 and ygld2. Two molecular markers co-segregated with these genes. ygld 1 co-segregated with the SSR marker wmc110 on chromosome 5AL and ygld 2 co-segregated with the SSR marker wmc28 on chromosome 5BL. These results will contribute to the gene cloning and the understanding of the mechanisms underlying chlorophyll metabolism and chloroplast development in wheat. PMID:23853511

Li, Ning; Jia, Jizeng; Xia, Chuan; Liu, Xu; Kong, Xiuying

2013-06-01

393

Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation.  

PubMed Central

When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle. Images

Shin, D Y; Matsumoto, K; Iida, H; Uno, I; Ishikawa, T

1987-01-01

394

Advantages of less-tech, less-than-lethal technologies  

NASA Astrophysics Data System (ADS)

This paper illustrates the advantages of developing less-tech technologies by reporting on two less-tech, less-than-lethal prototype law enforcement tools developed at the Idaho National Engineering Laboratory. The devices were developed for the National Institute of Justice, less- than-lethal weapons program: 1) an air bag restraint device for use in restraining suspects who become violent during transport in patrol vehicles, and 2) a retractable spiked barrier strip for stopping fleeing vehicles during high-speed pursuit. The success of both projects relied on developing design requirements in conjunction with the actual users of the devices.

Marts, Donna J.; Overlin, Trudy K.

1995-05-01

395

Palliative care for prenatally diagnosed lethal fetal abnormality  

PubMed Central

Diagnosis of lethal fetal abnormality raises challenging decisions for parents and clinicians. Most parents opt for termination, which may include feticide. Advances in imaging seem unlikely to lead to earlier diagnoses. Perinatal palliative care offers an alternative. Parental decision making and the clinical aspects of perinatal palliative care were studied after a prenatal diagnosis of lethal fetal abnormality in 20 pregnancies. 40% of parents chose to continue the pregnancy and pursue perinatal palliative care. Six of these eight babies were liveborn and lived for between 1½?h and 3?weeks.

Breeze, A C G; Lees, C C; Kumar, A; Missfelder-Lobos, H H; Murdoch, E M

2007-01-01

396

[Prospective study of lethal blood concentration of organophosphorous in humans].  

PubMed

Total cases of organophosphorous (dichlorvos, methamidophos, dimethoate) poisoning outpatients from six hospitals during four years were collected consecutively for lethal blood concentration study. Blood samples were detected with gas chromatography. The probabilities of death, coma were analyzed with Bliss method and their linear regressive equations of probit were obtained respectively, their 50% lethal concentrations (LC50) and 50% coma concentrations(CC50) were calculated by the formulas above. As the death rate was influenced by therapy, its natural death probability has been discussed and estimated their natural LC50 were between the LC50 and CC50 themselves. Combined LC50 and CC50, their natural LC50 were calculated. PMID:12533887

Bu, J; Yan, L; Chen, Y; Chu, J X; Xie, X F; Chen, T P

2001-02-01

397

Developmental consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis.  

PubMed

In order to recover mutations affecting imaginal discs in a way which would allow the relevant genes to be readily cloned, a hybrid dysgenic screen was performed for mutations causing late larval/early pupal lethality. This paper describes that mutagenesis procedure and the phenotypes caused by the mutations that were recovered. Of 81 late larval/pupal lethal mutations that were recovered, 20 cause imaginal disc defects. These 20 mutations define 12 different genes. This paper also includes a description of the developmental defects caused by a mutation in one of those 12 genes which we have named abnormal wing discs (awd); the following paper (C. Dearolf, N. Tripoulas, J. Biggs, and A. Shearn, 1988, Dev. Biol. 129, 169-178) describes the cloning of the awd gene and an analysis of its pattern of transcription. awdb3 homozygotes develop at a normal rate until the end of the second larval instar, when their rate of development is reduced. After an extended third larval instar, they form puparia and die. Mutant wing discs have an abnormal morphology and extensive cell death. These abnormal wing discs, and also the leg and eye-antenna discs which appear to be morphologically normal, differentiate poorly or not at all when injected into metamorphosing larvae. Analysis of genetic mosaics indicates that the awdb3 mutation is expressed in a cell-autonomous manner in wing, leg, and eye-antenna discs. The larval brain and proventriculus in awdb3 homozygous third-instar larvae appear to be vacuolated due to the accumulation of lipid droplets. Mutant ovaries are unable to develop when injected into wild-type larvae, although mutant germ cells are capable of producing normal eggs. PMID:3137111

Dearolf, C R; Hersperger, E; Shearn, A

1988-09-01

398

Investigation of new dominant-negative inhibitors of anthrax protective antigen mutants for use in therapy and vaccination.  

PubMed

The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 microg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1 beta (IL-1 beta) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections. PMID:19620345

Cao, Sha; Guo, Aizhen; Liu, Ziduo; Tan, Yadi; Wu, Gaobing; Zhang, Chengcai; Zhao, Yaxing; Chen, Huanchun

2009-07-20

399

Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine.  

PubMed

Cocaine is a widely abused drug without a U.S. Food and Drug Administration-approved medication. There is a recognized, promising anticocaine medication to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway [i.e., cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma]. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against (-)-cocaine but also certain selectivity for (-)-cocaine over neurotransmitter acetylcholine (ACh), such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. The present study accounting for the mutation-caused changes of the catalytic activities of BChE against both (-)-cocaine and ACh by means of molecular modeling and site-directed mutagenesis has led to identification of three BChE mutants that have not only a considerably improved catalytic efficiency against (-)-cocaine but also the desirable selectivity for (-)-cocaine over ACh. Two representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg). Pretreatment with the BChE mutant (i.e., 1 min before cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. In particular, all mice pretreated with the mutant (e.g., 0.02 mg or more of A199S/F227A/S287G/A328W/E441D BChE) survived. The in vivo data reveal the primary factor (i.e., the relative catalytic efficiency), determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment. PMID:20971807

Xue, Liu; Ko, Mei-Chuan; Tong, Min; Yang, Wenchao; Hou, Shurong; Fang, Lei; Liu, Junjun; Zheng, Fang; Woods, James H; Tai, Hsin-Hsiung; Zhan, Chang-Guo

2010-10-22

400

Rescue of mice homozygous for lethal albino deletions: implications for an animal model for the human liver disease tyrosinemia type 1.  

PubMed

Mice homozygous for specific deletions around the albino locus on chromosome 7 die within the first few hours of birth. They have a complex phenotype in liver and kidney, which includes multiple changes in gene expression and ultrastructural abnormalities. On the basis of this phenotype, it was proposed that these deletions remove a regulatory locus, alf or hsdr-1. Recently, we and others showed that the gene for fumarylacetoacetate hydrolase (Fah), an enzyme involved in tyrosine catabolism, was disrupted by the lethal albino deletion c14CoS. The finding that the Fah gene in wild-type mice is highly expressed only in cell types that develop a phenotype in mutants, and the fact that Fah deficiency determines the human liver disease hereditary tyrosinemia type 1 (HT1), suggested that disruption of the Fah gene was responsible for the lethal albino phenotype. To test this hypothesis, we have created lines of mice carrying Fah transgenes. We find that c14CoS homozygotes which express transgenic Fah are complemented for all aspects of the complex lethal albino phenotype. Moreover, the degree to which the phenotype is corrected depends on the level of transgenic Fah expression. These results unequivocally establish Fah as the gene mapping at alf/hsdr-1 and prove that the phenotype depends ultimately on the blockage of tyrosine metabolism. Finally, they suggest lethal albino mice as an animal model for HT1. PMID:8253377

Kelsey, G; Ruppert, S; Beermann, F; Grund, C; Tanguay, R M; Schütz, G

1993-12-01

401

Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung  

PubMed Central

A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immun