Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

PubMed Central

Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

2013-01-01

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

Cry64Ba and Cry64Ca, Two ETX/MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests.

PubMed

Liu, Yonglei; Wang, Yinglong; Shu, Changlong; Lin, Kejian; Song, Fuping; Bravo, Alejandra; Soberón, Mario; Zhang, Jie

2018-02-01

Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from Bt strain 1012 that showed toxicity against two important hemipteran rice pests, Laodelphax striatellus and Sogatella furcifera Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the β-pore-forming toxins. Coexpression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73 - resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis , Plutella xylostella , or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of the Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles isolated from L. striatellus Cry64Ba and Cry64Ca are Bt Cry toxins highly effective against hemipteran pests and could provide a novel strategy for the environmentally friendly biological control of rice planthoppers in transgenic plants. IMPORTANCE In Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been shown to have activity against rice planthoppers. We cloned two Bt toxin genes from Bt strain 1012 that showed toxicity against small brown planthoppers ( Laodelphax striatellus ) and white-backed planthoppers ( Sogatella furcifera ). To our knowledge, the proteins encoded by the cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins with activity against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some lepidopteran or coleopteran pests. These two proteins should be able to be used for integrated hemipteran pest management. Copyright © 2018 American Society for Microbiology.

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

PubMed

Agaisse, H; Lereclus, D

1994-08-01

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.

Hypothesis driven single cell dual oscillator mathematical model of circadian rhythms

PubMed Central

S, Shiju

2017-01-01

Molecular mechanisms responsible for 24 h circadian oscillations, entrainment to external cues, encoding of day length and the time-of-day effects have been well studied experimentally. However, it is still debated from the molecular network point of view whether each cell in suprachiasmatic nuclei harbors two molecular oscillators, where one tracks dawn and the other tracks dusk activities. A single cell dual morning and evening oscillator was proposed by Daan et al., based on the molecular network that has two sets of similar non-redundant per1/cry1 and per2/cry2 circadian genes and each can independently maintain their endogenous oscillations. Understanding of dual oscillator dynamics in a single cell at molecular level may provide insight about the circadian mechanisms that encodes day length variations and its response to external zeitgebers. We present here a realistic dual oscillator model of circadian rhythms based on the series of hypotheses proposed by Daan et al., in which they conjectured that the circadian genes per1/cry1 track dawn while per2/cry2 tracks dusk and they together constitute the morning and evening oscillators (dual oscillator). Their hypothesis also provides explanations about the encoding of day length in terms of molecular mechanisms of per/cry expression. We frame a minimal mathematical model with the assumption that per1 acts a morning oscillator and per2 acts as an evening oscillator and to support and interpret this assumption we fit the model to the experimental data of per1/per2 circadian temporal dynamics, phase response curves (PRC's), and entrainment phenomena under various light-dark conditions. We also capture different patterns of splitting phenomena by coupling two single cell dual oscillators with neuropeptides vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) as the coupling agents and provide interpretation for the occurrence of splitting in terms of ME oscillators, though they are not required to explain the morning and evening oscillators. The proposed dual oscillator model based on Daan's hypothesis supports per1 and per2 playing the role of morning and evening oscillators respectively and this may be the first step towards the understanding of the core molecular mechanism responsible for encoding the day length. PMID:28486525

Hypothesis driven single cell dual oscillator mathematical model of circadian rhythms.

PubMed

S, Shiju; Sriram, K

2017-01-01

Molecular mechanisms responsible for 24 h circadian oscillations, entrainment to external cues, encoding of day length and the time-of-day effects have been well studied experimentally. However, it is still debated from the molecular network point of view whether each cell in suprachiasmatic nuclei harbors two molecular oscillators, where one tracks dawn and the other tracks dusk activities. A single cell dual morning and evening oscillator was proposed by Daan et al., based on the molecular network that has two sets of similar non-redundant per1/cry1 and per2/cry2 circadian genes and each can independently maintain their endogenous oscillations. Understanding of dual oscillator dynamics in a single cell at molecular level may provide insight about the circadian mechanisms that encodes day length variations and its response to external zeitgebers. We present here a realistic dual oscillator model of circadian rhythms based on the series of hypotheses proposed by Daan et al., in which they conjectured that the circadian genes per1/cry1 track dawn while per2/cry2 tracks dusk and they together constitute the morning and evening oscillators (dual oscillator). Their hypothesis also provides explanations about the encoding of day length in terms of molecular mechanisms of per/cry expression. We frame a minimal mathematical model with the assumption that per1 acts a morning oscillator and per2 acts as an evening oscillator and to support and interpret this assumption we fit the model to the experimental data of per1/per2 circadian temporal dynamics, phase response curves (PRC's), and entrainment phenomena under various light-dark conditions. We also capture different patterns of splitting phenomena by coupling two single cell dual oscillators with neuropeptides vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) as the coupling agents and provide interpretation for the occurrence of splitting in terms of ME oscillators, though they are not required to explain the morning and evening oscillators. The proposed dual oscillator model based on Daan's hypothesis supports per1 and per2 playing the role of morning and evening oscillators respectively and this may be the first step towards the understanding of the core molecular mechanism responsible for encoding the day length.

A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy

PubMed Central

Zeng, Xianxu; Tate, Rebecca E.; McKee, Mary L.; Capen, Diane E.; Zhang, Zhan; Tanzi, Rudolph E.; Zhou, Chao

2015-01-01

Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function. PMID:26348211

Cross-resistance to purified Bt proteins, Bt corn and Bt cotton in a Cry2Ab2-corn resistant strain of Spodoptera frugiperda.

PubMed

Yang, Fei; Kerns, David L; Head, Graham P; Price, Paula; Huang, Fangneng

2017-12-01

Gene-pyramiding by combining two or more dissimilar Bacillus thuringiensis (Bt) proteins into a crop has been used to delay insect resistance. The durability of gene-pyramiding can be reduced by cross-resistance. Fall armyworm, Spodoptera frugiperda, is a major target pest of the Cry2Ab2 protein used in pyramided Bt corn and cotton. Here, we provide the first experimental evaluation of cross-resistance in S. frugiperda selected with Cry2Ab2 corn to multiple Bt sources including purified Bt proteins, Bt corn and Bt cotton. Concentration - response bioassays showed that resistance ratios for Cry2Ab2-resistant (RR) relative to Cry2Ab2-susceptible (SS) S. frugiperda were -1.4 for Cry1F, 1.2 for Cry1A.105, >26.7 for Cry2Ab2, >10.0 for Cry2Ae and -1.1 for Vip3A. Larvae of Cry2Ab2-heterozygous (RS), SS and RR S. frugiperda were all susceptible to Bt corn and Bt cotton containing Cry1 (Cry1F or Cry1A.105) and/or Vip3A proteins. Pyramided Bt cotton containing Cry1Ac + Cry2Ab2 or Cry1Ab + Cry2Ae were also effective against SS and RS, but not RR. These findings suggest that Cry2Ab2-corn-selected S. frugiperda is not cross-resistant to Cry1F, Cry1A.105 or Vip3A protein, or corn and cotton plants containing these Bt proteins, but it can cause strong cross-resistance to Cry2Ae and Bt crops expressing similar Bt proteins. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

The distinct properties of natural and GM cry insecticidal proteins.

PubMed

Latham, Jonathan R; Love, Madeleine; Hilbeck, Angelika

2017-04-01

The Cry toxins are a family of crystal-forming proteins produced by the bacterium Bacillus thuringiensis. Their mode of action is thought to be to create pores that disrupt the gut epithelial membranes of juvenile insects. These pores allow pathogen entry into the hemocoel, thereby killing the insect. Genes encoding a spectrum of Cry toxins, including Cry mutants, Cry chimaeras and other Cry derivatives, are used commercially to enhance insect resistance in genetically modified (GM) crops. In most countries of the world, such GM crops are regulated and must be assessed for human and environmental safety. However, such risk assessments often do not test the GM crop or its tissues directly. Instead, assessments rely primarily on historical information from naturally occurring Cry proteins and on data collected on Cry proteins (called 'surrogates') purified from laboratory strains of bacteria engineered to express Cry protein. However, neither surrogates nor naturally occurring Cry proteins are identical to the proteins to which humans or other nontarget organisms are exposed by the production and consumption of GM plants. To-date there has been no systematic survey of these differences. This review fills this knowledge gap with respect to the most commonly grown GM Cry-containing crops approved for international use. Having described the specific differences between natural, surrogate and GM Cry proteins this review assesses these differences for their potential to undermine the reliability of risk assessments. Lastly, we make specific recommendations for improving risk assessments.

Expression of a germline human kappa chain-associated cross-reactive idiotype after in vitro and in vivo infection with Epstein-Barr virus.

PubMed

Silverman, G J; Carson, D A; Patrick, K; Vaughan, J H; Fong, S

1987-06-01

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.

Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection.

PubMed Central

Cerón, J; Ortíz, A; Quintero, R; Güereca, L; Bravo, A

1995-01-01

In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper. PMID:8526493

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii[W

PubMed Central

Beel, Benedikt; Prager, Katja; Spexard, Meike; Sasso, Severin; Weiss, Daniel; Müller, Nico; Heinnickel, Mark; Dewez, David; Ikoma, Danielle; Grossman, Arthur R.; Kottke, Tilman; Mittag, Maria

2012-01-01

Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light–activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities. PMID:22773746

Laboratory evaluation of transgenic Populus davidiana×Populus bolleana expressing Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3 genes against gypsy moth and fall webworm.

PubMed

Ding, Liping; Chen, Yajuan; Wei, Xiaoli; Ni, Mi; Zhang, Jiewei; Wang, Hongzhi; Zhu, Zhen; Wei, Jianhua

2017-01-01

Transgenic poplar lines 'Shanxin' (Populus davidiana×Populus bolleana) were generated via Agrobacterium-mediated transformation. The transgenic lines carried the expression cassettes of Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3, respectively. The expression levels of the exogenous insect resistance genes in the transgenic lines were determined by Q-PCR and Western blot. Leaves of the transgenic lines were used for insect feeding bioassays on first instar larvae of the gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). At 5 d of feeding, the mean mortalities of larvae feeding on Cry1Ac + SCK and Cry1Ah3 transgenic poplars leaves were 97% and 91%, while mortality on Cry9Aa3 transgenic lines was about 49%. All gypsy moth and fall webworm larvae were killed in 7-9 days after feeding on leaves from Cry1Ac + SCK or Cry1Ah3 transgenic poplars, while all the fall webworm larvae were killed in 11 days and about 80% of gypsy moth larvae were dead in 14 days after feeding on those from Cry9Aa3 transgenic lines. It was concluded that the transgenic lines of Cry1Ac + SCK and Cry1Ah3 were highly toxic to larvae of both insect species while lines with Cry9Aa3 had lower toxicity,and H. cunea larvae are more sensitive to the insecticidal proteins compared to L. dispar. Transgenic poplar lines toxic to L. dispar and H. cunea could be used to provide Lepidoptera pest resistance to selected strains of poplar trees.

Changes in growth conditions alter the male strobilus gene expression pattern in Cryptomeria japonica.

PubMed

Fukui, Mitsue

2003-11-01

Two-year old saplings grown from cuttings of Cryptomeria japonica D. Don initiate strobilus development following treatment with gibberellic acid under long-day photoperiods. At 25 degrees C with a 14-h photoperiod in a phytotron, male strobili initiated normally; however, they remained green and fell from the saplings prematurely. To examine the change in male strobilus development at the molecular level, three genes expressed specifically in male strobili were analyzed. Two were MADS box genes homologous to the B-function genes in angiosperms, CjMADS1 and CjMADS2, and the third was Cry j I, which encodes an allergen protein, and this gene is expressed mainly in microspores. Under phytotron growing conditions, the homeotic genes were expressed constantly, which reflected the extended early developmental stage of male strobili. On the other hand, Cry j I expression was detected after a long delay just before strobilus development ceased. These results indicate that the expression of the genes related to male reproductive development in C. japonica is regulated by a factor(s) that is sensitive to environmental signals.

Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin.

PubMed

Oppert, Brenda; Dowd, Scot E; Bouffard, Pascal; Li, Lewyn; Conesa, Ana; Lorenzen, Marcé D; Toutges, Michelle; Marshall, Jeremy; Huestis, Diana L; Fabrick, Jeff; Oppert, Cris; Jurat-Fuentes, Juan Luis

2012-01-01

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome.

Transcriptome Profiling of the Intoxication Response of Tenebrio molitor Larvae to Bacillus thuringiensis Cry3Aa Protoxin

PubMed Central

Oppert, Brenda; Dowd, Scot E.; Bouffard, Pascal; Li, Lewyn; Conesa, Ana; Lorenzen, Marcé D.; Toutges, Michelle; Marshall, Jeremy; Huestis, Diana L.; Fabrick, Jeff; Oppert, Cris; Jurat-Fuentes, Juan Luis

2012-01-01

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome. PMID:22558093

The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis

PubMed Central

Saleem, Faiza; Shakoori, Abdul Rauf

2017-01-01

The Cry (crystal) proteins from Bacillus thuringiensis are known to have toxicity against a variety of insects and have been exploited to control insect pests through transgenic plants and biopesticides. B. thuringiensis SBS BT-1 carrying the cry2 genes was isolated from soil samples in Pakistan. The 2-kb full length cry2Ac gene was cloned, sequenced, and submitted to the EMBL DNA database (Accession No. AM292031). For expression analysis, Escherichia coli DH5α was transformed with the fragment sub-cloned in pET22b expression vector using NdeI and HindIII restriction sites, and later confirmed by restriction endonuclease analysis. To assess the toxicity of Cry2Ac7 protein against lepidopteran and dipteran insects, BL21 (codon plus) strain of E. coli was further transformed with the recombinant plasmid. The 65-kDa protein was expressed in the form of inclusion bodies up to 180 OD units per liter of the medium. Inclusions were washed with a buffer containing 1.5% Triton-X 100 and >90% pure Cry2Ac7 was obtained. The inclusion bodies were dissolved in 50 mM K2CO3 (pH 11.5), dialyzed, and freeze-dried. This freeze-dried protein as well as inclusion bodies were used in bioassays against larvae of Helicoverpa armigera and Musca domestica. The freeze-dried protein was toxic to H. armigera larvae with an LC50 value of 131 ng/mL. However, Cry2Ac7 produced in E. coli did not show any mortality to M. domestica larvae. This is the first report of Cry2Ac protein toxic to H. armigera. PMID:29099767

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

PubMed

Ghosh, Gourab; Ganguly, Shreeparna; Purohit, Arnab; Chaudhuri, Rituparna Kundu; Das, Sampa; Chakraborti, Dipankar

2017-07-01

Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T 0 transformants were selected on the basis of PCR and protein expression profile. T 1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T 1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T 2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T 3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T 0 , T 1, and T 2 ). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

Toxin Gene Contents and Activity of Bacillus thuringiensis Strains Against Two Sugarcane Borer Species, Diatraea saccharalis (F.) and D. flavipennella (Box).

PubMed

Silva, L M; Silva, M C; Silva, S M F A; Alves, R C; Siqueira, H A A; Marques, E J

2018-04-01

Bacillus thuringiensis (Berliner) bears essential characteristics in the control of insect pests, such as its unique mode of action, which confers specificity and selectivity. This study assessed cry gene contents from Bt strains and their entomotoxicity against Diatraea saccharalis (F.) and Diatraea flavipennella (Box) (Lepidoptera: Crambidae). Bioassays with Bt strains were performed against neonates to evaluate their lethal and sublethal activities and were further analyzed by PCR, using primers to identify toxin genes. For D. saccharalis and D. flavipennella, 16 and 18 strains showed over 30% larval mortality in the 7th day, respectively. The LC 50 values of strains for D. saccharalis varied from 0.08 × 10 5 (LIIT-0105) to 4104 × 10 5 (LIIT-2707) spores + crystals mL -1 . For D. flavipennella, the LC 50 values of strains varied from 0.40 × 10 5 (LIIT-2707) to 542 × 10 5 (LIIT-2109) spores + crystals mL -1 . For the LIIT-0105 strain, which was the most toxic to D. saccharalis, the genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, cry8, and cry9C were detected, whereas for the strain LIIT-2707, which was the most toxic to D. flavipennella, detected genes were cry1Aa, cry1Ab, cry1Ac, cry1B, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, and cry9. The toxicity data and toxin gene content in these strains of Bt suggest a great variability of activity with potential to be used in the development of novel biopesticides or as source of resistance genes that can be expressed in plants to control pests.

A Study of Gibberellin Homeostasis and Cryptochrome-Mediated Blue Light Inhibition of Hypocotyl Elongation1[W][OA

PubMed Central

Zhao, Xiaoying; Yu, Xuhong; Foo, Eloise; Symons, Gregory M.; Lopez, Javier; Bendehakkalu, Krishnaprasad T.; Xiang, Jing; Weller, James L.; Liu, Xuanming; Reid, James B.; Lin, Chentao

2007-01-01

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA4 in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA4 content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs. PMID:17644628

Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

PubMed

Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

2016-01-01

Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.

Effects of water management practices on residue decomposition and degradation of Cry1Ac protein from crop-wild Bt rice hybrids and parental lines during winter fallow season.

PubMed

Xiao, Manqiu; Dong, Shanshan; Li, Zhaolei; Tang, Xu; Chen, Yi; Yang, Shengmao; Wu, Chunyan; Ouyang, Dongxin; Fang, Changming; Song, Zhiping

2015-12-01

Rice is the staple diet of over half of the world's population and Bacillus thuringiensis (Bt) rice expressing insecticidal Cry proteins is ready for deployment. An assessment of the potential impact of Bt rice on the soil ecosystem under varied field management practices is urgently required. We used litter bags to assess the residue (leaves, stems and roots) decomposition dynamics of two transgenic rice lines (Kefeng6 and Kefeng8) containing stacked genes from Bt and sck (a modified CpTI gene encoding a cowpea trypsin inhibitor) (Bt/CpTI), a non-transgenic rice near-isoline (Minghui86), wild rice (Oryza rufipogon) and crop-wild Bt rice hybrid under contrasting conditions (drainage or continuous flooding) in the field. No significant difference was detected in the remaining mass, total C and total N among cultivars under aerobic conditions, whereas significant differences in the remaining mass and total C were detected between Kefeng6 and Kefeng8 and Minghui86 under the flooded condition. A higher decomposition rate constant (km) was measured under the flooded condition compared with the aerobic condition for leaf residues, whereas the reverse was observed for root residues. The enzyme-linked immunosorbent assay (ELISA), which was used to monitor the changes in the Cry1Ac protein in Bt rice residues, indicated that (1) the degradation of the Cry1Ac protein under both conditions best fit first-order kinetics, and the predicted DT50 (50% degradation time) of the Cry1Ac protein ranged from 3.6 to 32.5 days; (2) the Cry1Ac protein in the residue degraded relatively faster under aerobic conditions; and (3) by the end of the study (~154 days), the protein was present at a low concentration in the remaining residues under both conditions. The degradation rate constant was negatively correlated with the initial carbon content and positively correlated with the initial Cry1Ac protein concentration, but it was only correlated with the mass decomposition rate constants under the flooded condition. No Cry1Ac protein was detected in the soils surrounding the buried residue. Our results did not reveal any evidence that the stacked genes (Bt/CpTI) or the presence of the Cry1Ac protein influenced the decomposition dynamics of the rice residues. Furthermore, our results suggested that field drainage after residue incorporation would promote Cry1Ac protein degradation. Copyright © 2015 Elsevier Inc. All rights reserved.

Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline.

PubMed

Ye, Weixing; Zhu, Lei; Liu, Yingying; Crickmore, Neil; Peng, Donghai; Ruan, Lifang; Sun, Ming

2012-07-01

We have designed a high-throughput system for the identification of novel crystal protein genes (cry) from Bacillus thuringiensis strains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence of B. thuringiensis using next-generation sequencing biotechnology, and (ii) to identify cry genes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data were de novo assembled, and a total of 113 candidate cry sequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to known cry genes, and eight full-length genes were obtained with PCR. Finally, three new cry-type genes (primary ranks) and five cry holotypes, which were designated cry8Ac1, cry7Ha1, cry21Ca1, cry32Fa1, and cry21Da1 by the B. thuringiensis Toxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novel cry genes.

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

PubMed

Ribeiro, Thuanne Pires; Arraes, Fabricio Barbosa Monteiro; Lourenço-Tessutti, Isabela Tristan; Silva, Marilia Santos; Lisei-de-Sá, Maria Eugênia; Lucena, Wagner Alexandre; Macedo, Leonardo Lima Pepino; Lima, Janaina Nascimento; Santos Amorim, Regina Maria; Artico, Sinara; Alves-Ferreira, Márcio; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

2017-08-01

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T 0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2 -ΔΔCt analyses revealed that T 0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T 0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g -1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T 0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T 1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g -1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

INSECTICIDAL TOXIN FROM BACILLUS THURINGIENSIS IS RELEASED FROM ROOTS OF TRANSGENIC BT CORN IN VITRO AND IN SITU. (R826107)

EPA Science Inventory

Abstract

The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a...

Cry-Bt identifier: a biological database for PCR detection of Cry genes present in transgenic plants.

PubMed

Singh, Vinay Kumar; Ambwani, Sonu; Marla, Soma; Kumar, Anil

2009-10-23

We describe the development of a user friendly tool that would assist in the retrieval of information relating to Cry genes in transgenic crops. The tool also helps in detection of transformed Cry genes from Bacillus thuringiensis present in transgenic plants by providing suitable designed primers for PCR identification of these genes. The tool designed based on relational database model enables easy retrieval of information from the database with simple user queries. The tool also enables users to access related information about Cry genes present in various databases by interacting with different sources (nucleotide sequences, protein sequence, sequence comparison tools, published literature, conserved domains, evolutionary and structural data). http://insilicogenomics.in/Cry-btIdentifier/welcome.html.

Identification of Cha o 3 homolog Cry j 4 from Cryptomeria japonica (Japanese cedar) pollen: Limitation of the present Japanese cedar-specific ASIT.

PubMed

Osada, Toshihiro; Tanaka, Yuki; Yamada, Akira; Sasaki, Eiji; Utsugi, Teruhiro

2018-03-07

About one-third of the Japanese population suffers from Japanese cedar pollinosis, which is frequently accompanied by Japanese cypress pollinosis. Recently, a novel major Japanese cypress pollen allergen, Cha o 3, was discovered. However, whether a Cha o 3 homolog is present in Japanese cedar pollen remains to be determined. Western blot analysis was performed using Cha o 3-specific antiserum. In addition, cloning of the gene encoding Cry j 4 was conducted using total cDNA from the male flower of Japanese cedar trees. Allergen potency and cross-reactivity were investigated using a T-cell proliferation assay, basophil activation test, and ImmunoCAP inhibition assay. A low amount of Cha o 3 homolog protein was detected in Japanese cedar pollen extract. The deduced amino acid sequence of Cry j 4 showed 84% identity to that of Cha o 3. Cross-reactivity between Cry j 4 and Cha o 3 was observed at the T cell and IgE levels. Cry j 4 was discovered as a counterpart allergen of Cha o 3 in Japanese cedar pollen, with a relationship similar to that between Cry j 1-Cha o 1 and Cry j 2-Cha o 2. Our findings also suggest that allergen-specific immunotherapy (ASIT) using Japanese cedar pollen extract does not induce adequate immune tolerance to Cha o 3 due to the low amount of Cry j 4 in Japanese cedar pollen. Therefore, ASIT using Cha o 3 or cypress pollen extract coupled with Japanese cedar pollen extract is required in order to optimally control allergy symptoms during Japanese cypress pollen season. Copyright © 2018 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

Non-canonical Phototransduction Mediates Synchronization of the Drosophila melanogaster Circadian Clock and Retinal Light Responses.

PubMed

Ogueta, Maite; Hardie, Roger C; Stanewsky, Ralf

2018-06-04

The daily light-dark cycles represent a key signal for synchronizing circadian clocks. Both insects and mammals possess dedicated "circadian" photoreceptors but also utilize the visual system for clock resetting. In Drosophila, circadian clock resetting is achieved by the blue-light photoreceptor cryptochrome (CRY), which is expressed within subsets of the brain clock neurons. In addition, rhodopsin-expressing photoreceptor cells contribute to light synchronization. Light resets the molecular clock by CRY-dependent degradation of the clock protein Timeless (TIM), although in specific subsets of key circadian pacemaker neurons, including the small ventral lateral neurons (s-LNvs), TIM and Period (PER) oscillations can be synchronized by light independent of CRY and canonical visual Rhodopsin phototransduction. Here, we show that at least three of the seven Drosophila rhodopsins can utilize an alternative transduction mechanism involving the same α-subunit of the heterotrimeric G protein operating in canonical visual phototransduction (Gq). Surprisingly, in mutants lacking the canonical phospholipase C-β (PLC-β) encoded by the no receptor potential A (norpA) gene, we uncovered a novel transduction pathway using a different PLC-β encoded by the Plc21C gene. This novel pathway is important for behavioral clock resetting to semi-natural light-dark cycles and mediates light-dependent molecular synchronization within the s-LNv clock neurons. The same pathway appears to be responsible for norpA-independent light responses in the compound eye. We show that Rhodopsin 5 (Rh5) and Rh6, present in the R8 subset of retinal photoreceptor cells, drive both the long-term circadian and rapid light responses in the eye. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Clock genes and salt-sensitive hypertension: a new type of aldosterone-synthesizing enzyme controlled by the circadian clock and angiotensin II.

PubMed

Okamura, Hitoshi; Doi, Masao; Goto, Kaoru; Kojima, Rika

2016-10-01

With the current societal norm of shiftwork and long working hours, maintaining a stable daily life is becoming very difficult. An irregular lifestyle disrupts circadian rhythms, resulting in the malfunction of body physiology and ultimately leading to lifestyle-related diseases, including hypertension. By analyzing completely arrhythmic Cry1/Cry2 double-knockout (Cry-null) mice, we found salt-sensitive hypertension accompanied by hyperaldosteronism. On the basis of a DNA microarray analysis of the adrenal gland and subsequent biochemical analyses, we discovered that Hsd3b6/HSD3B1, a subtype of 3β-HSD, is markedly overexpressed in aldosterone-producing cells in the Cry-null adrenal cortex. In addition, we found that Hsd3b6/HSD3B1, which converts pregnenolone to progesterone, is a clock-controlled gene and might also be a key enzyme for the regulation of aldosterone biosynthesis, in addition to the previously established CYP11B2, which synthesizes aldosterone from deoxycorticosterone. Importantly, angiotensin II induces HSD3B1 via the transcription factor NGFIB in human adrenocortical H295R cells, similarly to CYP11B2. As HSD3B1 levels are abnormally high in the adrenal aldosterone-producing cells of idiopathic hyperaldosteronism (IHA), the temporal component of this system in the pathophysiology of IHA is a promising area for future research.

Differential regulation of mammalian Period genes and circadian rhythmicity by cryptochromes 1 and 2

PubMed Central

Vitaterna, Martha Hotz; Selby, Christopher P.; Todo, Takeshi; Niwa, Hitoshi; Thompson, Carol; Fruechte, Ethan M.; Hitomi, Kenichi; Thresher, Randy J.; Ishikawa, Tomoko; Miyazaki, Junichi; Takahashi, Joseph S.; Sancar, Aziz

1999-01-01

Cryptochromes regulate the circadian clock in animals and plants. Humans and mice have two cryptochrome (Cry) genes. A previous study showed that mice lacking the Cry2 gene had reduced sensitivity to acute light induction of the circadian gene mPer1 in the suprachiasmatic nucleus (SCN) and had an intrinsic period 1 hr longer than normal. In this study, Cry1−/− and Cry1−/−Cry2−/− mice were generated and their circadian clocks were analyzed at behavioral and molecular levels. Behaviorally, the Cry1−/− mice had a circadian period 1 hr shorter than wild type and the Cry1−/−Cry2−/− mice were arrhythmic in constant darkness (DD). Biochemically, acute light induction of mPer1 mRNA in the SCN was blunted in Cry1−/− and abolished in Cry1−/−Cry2−/− mice. In contrast, the acute light induction of mPer2 in the SCN was intact in Cry1−/− and Cry1−/−Cry2−/− animals. Importantly, in double mutants, mPer1 expression was constitutively elevated and no rhythmicity was detected in either 12-hr light/12-hr dark or DD, whereas mPer2 expression appeared rhythmic in 12-hr light/12-hr dark, but nonrhythmic in DD with intermediate levels. These results demonstrate that Cry1 and Cry2 are required for the normal expression of circadian behavioral rhythms, as well as circadian rhythms of mPer1 and mPer2 in the SCN. The differential regulation of mPer1 and mPer2 by light in Cry double mutants reveals a surprising complexity in the role of cryptochromes in mammals. PMID:10518585

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

PubMed Central

Song, Xiaozhao; Kain, Wendy; Cassidy, Douglas

2015-01-01

The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism. PMID:26025894

Evaluation of Bt Corn with Pyramided Genes on Efficacy and Insect Resistance Management for the Asian Corn Borer in China.

PubMed

Jiang, Fan; Zhang, Tiantao; Bai, Shuxiong; Wang, Zhenying; He, Kanglai

2016-01-01

A Bt corn hybrid (AcIe) with two Bt genes (cry1Ie and cry1Ac) was derived by breeding stack from line expressing Cry1Ie and a line expressing Cry1Ac. Efficacy of this pyramided Bt corn hybrid against the Asian corn borer (ACB), Ostrinia furnacalis, was evaluated. We conducted laboratory bioassays using susceptible and resistant ACB strains fed on artificial diet or fresh plant tissues. We also conducted field trials with artificial infestations of ACB neonates at the V6 and silk stages. The toxin-diet bioassay data indicated that mixtures of Cry1Ac and Cry1Ie proteins had synergistic insecticidal efficacy. The plant tissue bioassay data indicated that Bt corn hybrids expressing either a single toxin (Cry1Ac or Cry1Ie) or two toxins had high efficacy against susceptible ACB. Damage ratings in the field trials indicated that the Bt corn hybrids could effectively protect against 1st and the 2nd generation ACB in China. The hybrid line with two Bt genes showed a higher efficacy against ACB larvae resistant to Cry1Ac or CryIe than the hybrid containing one Bt gene, and the two gene hybrid would have increased potential for managing or delaying the evolution of ACB resistance to Bt corn plants.

The midgut cadherin-like gene is not associated with resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella (L.).

PubMed

Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

2015-03-01

The Gram-positive bacterium Bacillus thuringiensis (Bt) produces Cry toxins that have been used to control important agricultural pests. Evolution of resistance in target pests threatens the effectiveness of these toxins when used either in sprayed biopesticides or in Bt transgenic crops. Although alterations of the midgut cadherin-like receptor can lead to Bt Cry toxin resistance in many insects, whether the cadherin gene is involved in Cry1Ac resistance of Plutella xylostella (L.) remains unclear. Here, we present experimental evidence that resistance to Cry1Ac or Bt var. kurstaki (Btk) in P. xylostella is not due to alterations of the cadherin gene. The bona fide P. xylostella cadherin cDNA sequence was cloned and analyzed, and comparisons of the cadherin cDNA sequence among susceptible and resistant P. xylostella strains confirmed that Cry1Ac resistance was independent of mutations in this gene. In addition, real-time quantitative PCR (qPCR) indicated that cadherin transcript levels did not significantly differ among susceptible and resistant P. xylostella strains. RNA interference (RNAi)-mediated suppression of cadherin gene expression did not affect larval susceptibility to Cry1Ac toxin. Furthermore, genetic linkage assays using four cadherin gDNA allelic biomarkers confirmed that the cadherin gene is not linked to resistance against Cry1Ac in P. xylostella. Taken together, our findings demonstrate that Cry1Ac resistance of P. xylostella is independent of the cadherin gene. Copyright © 2015 Elsevier Inc. All rights reserved.

Performance and cross-crop resistance of Cry1F-maize selected Spodoptera frugiperda on transgenic Bt cotton: implications for resistance management

PubMed Central

Yang, Fei; Kerns, David L.; Brown, Sebe; Kurtz, Ryan; Dennehy, Tim; Braxton, Bo; Head, Graham; Huang, Fangneng

2016-01-01

Transgenic crops producing Bacillus thuringiensis (Bt) proteins have become a primary tool in pest management. Due to the intensive use of Bt crops, resistance of the fall armyworm, Spodoptera frugiperda, to Cry1F maize has occurred in Puerto Rico, Brazil, and some areas of the southeastern U.S. The sustainability of Bt crops faces a great challenge because the Cry1F-maize resistant S. frugiperda may also infest other Bt crops in multiple cropping ecosystems. Here we examined the survival and plant injury of a S. frugiperda population selected with Cry1F maize on three single-gene and five pyramided Bt cotton products. Larvae of Cry1F-susceptible (SS), -heterozygous (RS), and -resistant (RR) genotypes of S. frugiperda were all susceptible to the pyramided cotton containing Cry1Ac/Cry2Ab, Cry1Ac/Cry1F/Vip3A, Cry1Ab/Cry2Ae, or Cry1Ab/Cry2Ae/Vip3A, and the single-gene Cry2Ae cotton. Pyramided cotton containing Cry1Ac/Cry1F was effective against SS and RS, but not for RR. These findings show that the Cry1F-maize selected S. frugiperda can cause cross-crop resistance to other Bt crops expressing similar insecticidal proteins. Resistance management and pest management programs that utilize diversify mortality factors must be implemented to ensure the sustainability of Bt crops. This is especially important in areas where resistance to single-gene Bt crops is already widespread. PMID:27301612

Performance and cross-crop resistance of Cry1F-maize selected Spodoptera frugiperda on transgenic Bt cotton: implications for resistance management.

PubMed

Yang, Fei; Kerns, David L; Brown, Sebe; Kurtz, Ryan; Dennehy, Tim; Braxton, Bo; Head, Graham; Huang, Fangneng

2016-06-15

Transgenic crops producing Bacillus thuringiensis (Bt) proteins have become a primary tool in pest management. Due to the intensive use of Bt crops, resistance of the fall armyworm, Spodoptera frugiperda, to Cry1F maize has occurred in Puerto Rico, Brazil, and some areas of the southeastern U.S. The sustainability of Bt crops faces a great challenge because the Cry1F-maize resistant S. frugiperda may also infest other Bt crops in multiple cropping ecosystems. Here we examined the survival and plant injury of a S. frugiperda population selected with Cry1F maize on three single-gene and five pyramided Bt cotton products. Larvae of Cry1F-susceptible (SS), -heterozygous (RS), and -resistant (RR) genotypes of S. frugiperda were all susceptible to the pyramided cotton containing Cry1Ac/Cry2Ab, Cry1Ac/Cry1F/Vip3A, Cry1Ab/Cry2Ae, or Cry1Ab/Cry2Ae/Vip3A, and the single-gene Cry2Ae cotton. Pyramided cotton containing Cry1Ac/Cry1F was effective against SS and RS, but not for RR. These findings show that the Cry1F-maize selected S. frugiperda can cause cross-crop resistance to other Bt crops expressing similar insecticidal proteins. Resistance management and pest management programs that utilize diversify mortality factors must be implemented to ensure the sustainability of Bt crops. This is especially important in areas where resistance to single-gene Bt crops is already widespread.

Comparisons of Transcriptional Profiles of Gut Genes between Cry1Ab-Resistant and Susceptible Strains of Ostrinia nubilalis Revealed Genes Possibly Related to the Adaptation of Resistant Larvae to Transgenic Cry1Ab Corn.

PubMed

Yao, Jianxiu; Zhu, Yu-Cheng; Lu, Nanyan; Buschman, Lawrent L; Zhu, Kun Yan

2017-01-30

A microarray developed on the basis of 2895 unique transcripts from larval gut was used to compare gut gene expression profiles between a laboratory-selected Cry1Ab-resistant (R) strain and its isoline susceptible (S) strain of the European corn borer (Ostrinia nubilalis) after the larvae were fed the leaves of transgenic corn (MON810) expressing Cry1Ab or its non-transgenic isoline for 6 h. We revealed 398 gut genes differentially expressed (i.e., either up- or down-regulated genes with expression ratio ≥2.0) in S-strain, but only 264 gut genes differentially expressed in R-strain after being fed transgenic corn leaves. Although the percentages of down-regulated genes among the total number of differentially expressed genes (50% in S-strain and 45% in R-strain) were similar between the R- and S-strains, the expression ratios of down-regulated genes were much higher in S-strain than in R-strain. We revealed that 17 and 9 significantly up- or down-regulated gut genes from S and R-strain, respectively, including serine proteases and aminopeptidases. These genes may be associated with Cry1Ab toxicity by degradation, binding, and cellular defense. Overall, our study suggests enhanced adaptation of Cry1Ab-resistant larvae on transgenic Cry1Ab corn as revealed by lower number and lower ratios of differentially expressed genes in R-strain than in S-strain of O. nubilalis.

Midgut transcriptome response to a Cry toxin in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

PubMed

Lei, Yanyuan; Zhu, Xun; Xie, Wen; Wu, Qingjun; Wang, Shaoli; Guo, Zhaojiang; Xu, Baoyun; Li, Xianchun; Zhou, Xuguo; Zhang, Youjun

2014-01-01

To investigate the response of Plutella xylostella transcriptome in defending against a Bt toxin, high-throughput RNA-sequencing was carried out to examine Cry1Ac-susceptible and -resistant strains. The comparative analysis indentified over 2900 differentially expressed unigenes (DEUs) between these two strains. Gene Ontology analysis placed these unigenes primarily into cell, cell part, organelle, binding, catalytic, cellular process, metabolic process, and response to stimulus categories. Based on pathway analyses, DEUs were enriched in oxidoreductase activity and membrane lipid metabolic processes, and they were also significantly enriched in pathways related to the metabolic and biosynthesis of secondary metabolites. Most of the unigenes involved in the metabolic pathway were up-regulated in resistant strains. Within the ABC transporter pathway, majority of the down-regulated unigenes belong to ABCC2 and ABCC10, respectively, while up-regulated unigenes were mainly categorized as ABCG2. Furthermore, two aminopeptidases, and four cadherins encoding genes were significantly elevated as well. This study provides a transcriptome foundation for the identification and functional characterization of genes involved in the Bt resistance in an agriculturally important insect pest, P. xylostella. © 2013 Elsevier B.V. All rights reserved.

Adsorption of Insecticidal Crystal Protein Cry11Aa onto Nano-Mg(OH)2: Effects on Bioactivity and Anti-Ultraviolet Ability.

PubMed

Pan, Xiaohong; Xu, Zhangyan; Li, Lan; Shao, Enshi; Chen, Saili; Huang, Tengzhou; Chen, Zhi; Rao, Wenhua; Huang, Tianpei; Zhang, Lingling; Wu, Songqing; Guan, Xiong

2017-11-01

The traditional Bacillus thuringiensis (Bt) formulations for field applications are not resistant to harsh environmental conditions. Hence, the active ingredients of the Bt bioinsecticides could degrade quickly and has low anti-ultraviolet ability in the field, which significantly limits its practical application. In the present study, we developed an efficient and stable delivery system for Bt Cry11Aa toxins. We coated Cry11Aa proteins with Mg(OH) 2 nanoparticles (MHNPs), and then assessed the effects of MHNPs on bioactivity and anti-ultraviolet ability of the Cry11Aa proteins. Our results indicated that MHNPs, like "coating clothes", could effectively protect the Cry protein and enhance the insecticidal bioactivity after UV radiation (the degradation rate was decreased from 64.29% to 16.67%). In addtion, MHNPs could improve the proteolysis of Cry11Aa in the midgut and aggravate the damage of the Cry protein to the gut epithelial cells, leading to increased insecticidal activity against Culex quinquefasciatus. Our results revealed that MHNPs, as an excellent nanocarrier, could substantially improve the insecticidal bioactivity and anti-ultraviolet ability of Cry11Aa.

Expression of Cry1Ac toxin-binding region in Plutella xyllostella cadherin-like receptor and studying their interaction mode by molecular docking and site-directed mutagenesis.

PubMed

Hu, Xiaodan; Zhang, Xiao; Zhong, Jianfeng; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Lin, Manman; Xu, Chongxin; Lu, Lina; Zhu, Qing; Liu, Xianjin

2018-05-01

Cadherin-like protein has been identified as the primary Bacillus thuringiensis (Bt) Cry toxin receptor in Lepidoptera pests and plays a key role in Cry toxin insecticidal. In this study, we successfully expressed the putative Cry1Ac toxin-binding region (CR7-CR11) of Plutella xylostella cadherin-like in Escherichia coli BL21 (DE3). The expressed CR7-CR11 fragment showed binding ability to Cry1Ac toxin under denaturing (Ligand blot) and non-denaturing (ELISA) conditions. The three-dimensional structure of CR7-CR11 was constructed by homology modeling. Molecular docking results of CR7-CR11 and Cry1Ac showed that domain II and domain III of Cry1Ac were taking part in binding to CR7-CR11, while CR7-CR8 was the region of CR7-CR11 in interacting with Cry1Ac. The interaction of toxin-receptor complex was found to arise from hydrogen bond and hydrophobic interaction. Through the computer-aided alanine mutation scanning, amino acid residues of Cry1Ac (Met341, Asn442 and Ser486) and CR7-CR11 (Asp32, Arg101 and Arg127) were predicted as the hot spot residues involved in the interaction of the toxin-receptor complex. At last, we verified the importance role of these key amino acid residues by binding assay. These results will lay a foundation for further elucidating the insecticidal mechanism of Cry toxin and enhancing Cry toxin insecticidal activity by molecular modification. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular characterization of Cry1D-133 toxin from Bacillus thuringiensis strain HD133 and its toxicity against Spodoptera littoralis.

PubMed

BenFarhat-Touzri, Dalel; Driss, Fatma; Jemli, Sonia; Tounsi, Slim

2018-06-01

Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces bipyramidal crystals encompassing the insecticidal proteins Cry1Ab, Cry1Ca and Cry1D-133 in the proportions 60:37:3, respectively. In this study, we dealt with the relevance of the low rate of Cry1D-133. The cry1D-133 gene from HD133 was cloned and sequenced. Both nucleotide and amino acid sequence similarity analyses with the cry1D genes available in the GenBank database revealed that cry1D-133 is a new variant of cry1Da-type genes with 99% identity with cry1Da1. Molecular modeling of the Cry1D-133 toxin showed that its higher toxicity is correlated to a higher number of toxin-receptor interactions. Optimal culture conditions of 4 h post-induction time, 160 rpm agitation and 37 °C post-induction temperature were determined and adopted to overproduce Cry1D-133 toxin at adequate amounts to carryout bioassays. A gradual increase of the proportion of Cry1D-133 to the HD133 insecticidal proteins forming the crystal (Cry1D-133, Cry1Ca and Cry1Ab) showed an improvement of the toxicity against Spodoptera littoralis larvae. Therefore, the potential of Cry1D-133 to enhance HD133 toxicity could promote its combination with other B. thuringiensis insecticidal proteins toxins in order to increase target range or to delay the emergence of resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.

PubMed

Shu, Changlong; Zhang, Jingtao; Chen, Guihua; Liang, Gemei; He, Kanglai; Crickmore, Neil; Huang, Dafang; Zhang, Jie; Song, Fuping

2013-09-01

A pooled clone method was developed to screen for cry2A genes. This metagenomic method avoids the need to analyse isolated Bacillus thuringiensis strains by performing gene specific PCR on plasmid-enriched DNA prepared from a pooled soil sample. Using this approach the novel holotype gene cry2Ah1 was cloned and characterized. The toxin gene was over-expressed in Escherichia coli Rosetta (DE3) and the expressed toxin accumulated in both the soluble and insoluble fractions. The soluble Cry2Ah1 was found to have a weight loss activity against Ostrinia furnacalis, and a growth inhibitory activity to both Cry1Ac-susceptible and resistant Helicoverpa armigera populations. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica).

PubMed

Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Rui-Fen; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

2013-04-01

MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays. Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.

Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.).

PubMed

Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

2015-04-01

Biopesticides or transgenic crops based on Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) effectively control agricultural insect pests. The sustainable use of Bt biopesticides and Bt crops is threatened, however, by the development of Cry resistance in the target pests. The diamondback moth, Plutella xylostella (L.), is the first pest that developed resistance to a Bt biopesticide in the field, and a recent study has shown that the resistance of P. xylostella to Cry1Ac is caused by a mutation in an ATP-binding cassette (ABC) transporter gene (ABCC2). In this study, we report that down-regulation of a novel ABC transporter gene from ABCG subfamily (Pxwhite) is associated with Cry1Ac resistance in P. xylostella. The full-length cDNA sequence of Pxwhite was cloned and analyzed. Spatial-temporal expression detection revealed that Pxwhite was expressed in all tissues and developmental stages, and highest expressed in Malpighian tubule tissue and in egg stage. Sequence variation analysis of Pxwhite indicated the absence of constant non-synonymous mutations between susceptible and resistant strains, whereas midgut transcript analysis showed that Pxwhite was remarkably reduced in all resistant strains and further reduced when larvae of the moderately resistant SZ-R strain were subjected to selection with Cry1Ac toxin. Furthermore, RNA interference (RNAi)-mediated suppression of Pxwhite gene expression significantly reduced larval susceptibility to Cry1Ac toxin, and genetic linkage analysis confirmed that down-regulation of Pxwhite gene is tightly linked to Cry1Ac resistance in P. xylostella. To our knowledge, this is the first report indicating that Pxwhite gene is involved in Cry1Ac resistance in P. xylostella. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular studies of segmental aneusomy: FISHing for the atypical cry in del(5)(p15.3).

PubMed

Hodge, J C; Lawson-Yuen, A; Stoler, J M; Ligon, A H

2007-01-01

We report a newborn male with multiple congenital anomalies including growth retardation, hypotonia, dysmorphic facies, widely-spaced nipples, micropenis, cryptorchidism, optic nerve hypoplasia, heart disease, and a striking, high-pitched cry. Chromosome analysis revealed de novo partial trisomy 11q due to a der(5)t(5;11)(p15.3;q22). Fluorescence in situ hybridization (FISH) showed loss of the 5p telomere signal on the der(5) chromosome, indicating the infant has partial monosomy 5p in addition to partial trisomy 11q. Among cases involving trisomy 11q, an unusual cry has only been documented in the presence of a der(5)t(5p;11q). This apparent dependence of the abnormal cry on monosomy 5p suggested the same genetic mechanism that occurs in Cri du chat syndrome (CDCS) may be responsible for the atypical cry in der(5)t(5p;11q) individuals. Neither a commercial CDCS probe (LSI D5S23, D5S721) nor a series of BAC clones encompassing distal regions implicated in the CDCS-associated cat-cry were deleted in our patient. These results suggest a second cry-modifying locus maps telomeric to BAC RP11-94J21 in band 5p15.33. This locus may not only cause the abnormal cry in individuals with a der(5)t(5p;11q) but could also contribute to the phenotypic variability and discordant mapping studies observed for CDCS. Copyright (c) 2007 S. Karger AG, Basel.

Comparative performance of modified full-length and truncated Bacillus thuringiensis-cry1Ac genes in transgenic tomato.

PubMed

Koul, Bhupendra; Yadav, Reena; Sanyal, Indraneel; Amla, Devindra Vijay

2015-01-01

Bt-cry1Ac gene has been reputedly effective against Helicoverpa armigera a notorious lepidopteran pest. Reports on the expression of full-length and truncated cry1Ac genes in plants for effective resistance against Helicoverpa sp. have been documented however, their performance is still ambiguous. Moreover, the question remains to be addressed that truncation of 3' end of the native gene was documented and suggested for active insecticidal toxin production while the most successful transgenic event(s) of commercialized-cotton are based on full-length of the cry gene. Therefore, we performed a comparative study on the efficacy of the two versions of cry1Ac genes (full-length: 3,510 bp and truncated: 1,845 bp) in T0 and T1 transgenic tomato plants and analyzed the extent of protection against H. armigera and also compared the results with our previous findings related to a successful transgenic tomato line Ab25E, expressing cry1Ab gene. The integration of cry1Ac gene(s) in T0 transgenic plants and its inheritance in T1 progeny was observed by PCR, RT-PCR and Southern blot hybridization analysis while, the toxin integrity, expression and toxicity was monitored by Western immunoassay, DAS-ELISA and insect bioassay respectively. An average transformation frequency and Bt-Cry protein content of 16.93 ± 2.10 and 0.0020-0.0128% of total soluble protein (TSP) was obtained with pRD400 vector (Trcry1Ac) while, a much lower value of 9.30 ± 2.041 and 0.0001 - 0.0026% of TSP was observed with pNBRI-1 vector (Flcry1Ac), respectively. The promising Trcry1Ac T0 transgenic plants and their T1 progeny gave full protection from H. armigera. Although Flcry1Ac gene showed lower transformation frequency and lower expression, it showed higher toxicity to H. armigera when compared with truncated Trcry1Ac gene. The full-length cry1Ac gene can be redesigned for higher expression and performance in dicots or a hybrid gene could be designed having a blend of strong receptor binding and stable expression characteristics for enhanced efficacy and toxicity to the susceptible insects.

Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm (Spodoptera frugiperda).

PubMed

Martins, E S; Aguiar, R W D S; Martins, N F; Melatti, V M; Falcão, R; Gomes, A C M M; Ribeiro, B M; Monnerat, R G

2008-05-01

To evaluate the activity of cry1Ia gene against cotton pests, Spodoptera frugiperda and Anthonomus grandis. Had isolated and characterized a toxin gene from the Bacillus thuringiensis S1451 strain which have been previously shown to be toxic to S. frugiperda and A. grandis. The toxin gene (cry1Ia) was amplified by PCR, sequenced, and cloned into the genome of a baculovirus. The Cry1Ia protein was expressed in baculovirus infected insect cells, producing protein inclusions in infected cells. The Cry1Ia protein has used in bioassays against to S. frugiperda and A. grandis. Bioassays using the purified recombinant protein showed high toxicity to S. frugiperda and A. grandis larvae. Molecular modelling of the Cry1Ia protein translated from the DNA sequence obtained in this work, showed that this protein possibly posses a similar structure to the Cry3A protein. Ultrastructural analysis of midgut cells from A. grandis incubated with the Cry1Ia toxin, showed loss of microvilli integrity. The results indicate that the cry1Ia is a good candidate for the construction of transgenic plants resistant to these important cotton pests.

Genetic and biochemical characterization of field-evolved resistance to Bacillus thuringiensis toxin Cry1Ac in the diamondback moth, Plutella xylostella.

PubMed

Sayyed, Ali H; Raymond, Ben; Ibiza-Palacios, M Sales; Escriche, Baltasar; Wright, Denis J

2004-12-01

The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.

Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

PubMed Central

Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

2014-01-01

The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

PubMed

Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

2014-08-27

The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

Influence of one or two Bt genes transgenic cotton free living nitrogen fixers and p-solubilising microorganisms in vertisols and alfisols

NASA Astrophysics Data System (ADS)

Sudha, T.; Babu, R.; Biradar, D. P.; Patil, V. C.; Shirnalli, G.

2012-04-01

India, the largest cotton grower in the world benefited from the cultivation of genetically modified Bt transgenic cotton. Bt cotton with the single gene (cry 1Ac) contributed to increased productivity over the last eight years. But in the recent years, there has been an increasing trend to adopt two genes (cry 1Ac and cry 2Ab) transgenic cotton in India. The two gene Bt cotton hybrids were planted over a large area (57%) during 2009 than the single gene Bt cotton hybrids. In this context, the field experiments were conducted in farmers field in both Vertisols and Alfisols during monsoon season of 2009 to study the effect of a single gene Bt hybrid (RCH-2Bt, JK-99Bt, Mallika Bt, MRC-6918 Bt, Brahma Bt, RCH-708 Bt, Bunny Bt) as well as two gene Bt hybrids (RCH-2 BGII Bt, Bunny BGII Bt) compared with the non genetically modified (non-Bt) hybrid (DHH-11) on the population of free living nitrogen fixing microorganisms (Azospirillum and methylotrophs) and P-solubilizers in two different soil types under rainfed situation. Observations on microbial population were recorded at flowering and at harvest in both the soil types. Results indicated a higher population of Azospirillum, methylotrophs and P-solubilisers in the rhizosphere grown with single or two gene Bt hybrid and non-Bt hybrid at flowering stage in both the soil types. In Vertisol, significantly higher population of methylotrophs in MRC-6918 Bt (30 x 102/g of soil), P-solubilizers in RCH-2 Bt (31x103/g of soil) and Azospirillum in RCH-708 Bt (0.79 x 106 /g of soil) was recorded as compared to non-Bt hybrid DHH-11 (2 x 102/g of soil, 12 x 103/g of soil, 0.54 x 106/g of soil), respectively. Whereas, in Alfisol, significantly higher population of methylotrophs in RCH-2 Bt (13x 102/g of soil), P-solubilisers in JK-99 Bt (38 x 103/g of soil) and Azospirillum in RCH-2Bt (0.57 x 106/g of soil) was recorded over non Bt hybrid DHH-11 (2x 102/g of soil, 13x 103/g of soil and 0.17 x106/g of soil) respectively. Our results indicate no negative impact of Bt hybrids expressing single or two gene on the population of nitrogen fixers and P-solubilisers in rhizosphere soil. Variations in the population of the microorganisms observed among different Bt hybrids expressing single or two gene and non-Bt hybrid may be due to the genotype related effect rather than the cry toxins. However, impact of Bt hybrids expressing single or two gene on the population of the microorganisms in the rhizosphere soil over longer period needs to be investigated.

Multitrophic Cry-protein flow in Bt-cotton

USDA-ARS?s Scientific Manuscript database

Although most genetically engineered cotton plants grown today produce the insecticidal Cry-proteins Cry1Ac and Cry2Ab, studies are lacking on multitrophic Cry-protein acquisition in dual-gene cotton fields. Such field data are important for the design and interpretation of laboratory risk assessmen...

Safety and long-term immunological effects of CryJ2-LAMP plasmid vaccine in Japanese red cedar atopic subjects: A phase I study.

PubMed

Su, Yan; Romeu-Bonilla, Eliezer; Anagnostou, Athanasia; Fitz-Patrick, David; Hearl, William; Heiland, Teri

2017-12-02

Japanese Red Cedar (JRC) pollen induced allergy affects one third of Japanese and the development of effective therapies remains an unachieved challenge. We designed a DNA vaccine encoding CryJ2 allergen from the JRC pollen and Lysosomal Associated Membrane Protein 1 (LAMP-1) to treat JRC allergy. These Phase IA and IB trials assessed safety and immunological effects of the investigational CryJ2-LAMP DNA vaccine in both non-sensitive and sensitive Japanese expatriates living in Honolulu, Hawaii. In the Phase IA trial, 6 JRC non-sensitive subjects and 9 JRC and/or Mountain Cedar (MC) sensitive subjects were given 4 vaccine doses (each 4mg/1ml) intramuscularly (IM) at 14-day intervals. Nine JRC and/or MC sensitive subjects were given 4 doses (2 mg/0.5 ml) IM at 14-day intervals. The safety and functional biomarkers were followed for 132 d. Following this, 17 of 24 subjects were recruited into the IB trial and received one booster dose (2 mg/0.5 ml) IM approximately 300 d after the first vaccination dose to which they were randomized in the first phase of the trial. All safety endpoints were met and all subjects tolerated CryJ2-LAMP vaccinations well. At the end of the IA trial, 10 out of 12 JRC sensitive and 6 out of 11 MC sensitive subjects experienced skin test negative conversion, possibly related to the CryJ2-LAMP vaccinations. Collectively, these data suggested that the CryJ2-LAMP DNA vaccine is safe and may be immunologically effective in treating JRC induced allergy.

Tradeoff between reproduction and resistance evolution to Bt-toxin in Helicoverpa armigera: regulated by vitellogenin gene expression.

PubMed

Zhang, W N; Xiao, H J; Liang, G M; Guo, Y Y; Wu, K M

2014-08-01

Evolution of resistance to insecticides usually has fitness tradeoffs associated with adaptation to the stress. The basic regulation mechanism of tradeoff between reproduction and resistance evolution to Bacillus thuringiensis (Bt) toxin in the cotton bollworm, Helicoverpa armigera (Ha), based on the vitellogenin (Vg) gene expression was analyzed here. The full-length cDNA of the Vg gene HaVg (JX504706) was cloned and identified. HaVg has 5704 base pairs (bp) with an open reading frame (ORF) of 5265 bp, which encoded 1756 amino acid protein with a predicted molecular mass of 197.28 kDa and a proposed isoelectric point of 8.74. Sequence alignment analysis indicated that the amino acid sequence of HaVg contained all of the conserved domains detected in the Vgs of the other insects and had a high similarity with the Vgs of the Lepidoptera insects, especially Noctuidae. The resistance level to Cry1Ac Bt toxin and relative HaVg mRNA expression levels among the following four groups: Cry1Ac-susceptible strain (96S), Cry1Ac-resistant strain fed on artificial diet with Bt toxin for 135 generations (BtR stands for the Cry1Ac Bt resistance), progeny of the Cry1Ac-resistant strain with a non-Bt-toxin artificial diet for 38 generations (CK1) and the direct descendants of the 135th-generation resistant larvae which were fed on an artificial diet without the Cry1Ac protein (CK2) were analyzed. Compared with the 96S strain, the resistance ratios of the BtR strain, the CK1 strain and the CK2 strain were 2917.15-, 2.15- and 2037.67-fold, respectively. The maximum relative HaVg mRNA expression levels of the BtR strain were approximately 50% less than that of the 96S strain, and the coming of maximum expression was delayed for approximately 4 days. The overall trend of the HaVg mRNA expression levels in the CK1 strain was similar to that in the 96S strain, and the overall trend of the HaVg mRNA expression levels in the CK2 strain was similar to that in the BtR strain. Our results suggest that the changes in reproduction due to the Bt-toxin resistance evolution in the BtR strain may be regulated by the Vg gene expression. The down-regulation of HaVg at the early stages resulted in a period of delayed reproduction and decreased fecundity in the BtR strain. This performance disappeared when the Bt-toxin selection pressure was lost.

Circadian Clock Gene Expression in the Coral Favia fragum over Diel and Lunar Reproductive Cycles

PubMed Central

Hoadley, Kenneth D.; Szmant, Alina M.; Pyott, Sonja J.

2011-01-01

Natural light cycles synchronize behavioral and physiological cycles over varying time periods in both plants and animals. Many scleractinian corals exhibit diel cycles of polyp expansion and contraction entrained by diel sunlight patterns, and monthly cycles of spawning or planulation that correspond to lunar moonlight cycles. The molecular mechanisms for regulating such cycles are poorly understood. In this study, we identified four molecular clock genes (cry1, cry2, clock and cycle) in the scleractinian coral, Favia fragum, and investigated patterns of gene expression hypothesized to be involved in the corals' diel polyp behavior and lunar reproductive cycles. Using quantitative PCR, we measured fluctuations in expression of these clock genes over both diel and monthly spawning timeframes. Additionally, we assayed gene expression and polyp expansion-contraction behavior in experimental corals in normal light:dark (control) or constant dark treatments. Well-defined and reproducible diel patterns in cry1, cry2, and clock expression were observed in both field-collected and the experimental colonies maintained under control light:dark conditions, but no pattern was observed for cycle. Colonies in the control light:dark treatment also displayed diel rhythms of tentacle expansion and contraction. Experimental colonies in the constant dark treatment lost diel patterns in cry1, cry2, and clock expression and displayed a diminished and less synchronous pattern of tentacle expansion and contraction. We observed no pattern in cry1, cry2, clock, or cycle expression correlated with monthly spawning events suggesting these genes are not involved in the entrainment of reproductive cycles to lunar light cycles in F. fragum. Our results suggest a molecular clock mechanism, potentially similar to that in described in fruit flies, exists within F. fragum. PMID:21573070

Existence of a photoinducible phase for ovarian development and photoperiod-related alteration of clock gene expression in a damselfish.

PubMed

Takeuchi, Yuki; Hada, Noriko; Imamura, Satoshi; Hur, Sung-Pyo; Bouchekioua, Selma; Takemura, Akihiro

2015-10-01

The sapphire devil, Chrysiptera cyanea, is a reef-associated damselfish and their ovarian development can be induced by a long photoperiod. In this study, we demonstrated the existence of a photoinducible phase for the photoperiodic ovarian development in the sapphire devil. Induction of ovarian development under night-interruption light schedules and Nanda-Hamner cycles revealed that the photoinducible phase appeared in a circadian manner between ZT12 and ZT13. To characterize the effect of photoperiod on clock gene expression in the brain of this species, we determined the expression levels of the sdPer1, sdPer2, sdCry1, and sdCry2 clock genes under constant light and dark conditions (LL and DD) and photoperiodic (short and long photoperiods). The expression of sdPer1 exhibited clear circadian oscillation under both LL and DD conditions, while sdPer2 and sdCry1 expression levels were lower under DD than under LL conditions and sdCry2 expression was lower under LL than under DD conditions. These results suggest a key role for sdPer1 in circadian clock cycling and that sdPer2, sdCry1, and sdCry2 are light-responsive clock genes in the sapphire devil. After 1 week under a long photoperiod, we observed photoperiod-related changes in sdPer1, sdPer2, and sdCry2 expression, but not in sdCry1 expression. These results suggest that the expression patterns of some clock genes exhibit seasonal variation according to seasonal changes in day length and that such seasonal alteration of clock gene expression may contribute to seasonal recognition by the sapphire devil. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of a Novel Aminopeptidase P-Like Gene (OnAPP) Possibly Involved in Bt Toxicity and Resistance in a Major Corn Pest (Ostrinia nubilalis)

PubMed Central

Khajuria, Chitvan; Buschman, Lawrent L.; Chen, Ming-Shun; Siegfried, Blair D.; Zhu, Kun Yan

2011-01-01

Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study, we identified 10 aminopeptidase-like genes, which have previously been identified as putative Bt toxin receptors in other insects and examined their expression in relation to Cry1Ab toxicity and resistance. Expression analysis for the 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut, but there was no difference in the expression of these genes in Cry1Ab resistant and susceptible strains. This suggested that altered expression of these genes was unlikely to be responsible for resistance in these ECB strains. However, we found that there were changes in two amino acid residues of the aminopeptidase-P like gene (OnAPP) involving Glu305 to Lys305 and Arg307 to Leu307 in the two Cry1Ab-resistant strains as compared with three Cry1Ab-susceptible strains. The mature OnAPP contains 682 amino acid residues and has a putative signal peptide at the N-terminus, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. We used a feeding based-RNA interference assay to examine the role of the OnAPP gene in Cry1Ab toxicity and resistance. Bioassays of Cry1Ab in larvae fed diet containing OnAPP dsRNA resulted in a 38% reduction in the transcript level of OnAPP and a 25% reduction in the susceptibility to Cry1Ab as compared with larvae fed GFP dsRNA or water. These results strongly suggest that the OnAPP gene could be involved in binding the Cry1Ab toxin in the ECB larval midgut and that mutations in this gene may be associated with Bt resistance in these two ECB strains. PMID:21887358

Cry1Ab-expressing rice did not influence expression of fecundity-related genes in the wolf spider Pardosa pseudoannulata.

PubMed

Wang, Juan; Peng, Yuan-De; He, Chao; Wei, Bao-Yang; Liang, Yun-Shan; Yang, Hui-Lin; Wang, Zhi; Stanley, David; Song, Qi-Sheng

2016-10-30

The impact of Bacillus thuringiensis (Bt) toxin proteins on non-target predatory arthropods is not well understood at the cellular and molecular levels. Here, we investigated the potential effects of Cry1Ab expressing rice on fecundity of the wolf spider, Pardosa pseudoannulata, and some of the underlying molecular mechanisms. The results indicated that brown planthoppers (BPHs) reared on Cry1Ab-expressing rice accumulated the Cry toxin and that reproductive parameters (pre-oviposition period, post-oviposition stage, number of eggs, and egg hatching rate) of the spiders that consumed BPHs reared on Bt rice were not different from those that consumed BPHs reared on the non-Bt control rice. The accumulated Cry1Ab did not influence several vitellin (Vt) parameters, including stored energy and amino acid composition, during one generation. We considered the possibility that the Cry toxins exert their influence on beneficial predators via more subtle effects detectable at the molecular level in terms of gene expression. This led us to transcriptome analysis to detect differentially expressed genes in the ovaries of spiders exposed to dietary Cry1Ab and their counterpart control spiders. Eight genes, associated with vitellogenesis, vitellogenin receptor activity, and vitellin membrane formation were not differentially expressed between ovaries from the treated and control spiders, confirmed by qPCR analysis. We infer that dietary Cry1Ab expressing rice does not influence fecundity, nor expression levels of Vt-associated genes in P. pseudoannulata. Copyright © 2016. Published by Elsevier B.V.

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock.

PubMed

Rubin, Elad B; Shemesh, Yair; Cohen, Mira; Elgavish, Sharona; Robertson, Hugh M; Bloch, Guy

2006-11-01

The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

Bt proteins Cry1Ah and Cry2Ab do not affect cotton aphid Aphis gossypii and ladybeetle Propylea japonica

PubMed Central

Zhao, Yao; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lv, Li-Min; Wang, Xiao-Ping; Cui, Jin-Jie; Lei, Chao-Liang

2016-01-01

Plant varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins Cry1Ah and Cry2Ab have potential commercialization prospects in China. However, their potential effects on non-target arthropods (NTAs) remain uncharacterized. The cotton aphid Aphis gossypii is a worldwide pest that damages various important crops. The ladybeetle Propylea japonica is a common and abundant natural enemy in many cropping systems in East Asia. In the present study, the effects of Cry1Ah and Cry2Ab proteins on A. gossypii and P. japonica were assessed from three aspects. First, neither of the Cry proteins affected the growth or developmental characteristics of the two test insects. Second, the expression levels of the detoxification-related genes of the two test insects did not change significantly in either Cry protein treatment. Third, neither of the Cry proteins had a favourable effect on the expression of genes associated with the amino acid metabolism of A. gossypii and the nutrition utilization of P. japonica. In conclusion, the Cry1Ah and Cry2Ab proteins do not appear to affect the cotton aphid A. gossypii or the ladybeetle P. japonica. PMID:26829252

A Plant Cryptochrome Controls Key Features of the Chlamydomonas Circadian Clock and Its Life Cycle.

PubMed

Müller, Nico; Wenzel, Sandra; Zou, Yong; Künzel, Sandra; Sasso, Severin; Weiß, Daniel; Prager, Katja; Grossman, Arthur; Kottke, Tilman; Mittag, Maria

2017-05-01

Cryptochromes are flavin-binding proteins that act as blue light receptors in bacteria, fungi, plants, and insects and are components of the circadian oscillator in mammals. Animal and plant cryptochromes are evolutionarily divergent, although the unicellular alga Chlamydomonas reinhardtii ( Chlamydomonas throughout) has both an animal-like cryptochrome and a plant cryptochrome (pCRY; formerly designated CPH1). Here, we show that the pCRY protein accumulates at night as part of a complex. Functional characterization of pCRY was performed based on an insertional mutant that expresses only 11% of the wild-type pCRY level. The pcry mutant is defective for central properties of the circadian clock. In the mutant, the period is lengthened significantly, ultimately resulting in arrhythmicity, while blue light-based phase shifts show large deviations from what is observed in wild-type cells. We also show that pCRY is involved in gametogenesis in Chlamydomonas pCRY is down-regulated in pregametes and gametes, and in the pcry mutant, there is altered transcript accumulation under blue light of the strictly light-dependent, gamete-specific gene GAS28 pCRY acts as a negative regulator for the induction of mating ability in the light and for the loss of mating ability in the dark. Moreover, pCRY is necessary for light-dependent germination, during which the zygote undergoes meiosis that gives rise to four vegetative cells. In sum, our data demonstrate that pCRY is a key blue light receptor in Chlamydomonas that is involved in both circadian timing and life cycle progression. © 2017 American Society of Plant Biologists. All Rights Reserved.

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

PubMed Central

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-01-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression. PMID:22434878

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

PubMed

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-07-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

What are farmers really planting? Measuring the presence and effectiveness of Bt cotton in Pakistan.

PubMed

Spielman, David J; Zaidi, Fatima; Zambrano, Patricia; Khan, Asif Ali; Ali, Shaukat; Cheema, H Masooma Naseer; Nazli, Hina; Khan, Rao Sohail Ahmad; Iqbal, Arshad; Zia, Muhammad Amir; Ali, Ghulam Muhammad

2017-01-01

Genetically modified, insect-resistant Bacillus thuringiensis (Bt) cotton is cultivated extensively in Pakistan. Past studies, however, have raised concerns about the prevalence of Bt cotton varieties possessing weak or nonperforming insect-resistance traits conferred by the cry gene. We examine this issue using data drawn from a representative sample of cotton-growing households that were surveyed in six agroclimatic zones spanning 28 districts in Pakistan in 2013, as well as measurements of Cry protein levels in cotton tissue samples collected from the sampled households' main fields. The resultant dataset combines information from 593 sampled households with corresponding plant tissue diagnostics from 70 days after sowing, as well as information from 589 sampled households with corresponding diagnostics from 120 days after sowing. Our analysis indicates that 11 percent of farmers believed they were cultivating Bt cotton when, in fact, the Cry toxin was not present in the tested tissue at 70 days after sowing (i.e., a Type I error). The analysis further indicates that 5 percent of farmers believed they were cultivating non-Bt cotton when, in fact, the Cry toxin was present in the tested tissue (i.e., a Type II error). In addition, 17 percent of all sampled farmers were uncertain whether or not they were cultivating Bt cotton. Overall, 33 percent of farmers either did not know or were mistaken in their beliefs about the presence of the cry gene in the cotton they cultivated. Results also indicate that toxic protein levels in the plant tissue samples occurred below threshold levels for lethality in a significant percentage of cases, although these measurements may also be affected by factors related to tissue sample collection, handling, storage, and testing procedures. Nonetheless, results strongly suggest wide variability both in farmers' beliefs and in gene expression. Such variability has implications for policy and regulation in Pakistan's transgenic cotton seed market.

Synthesis and antibacterial activity of novel 15-membered macrolide derivatives: 4''-carbamate, 11,12-cyclic carbonate-4''-carbamate and 11,4''-di-O-arylcarbamoyl analogs of azithromycin.

PubMed

Ma, Shutao; Ma, Ruixin; Liu, Zhaopeng; Ma, Chenchen; Shen, Xuecui

2009-10-01

4''-Carbamate, 11,12-cyclic carbonate-4''-carbamate and 11,4''-di-O-arylcarbamoyl analogs of azithromycin were designed, synthesized and evaluated. The 4''-carbamate analogs retained excellent activity against erythromycin-susceptible Staphylococcus pneumoniae and showed improved activity against erythromycin-resistant Staphylococcus pneumoniae. Compared with 4''-carbamate analogs, 11,12-cyclic carbonate-4''-carbamate analogs exhibited improved activity against erythromycin-resistant Staphylococcus pneumoniae encoded by the mef gene or the erm and mef genes, and 11,4''-di-O-arylalkylcarbamoyl analogs showed greatly improved activity (0.25-0.5 microg/mL) against erythromycin-resistant Staphylococcus pneumoniae encoded by the erm gene. Among them, the novel series of 11,4''-di-O-arylalkylcarbamoyl analogs 7a-k exhibited potent and balanced activity against susceptible and resistant bacteria. In particular, compounds 7f and 7k were the most effective against susceptible bacteria and resistant bacteria encoded by the erm gene or the mef gene.

Effects of 24-epibrassinolide and green light on plastid gene transcription and cytokinin content of barley leaves.

PubMed

Efimova, Marina V; Vankova, Radomira; Kusnetsov, Victor V; Litvinovskaya, Raisa P; Zlobin, Ilya E; Dobrev, Petre; Vedenicheva, Nina P; Savchuk, Alina L; Karnachuk, Raisa A; Kudryakova, Natalia V; Kuznetsov, Vladimir V

2017-04-01

In order to evaluate whether brassinosteroids (BS) and green light regulate the transcription of plastid genes in a cross-talk with cytokinins (CKs), transcription rates of 12 plastid genes (ndhF, rrn23, rpoB, psaA, psaB, rrn16, psbA, psbD, psbK, rbcL, atpB, and trnE/trnY) as well as the accumulation of transcripts of some photoreceptors (PHYA, CRY2, CRY1A, and CRY1B) and signaling (SERK and CAS) genes were followed in detached etiolated barley leaves exposed to darkness, green or white light ±1μm 24-epibrassinolide (EBL). EBL in the dark was shown to up-regulate the transcription of 12 plastid genes, while green light activated 10 genes and the EBL combined with the green light affected the transcription of only two genes (psaB and rpoB). Green light inhibited the expression of photoreceptor genes, except for CRY1A. Under the green light, EBL practically did not affect the expression of CRY1A, CAS and SERK genes, but it reduced the influence of white light on the accumulation of CAS, CRY1A, CRY1B, and SERK gene transcripts. The total content of BS in the dark and under white light remained largely unchanged, while under green light the total content of BRs (brassinolide, castasterone, and 6-deoxocastasterone) and HBRs (28-homobrassinolide, 28-homocastasterone, and 6-deoxo-28-homocastasterone) increased. The EBL-dependent up-regulation of plastome transcription in the dark was accompanied by a significant decrease in CK deactivation by O-glucosylation. However, no significant effect on the content of active CKs was detected. EBL combined with green light moderately increased the contents of trans-zeatin and isopentenyladenine, but had a negative effect on cis-zeatin. The most significant promotive effect of EBL on active CK bases was observed in white light. The data obtained suggest the involvement of CKs in the BS- and light-dependent transcription regulation of plastid genes. Copyright © 2016 Elsevier Inc. All rights reserved.

Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda

PubMed Central

Lemes, Ana Rita Nunes; Davolos, Camila Chiaradia; Legori, Paula Cristina Brunini Crialesi; Fernandes, Odair Aparecido; Ferré, Juan; Lemos, Manoel Victor Franco; Desiderio, Janete Apparecida

2014-01-01

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested. PMID:25275646

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis.

PubMed

Gao, Yulin; Hu, Yang; Fu, Qiang; Zhang, Jie; Oppert, Brenda; Lai, Fengxiang; Peng, Yufa; Zhang, Zhitao

2010-09-01

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC(50) of 0.24 and 0.30 microg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations. Copyright 2010 Elsevier Inc. All rights reserved.

Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

PubMed Central

Puspito, Agung N.; Rao, Abdul Q.; Hafeez, Muhammad N.; Iqbal, Muhammad S.; Bajwa, Kamran S.; Ali, Qurban; Rashid, Bushra; Abbas, Muhammad A.; Latif, Ayesha; Shahid, Ahmad A.; Nasir, Idrees A.; Husnain, Tayyab

2015-01-01

More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered “white gold” because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton. PMID:26617613

Comparative Proteomic and Nutritional Composition Analysis of Independent Transgenic Pigeon Pea Seeds Harboring cry1AcF and cry2Aa Genes and Their Nontransgenic Counterparts.

PubMed

Mishra, Pragya; Singh, Shweta; Rathinam, Maniraj; Nandiganti, Muralimohan; Ram Kumar, Nikhil; Thangaraj, Arulprakash; Thimmegowda, Vinutha; Krishnan, Veda; Mishra, Vagish; Jain, Neha; Rai, Vandna; Pattanayak, Debasis; Sreevathsa, Rohini

2017-02-22

Safety assessment of genetically modified plants is an important aspect prior to deregulation. Demonstration of substantial equivalence of the transgenics compared to their nontransgenic counterparts can be performed using different techniques at various molecular levels. The present study is a first-ever comprehensive evaluation of pigeon pea transgenics harboring two independent cry genes, cry2Aa and cry1AcF. The absence of unintended effects in the transgenic seed components was demonstrated by proteome and nutritional composition profiling. Analysis revealed that no significant differences were found in the various nutritional compositional analyses performed. Additionally, 2-DGE-based proteome analysis of the transgenic and nontransgenic seed protein revealed that there were no major changes in the protein profile, although a minor fold change in the expression of a few proteins was observed. Furthermore, the study also demonstrated that neither the integration of T-DNA nor the expression of the cry genes resulted in the production of unintended effects in the form of new toxins or allergens.

Field trials to evaluate the effects of transgenic cry1le maize on the community characteristics of arthropod natural enemies

USDA-ARS?s Scientific Manuscript database

Possible non-target effects of transgenic cry1Ie gene maize exerts on natural enemy community biodiversity in the field is unresolved. In the present study, a 2-yr study of transgenic cry1Ie gene maize (Event IE09S034, Bt maize) and its near isoline (Zong 31, non-Bt maize) on natural enemy community...

Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer.

PubMed

Cheng, X; Sardana, R; Kaplan, H; Altosaar, I

1998-03-17

Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.

MAPK Signaling Pathway Alters Expression of Midgut ALP and ABCC Genes and Causes Resistance to Bacillus thuringiensis Cry1Ac Toxin in Diamondback Moth

PubMed Central

Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhu, Xun; Baxter, Simon W.; Zhou, Xuguo; Jurat-Fuentes, Juan Luis; Zhang, Youjun

2015-01-01

Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella. PMID:25875245

Rhythm Defects Caused by Newly Engineered Null Mutations in Drosophila's cryptochrome Gene

PubMed Central

Dolezelova, Eva; Dolezel, David; Hall, Jeffrey C.

2007-01-01

Much of the knowledge about cryptochrome function in Drosophila stems from analyzing the cryb mutant. Several features of this variant's light responsiveness imply either that CRYb retains circadian-photoreceptive capacities or that additional CRY-independent light-input routes subserve these processes. Potentially to resolve these issues, we generated cry knock-out mutants (cry0's) by gene replacement. They behaved in an anomalously rhythmic manner in constant light (LL). However, cry0 flies frequently exhibited two separate circadian components in LL, not observed in most previous cryb analyses. Temperature-dependent circadian phenotypes exhibited by cry0 flies suggest that CRY is involved in core pacemaking. Further locomotor experiments combined cry0 with an externally blinding mutation (norpAP24), which caused the most severe decrements of circadian photoreception observed so far. cryb cultures were shown previously to exhibit either aperiodic or rhythmic eclosion in separate studies. We found cry0 to eclose in a solidly periodic manner in light:dark cycles or constant darkness. Furthermore, both cry0 and cryb eclosed rhythmically in LL. These findings indicate that the novel cry0 type causes more profound defects than does the cryb mutation, implying that CRYb retains residual activity. Because some norpAP24 cry0 individuals can resynchronize to novel photic regimes, an as-yet undetermined light-input route exists in Drosophila. PMID:17720919

Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa

PubMed Central

Chen, Lin; Jiang, Huang; Cheng, Qipeng; Chen, Junpeng; Wu, Gaobing; Kumar, Ashok; Sun, Ming; Liu, Ziduo

2015-01-01

Nematodes are known to be harmful to various crops, vegetables, plants and insects. The present study reports that, chitin upregulates the activity of chitinase (20%) and nematicidal potential (15%) of Pseudomonas aeruginosa. The chitinase gene (pachi) from P. aeruginosa was cloned, and its nematicidal activity of pachi protein against Caenorhabditis elegans was studied. The mortality rate induced by pachi increased by 6.3-fold when in association with Cry21Aa from Bacillus thuringiensis. Pachi efficiently killed C. elegans in its native state (LC50 = 387.3 ± 31.7 μg/ml), as well as in association with Cry21Aa (LC50 = 30.9 ± 4.1 μg/ml), by degrading the cuticle, egg shell and intestine in a relatively short time period of 24 h. To explore the nematidal potential of chitinase, six fusion proteins were constructed using gene engineering techniques. The CHACry showed higher activity against C. elegans than others owing to its high solubility. Notably, the CHACry showed a synergistic factor of 4.1 versus 3.5 a mixture [1:1] of pachi and Cry21Aa. The present study has identified eco-friendly biological routes (e.g., mixed proteins, fusion proteins) with potent nematicidal activity, which not only can help to prevent major crop losses but also strengthen the agro-economy and increase gross crop yield. PMID:26400097

Field-Evolved Resistance in Corn Earworm to Cry Proteins Expressed by Transgenic Sweet Corn.

PubMed

Dively, Galen P; Venugopal, P Dilip; Finkenbinder, Chad

2016-01-01

Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins. We present field monitoring data using Cry1Ab (1996-2016) and Cry1A.105+Cry2Ab2 (2010-2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab-event Bt11, and Cry1A.105+Cry2Ab2-event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood. After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt sweet corn provide strong evidence of field-evolved resistance in H. zea populations to multiple Cry toxins. The high adoption rate of Bt field corn and cotton, along with the moderate dose expression of Cry1Ab and related Cry toxins in these crops, and decreasing refuge compliance probably contributed to the evolution of resistance. Our results have important implications for resistance monitoring, refuge requirements and other regulatory policies, cross-resistance issues, and the sustainability of the pyramided Bt technology.

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms.

PubMed

Dinon, Andréia Z; Prins, Theo W; van Dijk, Jeroen P; Arisi, Ana Carolina M; Scholtens, Ingrid M J; Kok, Esther J

2011-05-01

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.

Transgenic rice expressing the cry2AX1 gene confers resistance to multiple lepidopteran pests.

PubMed

Chakraborty, M; Reddy, P Sairam; Mustafa, G; Rajesh, G; Narasu, V M Laxmi; Udayasuriyan, V; Rana, Debashis

2016-10-01

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34 µg g(-1) leaf fresh weight and with 80-92 % levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7-3.6 ng g(-1)). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.

Hypothalamic expression and moonlight-independent changes of Cry3 and Per4 implicate their roles in lunar clock oscillators of the lunar-responsive Goldlined spinefoot.

PubMed

Toda, Riko; Okano, Keiko; Takeuchi, Yuki; Yamauchi, Chihiro; Fukushiro, Masato; Takemura, Akihiro; Okano, Toshiyuki

2014-01-01

Lunar cycle-associated physiology has been found in a wide variety of organisms. Studies suggest the presence of a circalunar clock in some animals, but the location of the lunar clock is unclear. We previously found lunar-associated expression of transcripts for Cryptochrome3 gene (SgCry3) in the brain of a lunar phase-responsive fish, the Goldlined spinefoot (Siganus guttatus). Then we proposed a photoperiodic model for the lunar phase response, in which SgCry3 might function as a phase-specific light response gene and/or an oscillatory factor in unidentified circalunar clock. In this study, we have developed an anti-SgCRY3 antibody to identify SgCRY3-immunoreactive cells in the brain. We found immunoreactions in the subependymal cells located in the mediobasal region of the diencephalon, a crucial site for photoperiodic seasonal responses in birds. For further assessment of the lunar-responding mechanism and the circalunar clock, we investigated mRNA levels of Cry3 as well as those of the other clock(-related) genes, Period (Per2 and Per4), in S. guttatus reared under nocturnal moonlight interruption or natural conditions. Not only SgCry3 but SgPer4 mRNA levels showed lunar phase-dependent variations in the diencephalon without depending on light condition during the night. These results suggest that the expressions of SgCry3 and SgPer4 are not directly regulated by moonlight stimulation but endogenously mediated in the brain, and implicate that circadian clock(-related) genes may be involved in the circalunar clock locating within the mediobasal region of the diencephalon.

Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer

PubMed Central

Gao, Shiwu; Yang, Yingying; Wang, Chunfeng; Guo, Jinlong; Zhou, Dinggang; Wu, Qibin; Su, Yachun; Xu, Liping

2016-01-01

We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines. PMID:27093437

Expression of an engineered synthetic cry2Aa (D42/K63F/K64P) gene of Bacillus thuringiensis in marker free transgenic tobacco facilitated full-protection from cotton leaf worm (S. littoralis) at very low concentration.

PubMed

Gayen, Srimonta; Mandal, Chandi Charan; Samanta, Milan Kumar; Dey, Avishek; Sen, Soumitra Kumar

2016-04-01

Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae).

PubMed

Chen, Hao; Zhang, Guoan; Zhang, Qifa; Lin, Yongjun

2008-02-01

Ten transgenic Bacillus thuringiensis Bt rice, Oryza sativa L., lines with different Bt genes (two Cry1Ac lines, three Cry2A lines, and five Cry9C lines) derived from the same variety Minghui 63 were evaluated in both the laboratory and the field. Bioassays were conducted by using the first instars of two main rice lepidopteran insect species: yellow stem borer, Scirpophaga incertulas (Walker) and Asiatic rice borer, Chilo suppressalis (Walker). All transgenic lines exhibited high toxicity to these two rice borers. Field evaluation results also showed that all transgenic lines were highly insect resistant with both natural infestation and manual infestation of the neonate larvae of S. incertulas compared with the nontransformed Minghui63. Bt protein concentrations in leaves of 10 transgenic rice lines were estimated by the sandwich enzyme-linked immunosorbent assay. The cry9C gene had the highest expression level, next was cry2A gene, and the cry1Ac gene expressed at the lowest level. The feeding behavior of 7-d-old Asiatic rice borer to three classes of Bt transgenic rice lines also was detected by using rice culm cuttings. The results showed that 7-d-old larvae of Asiatic rice borer have the capacity to distinguish Bt and non-Bt culm cuttings and preferentially fed on non-Bt cuttings. When only Bt culm cuttings with three classes of different Bt proteins (CrylAc, Cry2A, and Cry9C) were fed, significant distribution difference of 7-d-old Asiatic rice borer in culm cuttings of different Bt proteins also was found. In the current study, we evaluate different Bt genes in the same rice variety in both the laboratory and the field, and also tested feeding behavior of rice insect to these Bt rice. These data are valuable for the further development of two-toxin Bt rice and establishment of appropriate insect resistance management in the future.

Oxytocin receptor gene and depressive symptoms associated with physiological reactivity to infant crying.

PubMed

Riem, Madelon M E; Pieper, Suzanne; Out, Dorothée; Bakermans-Kranenburg, Marian J; van Ijzendoorn, Marinus H

2011-06-01

Both the oxytocin receptor (OXTR) gene and depressive symptoms have been associated with parenting behaviour. The OXTR GG genotype has been suggested to be related to more sensitive parenting, whereas depressive symptoms may affect sensitivity negatively. We examined the role of OXTR and the influence of depressive symptoms in explaining differences in physiological reactivity to infant crying. Heart rate responses of 40 healthy females without children (age 19-47 years, randomly selected half of twin pairs) were measured during the presentation of three episodes of infant cry sounds. Participants with the presumably more efficient variant of the oxytonergic system gene (OXTR GG) had more pronounced physiological reactivity to repeated cry sounds, except when they showed more symptoms of depression. Results were replicated in the second half of the twin sample. This is the first study to suggest effects of OXTR genotype on physiological reactivity to infant crying. Depressive symptoms may however suppress the effect of the OXTR GG genotype.

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).

PubMed

Zheng, Si-Jun; Henken, Betty; de Maagd, Ruud A; Purwito, Agus; Krens, Frans A; Kik, Chris

2005-06-01

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae.

PubMed

Blázquez, M A; Gamo, F J; Gancedo, C

1995-12-18

Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose-ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307-315). We have isolated a dominant mutation, BPC1-1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1-1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cycle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1-1 on growth in glucose-ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1-1 is either allelic or closely linked to the mutation DGT1-1.

Parallel processing on the Livermore VAX 11/780-4 parallel processor system with compatibility to Cray Research, Inc. (CRI) multitasking. Version 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werner, N.E.; Van Matre, S.W.

1985-05-01

This manual describes the CRI Subroutine Library and Utility Package. The CRI library provides Cray multitasking functionality on the four-processor shared memory VAX 11/780-4. Additional functionality has been added for more flexibility. A discussion of the library, utilities, error messages, and example programs is provided.

Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

PubMed Central

Zhou, Dinggang; Guo, Jinlong; Xu, Liping; Gao, Shiwu; Lin, Qingliang; Wu, Qibin; Wu, Luguang; Que, Youxiong

2014-01-01

To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg2+, 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn2+ (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane. PMID:24810230

Bt Cry1Ie Toxin Does Not Impact the Survival and Pollen Consumption of Chinese Honey Bees, Apis cerana cerana (Hymenoptera, Apidae).

PubMed

Dai, Ping-Li; Jia, Hui-Ru; Jack, Cameron J; Geng, Li-Li; Liu, Feng; Hou, Chun-Sheng; Diao, Qing-Yun; Ellis, James D

2016-12-01

The cry1Ie gene may be a good candidate for the development of Bt maize because over-expression of Cry1Ie is highly toxic to Lepidopteran pests such as Heliothis armigera Hübner and Ostrinia furnacalis Guenée. The Bt cry1Ie gene also has no cross resistance with other insecticidal proteins such as Cry1Ab, Cry1Ac, Cry1Ah, or Cry1F. Chinese honey bees (Apis cerana cerana) are potentially exposed to insect-resistant genetically modified (IRGM) crops expressing Cry1Ie toxin via the collection of IRGM crop pollen. In this study, we tested whether Chinese honey bee workers are negatively affected by sugar syrup containing 20, 200, or 20,000 ng/ml Cry1Ie toxin and 48 ng/ml imidacloprid under controlled laboratory conditions. Our results demonstrated that the Cry1Ie toxin does not adversely impact survival and pollen consumption of Chinese honey bees. However, imidacloprid decreases Chinese honey bee survival and the total pollen consumption on the 5th, 6th, and 18th d of exposure. The described bioassay is suitable to assess the effects of GM expressed toxins against honey bee. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions.

PubMed

Breitler, Jean Christophe; Vassal, Jean Michel; del Mar Catala, Maria; Meynard, Donaldo; Marfà, Victoria; Melé, Enric; Royer, Monique; Murillo, Isabel; San Segundo, Blanca; Guiderdoni, Emmanuel; Messeguer, Joaquima

2004-09-01

Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.

Analysis of flour and food samples for cry9C from bioengineered corn.

PubMed

Orlandi, Palmer A; Lampel, Keith A; South, Paul K; Assar, Samir K; Carter, Laurenda; Levy, Dan D

2002-02-01

StarLink corn is a variety of yellow corn that has been genetically modified by the insertion of an altered cry9C gene into the plant genome. resulting in expression of the insecticidal Cry9C protein. The U.S. Environmental Protection Agency has approved StarLink corn for use in animal feed but not in food intended for human consumption. Therefore, under the U.S. Food, Drug, and Cosmetic Act, any food intended for human consumption in which the presence of StarLink corn is indicated by the presence of either the Cry9C protein or the cry9C gene would be considered adulterated. Extraction and PCR-based methods were used to detect the presence of the cry9C DNA initially in corn flour and corn meal, and then these methods were extended to the analysis of processed corn products, including taco shells, cereals, baby foods, party snacks, and chips, for the presence of this modified genetic material. In a survey of 63 products, the cry9C transgene was detected in 4 taco shells.

A role for cryptochromes in sleep regulation.

PubMed

Wisor, Jonathan P; O'Hara, Bruce F; Terao, Akira; Selby, Chris P; Kilduff, Thomas S; Sancar, Aziz; Edgar, Dale M; Franken, Paul

2002-12-20

The cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes. Under all three conditions, cry1,2-/- mice exhibit the hallmarks of high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS consolidation, and EEG delta power during NREMS). This unexpected phenotype was associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and albumin d-binding protein (dbp), which are known to be transcriptionally inhibited by CRY1,2. To further examine the relationship between circadian genes and sleep homeostasis, we examined wild type mice and rats following sleep deprivation and found increased levels of per1,2 mRNA and decreased levels of dbp mRNA specifically in the cerebral cortex; these changes subsided with recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and casein-kinase-1epsilon did not change with sleep deprivation. These results indicate that mice lacking cryptochromes are not simply a genetic model of circadian arrhythmicity in rodents and functionally implicate cryptochromes in the homeostatic regulation of sleep.

CRY1 circadian gene variant interacts with carbohydrate intake for insulin resistance in two independent populations: Mediterranean and North American

USDA-ARS?s Scientific Manuscript database

Dysregulation in the circadian system induced by variants of clock genes has been associated with type 2 diabetes. Evidence for the role of cryptochromes, core components of the system, in regulating glucose homeostasis is not supported by CRY1 candidate gene association studies for diabetes and ins...

Susceptibility of field populations of the fall armyworm (Lepidoptera: Noctuidae) from Florida and Puerto Rico to purified Cry1F protein and corn leaf tissue containing single and pyramided Bt genes

USDA-ARS?s Scientific Manuscript database

Larval survival of Cry1F-susceptible (FL), -resistant (PR and Cry1F-RR), and -heterozygous (FL x PR and Cry1F-RS) populations of the fall armyworm, Spodoptera frugiperda (J.E. Smith) to purified Cry1F protein and corn leaf tissue of seven Bacillus thuringiensis (Bt) corn hybrids and five non-Bt corn...

75 FR 57778 - Issuance of an Experimental Use Permit

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-22

...) necessary for its production in Event TC1507 corn. There are 10.213 kilograms (kg) of eCry3.1Ab, 11.14 grams of Cry1Ab, 8,145 grams of Cry34Ab1, 112.1 grams of Cry35Ab1, 280.6 grams of Vip3Aa20, 217.9 grams of mCry3A, and 177.5 grams of Cry1F proteins in 69,473 kg seeds on 7,311 acres (3,830 PIP and 3,481 non...

Bioinformatic analysis for allergenicity assessment of Bacillus thuringiensis Cry proteins expressed in insect-resistant food crops.

PubMed

Randhawa, Gurinder Jit; Singh, Monika; Grover, Monendra

2011-02-01

The novel proteins introduced into the genetically modified (GM) crops need to be evaluated for the potential allergenicity before their introduction into the food chain to address the safety concerns of consumers. At present, there is no single definitive test that can be relied upon to predict allergic response in humans to a new protein; hence a composite approach to allergic response prediction is described in this study. The present study reports on the evaluation of the Cry proteins, encoded by cry1Ac, cry1Ab, cry2Ab, cry1Ca, cry1Fa/cry1Ca hybrid, being expressed in Bt food crops that are under field trials in India, for potential allergenic cross-reactivity using bioinformatics search tools. The sequence identity of amino acids was analyzed using FASTA3 of AllergenOnline version 10.0 and BLASTX of NCBI Entrez to identify any potential sequence matches to allergen proteins. As a step further in the detection of allergens, an independent database of domains in the allergens available in the AllergenOnline database was also developed. The results indicated no significant alignment and similarity of Cry proteins at domain level with any of the known allergens revealing that there is no potential risk of allergenic cross-reactivity. Copyright © 2010 Elsevier Ltd. All rights reserved.

75 FR 3234 - Issuance of an Experimental Use Permit

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-20

... The quantity authorized equates to 68,066 pounds of corn seed [containing 62 grams (0.137 pounds) of Cry1F protein, 787 grams (1.74 pounds) of Cry34Ab1 and Cry35Ab1 proteins, 27 grams (0.060 pounds) of Cry1Ab protein, 347 grams (0.765 pounds) of Vip3Aa20 protein, and 11 grams (0.024 pounds) of mCry3A...

Assessment of the Antimicrobial Activity and the Entomocidal Potential of Bacillus thuringiensis Isolates from Algeria

PubMed Central

Djenane, Zahia; Nateche, Farida; Amziane, Meriam; Gomis-Cebolla, Joaquín; El-Aichar, Fairouz; Khorf, Hassiba; Ferré, Juan

2017-01-01

This work represents the first initiative to analyze the distribution of B. thuringiensis in Algeria and to evaluate the biological potential of the isolates. A total of 157 isolates were recovered, with at least one isolate in 94.4% of the samples. The highest Bt index was found in samples from rhizospheric soil (0.48) and from the Mediterranean area (0.44). Most isolates showed antifungal activity (98.5%), in contrast to the few that had antibacterial activity (29.9%). A high genetic diversity was made evident by the finding of many different crystal shapes and various combinations of shapes within a single isolate (in 58.4% of the isolates). Also, over 50% of the isolates harbored cry1, cry2, or cry9 genes, and 69.3% contained a vip3 gene. A good correlation between the presence of chitinase genes and antifungal activity was observed. More than half of the isolates with a broad spectrum of antifungal activity harbored both endochitinase and exochitinase genes. Interestingly, 15 isolates contained the two chitinase genes and all of the above cry family genes, with some of them harboring a vip3 gene as well. The combination of this large number of genes coding for entomopathogenic proteins suggests a putative wide range of entomotoxic activity. PMID:28406460

A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs.

PubMed

Guo, Jinchao; Chen, Lili; Liu, Xin; Gao, Ying; Zhang, Dabing; Yang, Litao

2012-06-01

Currently, the detection methods with lower cost and higher throughput are the major trend in screening genetically modified (GM) food or feed before specific identification. In this study, we developed a quadruplex degenerate PCR screening approach for more than 90 approved GMO events. This assay is consisted of four PCR systems targeting on nine DNA sequences from eight trait genes widely introduced into GMOs, such as CP4-EPSPS derived from Acetobacterium tumefaciens sp. strain CP4, phosphinothricin acetyltransferase gene derived from Streptomyceshygroscopicus (bar) and Streptomyces viridochromogenes (pat), and Cry1Ab, Cry1Ac, Cry1A(b/c), mCry3A, and Cry3Bb1 derived from Bacillus thuringiensis. The quadruplex degenerate PCR assay offers high specificity and sensitivity with the absolute limit of detection (LOD) of approximate 80targetcopies. Furthermore, the applicability of the quadruplex PCR assay was confirmed by screening either several artificially prepared samples or samples of Grain Inspection, Packers and Stockyards Administration (GIPSA) proficiency program. Copyright © 2011 Elsevier Ltd. All rights reserved.

CryJ-LAMP DNA Vaccines for Japanese Red Cedar Allergy Induce Robust Th1-Type Immune Responses in Murine Model

PubMed Central

Connolly, Michael; Marketon, Anthony

2016-01-01

Allergies caused by Japanese Red Cedar (JRC) pollen affect up to a third of Japanese people, necessitating development of an effective therapeutic. We utilized the lysosomal targeting property of lysosomal-associated membrane protein-1 (LAMP-1) to make DNA vaccines that encode LAMP-1 and the sequences of immunodominant allergen CryJ1 or CryJ2 from the JRC pollen. This novel strategy is designed to skew the CD4 T cell responses to the target allergens towards a nonallergenic Th1 response. CryJ1-LAMP and CryJ2-LAMP were administrated to BALB/c mice and antigen-specific Th1-type IgG2a and Th2-type IgG1 antibodies, as well as IgE antibodies, were assayed longitudinally. We also isolated different T cell populations from immunized mice and adoptively transferred them into naïve mice followed by CryJ1/CryJ2 protein boosts. We demonstrated that CryJ-LAMP immunized mice produce high levels of IFN-γ and anti-CryJ1 or anti-CryJ2 IgG2a antibodies and low levels of IgE antibodies, suggesting that a Th1 response was induced. In addition, we found that CD4+ T cells are the immunological effectors of DNA vaccination in this allergy model. Together, our results suggest the CryJ-LAMP Vaccine has a potential as an effective therapeutic for JRC induced allergy by skewing Th1/Th2 responses. PMID:27239481

Field-Evolved Resistance in Corn Earworm to Cry Proteins Expressed by Transgenic Sweet Corn

PubMed Central

Dively, Galen P.; Finkenbinder, Chad

2016-01-01

Background Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins. Methodology/Major Findings We present field monitoring data using Cry1Ab (1996–2016) and Cry1A.105+Cry2Ab2 (2010–2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab—event Bt11, and Cry1A.105+Cry2Ab2—event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood. Conclusions/Significance After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt sweet corn provide strong evidence of field-evolved resistance in H. zea populations to multiple Cry toxins. The high adoption rate of Bt field corn and cotton, along with the moderate dose expression of Cry1Ab and related Cry toxins in these crops, and decreasing refuge compliance probably contributed to the evolution of resistance. Our results have important implications for resistance monitoring, refuge requirements and other regulatory policies, cross-resistance issues, and the sustainability of the pyramided Bt technology. PMID:28036388

Mutational disruption of the ABCC2 gene in fall armyworm, Spodoptera frugiperda, confers resistance to the Cry1Fa and Cry1A.105 insecticidal proteins.

PubMed

Flagel, Lex; Lee, Young Wha; Wanjugi, Humphrey; Swarup, Shilpa; Brown, Alana; Wang, Jinling; Kraft, Edward; Greenplate, John; Simmons, Jeni; Adams, Nancy; Wang, Yanfei; Martinelli, Samuel; Haas, Jeffrey A; Gowda, Anilkumar; Head, Graham

2018-05-08

The use of Bt proteins in crops has revolutionized insect pest management by offering effective season-long control. However, field-evolved resistance to Bt proteins threatens their utility and durability. A recent example is field-evolved resistance to Cry1Fa and Cry1A.105 in fall armyworm (Spodoptera frugiperda). This resistance has been detected in Puerto Rico, mainland USA, and Brazil. A S. frugiperda population with suspected resistance to Cry1Fa was sampled from a maize field in Puerto Rico and used to develop a resistant lab colony. The colony demonstrated resistance to Cry1Fa and partial cross-resistance to Cry1A.105 in diet bioassays. Using genetic crosses and proteomics, we show that this resistance is due to loss-of-function mutations in the ABCC2 gene. We characterize two novel mutant alleles from Puerto Rico. We also find that these alleles are absent in a broad screen of partially resistant Brazilian populations. These findings confirm that ABCC2 is a receptor for Cry1Fa and Cry1A.105 in S. frugiperda, and lay the groundwork for genetically enabled resistance management in this species, with the caution that there may be several distinct ABCC2 resistances alleles in nature.

Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes.

PubMed

Fraiture, Marie-Alice; Vandamme, Julie; Herman, Philippe; Roosens, Nancy H C

2018-06-27

Recently, an integrated DNA walking strategy has been proposed to prove the presence of GMO via the characterisation of sequences of interest, including their transgene flanking regions and the unnatural associations of elements in their transgenic cassettes. To this end, the p35S, tNOS and t35S pCAMBIA elements have been selected as key targets, allowing the coverage of most of GMO, EU authorized or not. In the present study, a bidirectional DNA walking method anchored on the CryAb/c genes is proposed with the aim to cover additional GMO and additional sequences of interest. The performance of the proposed bidirectional DNA walking method anchored on the CryAb/c genes has been evaluated in a first time for its feasibility using several GM events possessing these CryAb/c genes. Afterwards, its sensitivity has been investigated through low concentrations of targets (as low as 20 HGE). In addition, to illustrate its applicability, the entire workflow has been tested on a sample mimicking food/feed matrices analysed in GMO routine analysis. Given the successful assessment of its performance, the present bidirectional DNA walking method anchored on the CryAb/c genes can easily be implemented in GMO routine analysis by the enforcement laboratories and allows completing the entire DNA walking strategy in targeting an additional transgenic element frequently found in GMO.

The Flavoproteins CryD and VvdA Cooperate with the White Collar Protein WcoA in the Control of Photocarotenogenesis in Fusarium fujikuroi

PubMed Central

Castrillo, Marta; Avalos, Javier

2015-01-01

Light stimulates carotenoid biosynthesis in the ascomycete fungus Fusarium fujikuroi through transcriptional activation of the structural genes of the pathway carRA, carB, and cart, but the molecular basis of this photoresponse is unknown. The F. fujikuroi genome contains genes for different predicted photoreceptors, including the WC protein WcoA, the DASH cryptochrome CryD and the Vivid-like flavoprotein VvdA. We formerly found that null mutants of wcoA, cryD or vvdA exhibit carotenoid photoinduction under continuous illumination. Here we show that the wild type exhibits a biphasic response in light induction kinetics experiments, with a rapid increase in carotenoid content in the first hours, a transient arrest and a subsequent slower increase. The mutants of the three photoreceptors show different kinetic responses: the wcoA mutants are defective in the rapid response, the cryD mutants are affected in the slower response, while the fast and slow responses were respectively enhanced and attenuated in the vvdA mutants. Transcriptional analyses of the car genes revealed a strong reduction of dark and light-induced transcript levels in the wcoA mutants, while minor or no reductions were found in the cryD mutants. Formerly, we found no change on carRA and carB photoinduction in vvdA mutants. Taken together, our data suggest a cooperative participation of WcoA and CryD in early and late stages of photoinduction of carotenoid biosynthesis in F. fujikuroi, and a possible modulation of WcoA activity by VvdA. An unexpected transcriptional induction by red light of vvdA, cryD and carRA genes suggest the participation of an additional red light-absorbing photoreceptor. PMID:25774802

Assessment of potential adjuvanticity of Cry proteins.

PubMed

Joshi, Saurabh S; Barnett, Brian; Doerrer, Nancy G; Glenn, Kevin; Herman, Rod A; Herouet-Guicheney, Corinne; Hunst, Penny; Kough, John; Ladics, Gregory S; McClain, Scott; Papineni, Sabitha; Poulsen, Lars K; Rascle, Jean-Baptiste; Tao, Ai-Lin; van Ree, Ronald; Ward, Jason; Bowman, Christal C

2016-08-01

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops. Copyright © 2016 Elsevier Inc. All rights reserved.

Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.

PubMed

Tanaka, Shiho; Miyamoto, Kazuhisa; Noda, Hiroaki; Endo, Haruka; Kikuta, Shingo; Sato, Ryoichi

2016-04-01

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins. Copyright © 2016. Published by Elsevier Inc.

Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas).

PubMed

Nayak, P; Basu, D; Das, S; Basu, A; Ghosh, D; Ramakrishnan, N A; Ghosh, M; Sen, S K

1997-03-18

Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

Multi-state trials of Bt sweet corn varieties for control of the corn earworm (Lepidoptera: Noctuidae).

PubMed

Shelton, A M; Olmstead, D L; Burkness, E C; Hutchison, W D; Dively, G; Welty, C; Sparks, A N

2013-10-01

Field tests in 2010-2011 were performed in New York, Minnesota, Maryland, Ohio, and Georgia to compare Bt sweet corn lines expressing Cry1A.105 + Cry2Ab2 and Cry1Ab with their non-Bt isolines, with and without the use of foliar insecticides. The primary insect pest in all locations during the trial years was Heliocoverpa zea (Boddie), which is becoming the most serious insect pest of sweet corn in the United States. At harvest, the ears were measured for marketability according to fresh market and processing standards. For fresh market and processing, least squares regression showed significant effects of protein expression, state, and insecticide frequency. There was a significant effect of year for fresh market but not for processing. The model also showed significant effects of H. zea per ear by protein expression. Sweet corn containing two genes (Cry1A.105 + Cry2Ab2) and a single gene (Cry1Ab) provided high marketability, and both Bt varieties significantly outperformed the traditional non-Bt isolines in nearly all cases regardless of insecticide application frequency. For pest suppression of H. zea, plants expressing Bt proteins consistently performed better than non-Bt isoline plants, even those sprayed at conventional insecticide frequencies. Where comparisons in the same state were made between Cry1A.105 + Cry2Ab2 and Cry1Ab plants for fresh market, the product expressing Cry1A.105 + Cry2Ab2 provided better protection and resulted in less variability in control. Overall, these results indicate Cry1A.105 + Cry2Ab2 and Cry1Ab plants are suitable for fresh market and processing corn production across a diversity of growing regions and years. Our results demonstrate that Bt sweet corn has the potential to significantly reduce the use of conventional insecticides against lepidopteran pests and, in turn, reduce occupational and environmental risks that arise from intensive insecticide use.

Gene expression analysis of enzymes of the carotenoid biosynthesis pathway involved in β-cryptoxanthin accumulation in wild raspberry, Rubus palmatus.

PubMed

Mizuno, Kouichi; Tokiwano, Tetsuo; Yoshizawa, Yuko

2017-03-18

β-cryptoxanthin (β-Cry), a xanthophyll, is unlike other abundant carotenoids, such as α-carotene, β-carotene, lycopene, lutein, and zeaxanthin. It is not found in most fruits or vegetables but is found only in specific fruits, such as hot chili pepper, persimmon, and citrus fruits. Because recent reports suggest that β-Cry intake is beneficial to human health, the xanthophyll requires further investigation. Although β-Cry accumulates in the fruit of wild raspberry, Rubus palmatus, it is not present in cultivated raspberry. In the present study, two wild raspberry species were studied-R. palmatus, which accumulates β-Cry in the fruit, and R. crataegifolius, which does not accumulate β-Cry. Four carotenoid biosynthetic enzymes derived from these two species were analyzed-phytoene synthase (PSY), lycopene β-cyclase (LCYb), β-carotene hydroxylase (HYb), and zeaxanthin epoxidase (ZEP). Expression levels of their genes were also assessed to elucidate mechanism underlying β-Cry accumulation. Partial gene sequences of RubPSY, RubLCYb, RubHYb, and RubZEP, isolated from immature raspberry fruits of R. palmatus, were used as probes for Northern blot analysis. RubZEP expression ceased as the fruits matured, possibly because of reduced production of zeaxanthin. β-Cry is considered to be an intermediate compound that accumulates in the mature fruits of R. palmatus. High expression of RubPSY was detectable in the mature fruits of R. crataegifolius, and the expression of RubLCYb, RubHYb, and RubZEP was detectable during all stages of fruit maturation. In contrast, β-Cry was absent in the mature fruits of R. crataegifolius. Copyright © 2017 Elsevier Inc. All rights reserved.

Systems-level analysis of risk genes reveals the modular nature of schizophrenia.

PubMed

Liu, Jiewei; Li, Ming; Luo, Xiong-Jian; Su, Bing

2018-05-19

Schizophrenia (SCZ) is a complex mental disorder with high heritability. Genetic studies (especially recent genome-wide association studies) have identified many risk genes for schizophrenia. However, the physical interactions among the proteins encoded by schizophrenia risk genes remain elusive and it is not known whether the identified risk genes converge on common molecular networks or pathways. Here we systematically investigated the network characteristics of schizophrenia risk genes using the high-confidence protein-protein interactions (PPI) from the human interactome. We found that schizophrenia risk genes encode a densely interconnected PPI network (P = 4.15 × 10 -31 ). Compared with the background genes, the schizophrenia risk genes in the interactome have significantly higher degree (P = 5.39 × 10 -11 ), closeness centrality (P = 7.56 × 10 -11 ), betweeness centrality (P = 1.29 × 10 -11 ), clustering coefficient (P = 2.22 × 10 -2 ), and shorter average shortest path length (P = 7.56 × 10 -11 ). Based on the densely interconnected PPI network, we identified 48 hub genes and 4 modules formed by highly interconnected schizophrenia genes. We showed that the proteins encoded by schizophrenia hub genes have significantly more direct physical interactions. Gene ontology (GO) analysis revealed that cell adhesion, cell cycle, immune system response, and GABR-receptor complex categories were enriched in the modules formed by highly interconnected schizophrenia risk genes. Our study reveals that schizophrenia risk genes encode a densely interconnected molecular network and demonstrates the modular nature of schizophrenia. Copyright © 2018 Elsevier B.V. All rights reserved.

The Two Cryptochrome/Photolyase Family Proteins Fulfill Distinct Roles in DNA Photorepair and Regulation of Conidiation in the Gray Mold Fungus Botrytis cinerea

PubMed Central

Cohrs, Kim C.

2017-01-01

ABSTRACT The plant-pathogenic leotiomycete Botrytis cinerea is known for the strict regulation of its asexual differentiation programs by environmental light conditions. Sclerotia are formed in constant darkness; black/near-UV (NUV) light induces conidiation; and blue light represses both differentiation programs. Sensing of black/NUV light is attributed to proteins of the cryptochrome/photolyase family (CPF). To elucidate the molecular basis of the photoinduction of conidiation, we functionally characterized the two CPF proteins encoded in the genome of B. cinerea as putative positive-acting components. B. cinerea CRY1 (BcCRY1), a cyclobutane pyrimidine dimer (CPD) photolyase, acts as the major enzyme of light-driven DNA repair (photoreactivation) and has no obvious role in signaling. In contrast, BcCRY2, belonging to the cry-DASH proteins, is dispensable for photorepair but performs regulatory functions by repressing conidiation in white and especially black/NUV light. The transcription of bccry1 and bccry2 is induced by light in a White Collar complex (WCC)-dependent manner, but neither light nor the WCC is essential for the repression of conidiation through BcCRY2 when bccry2 is constitutively expressed. Further, BcCRY2 affects the transcript levels of both WCC-induced and WCC-repressed genes, suggesting a signaling function downstream of the WCC. Since both CPF proteins are dispensable for photoinduction by black/NUV light, the origin of this effect remains elusive and may be connected to a yet unknown UV-light-responsive system. IMPORTANCE Botrytis cinerea is an economically important plant pathogen that causes gray mold diseases in a wide variety of plant species, including high-value crops and ornamental flowers. The spread of disease in the field relies on the formation of conidia, a process that is regulated by different light qualities. While this feature has been known for a long time, we are just starting to understand the underlying molecular mechanisms. Conidiation in B. cinerea is induced by black/near-UV light, whose sensing is attributed to the action of cryptochrome/photolyase family (CPF) proteins. Here we report on the distinct functions of two CPF proteins in the photoresponse of B. cinerea. While BcCRY1 acts as the major photolyase in photoprotection, BcCRY2 acts as a cryptochrome with a signaling function in regulating photomorphogenesis (repression of conidiation). PMID:28667107

The Blue Light-Dependent Polyubiquitination and Degradation of Arabidopsis Cryptochrome2 Requires Multiple E3 Ubiquitin Ligases.

PubMed

Liu, Qing; Wang, Qin; Liu, Bin; Wang, Wei; Wang, Xu; Park, Joon; Yang, Zhenming; Du, Xinglin; Bian, Mingdi; Lin, Chentao

2016-10-01

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Postnatal Ontogeny of the Circadian Expression of the Adrenal Clock Genes and Corticosterone Rhythm in Male Rats.

PubMed

Roa, Silvia Liliana Ruiz; Martinez, Edson Zangiacomi; Martins, Clarissa Silva; Antonini, Sonir Rauber; de Castro, Margaret; Moreira, Ayrton Custódio

2017-05-01

The postnatal synchronization of the circadian variation of the adrenal clock genes in mammals remains unknown. We evaluated the postnatal ontogeny of daily variation of clock genes (Clock/Bmal1/Per1/Per2/Per3/Cry1/Cry2/Rorα/Rev-Erbα) and steroidogenesis-related genes (Star and Mc2r) in rat adrenals and its relationship with the emergence of plasma corticosterone rhythm using cosinor analysis. Plasma corticosterone circadian rhythm was detected from postnatal day (P)1, with morning acrophase, between zeitgeber time (ZT)0 and ZT2. From P14, there was a nocturnal acrophase of corticosterone at ZT20, which was associated with pups' eye opening. From P3 there was a circadian variation of the mRNA expression of Bmal1, Per2, Per3, and Cry1 genes with morning acrophase, whereas Rev-Erbα had nocturnal acrophase. From P14, Bmal1, Per2, Per3, and Cry1 acrophases advanced by approximately 10 hours, as compared with early neonatal days, becoming vespertine-nocturnal. In all postnatal ages, Per2 and Cry1 circadian profiles were synchronized in phase with the circadian rhythm of plasma corticosterone, whereas Bmal1 was in antiphase. An adult-like Star circadian rhythm profile was observed only from P21. In conclusion, our original data demonstrated a progressive postnatal maturation of the circadian variation of the adrenal clock genes in synchrony with the development of the corticosterone circadian rhythm in rats. Copyright © 2017 Endocrine Society.

Moonlight controls lunar-phase-dependency and regular oscillation of clock gene expressions in a lunar-synchronized spawner fish, Goldlined spinefoot.

PubMed

Takeuchi, Yuki; Kabutomori, Ryo; Yamauchi, Chihiro; Miyagi, Hitomi; Takemura, Akihiro; Okano, Keiko; Okano, Toshiyuki

2018-04-18

Goldlined spinefoot, Siganus guttatus, inhabits tropical and subtropical waters and synchronizes its spawning around the first quarter moon likely using an hourglass-like lunar timer. In previous studies, we have found that clock genes (Cryptochrome3 and Period1) could play the role of state variable in the diencephalon when determining the lunar phase for spawning. Here, we identified three Cry, two Per, two Clock, and two Bmal genes in S. guttatus and investigated their expression patterns in the diencephalon and pituitary gland. We further evaluated the effect on their expression patterns by daily interruptions of moonlight stimuli for 1 lunar cycle beginning at the new moon. It significantly modified the expression patterns in many of the examined clock(-related) genes including Cry3 in the diencephalon and/or pituitary gland. Acute interruptions of moonlight around the waxing gibbous moon upregulated nocturnal expressions of Cry1b and Cry2 in the diencephalon and pituitary gland, respectively, but did not affect expression levels of the other clock genes. These results highlighted the importance of repetitive moonlight illumination for stable or lunar-phase-specific daily expression of clock genes in the next lunar cycle that may be important for the lunar-phase-synchronized spawning on the next first quarter moon.

Transgenic elite indica rice plants expressing CryIAc ∂-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas)

PubMed Central

Nayak, Pritilata; Basu, Debabrata; Das, Sampa; Basu, Asitava; Ghosh, Dipankar; Ramakrishnan, Neeliyath A.; Ghosh, Maloy; Sen, Soumitra K.

1997-01-01

Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding. PMID:9122157

[Influence of extremely low-frequency magnetic field on circadian rhythm of cryptochrome in mouse embryonic fibroblasts].

PubMed

Sun, Z Y; Geng, D Y; Chen, C F; Wang, P P; Song, T

2017-06-20

Objective: To investigate the influence of extremely low-frequency magnetic field on periodical expression of cryptochrome ( Cry ) gene in mouse embryonic fibroblast NIH3T3 cells. Methods: The NIH3T3 cells were divided into magnetic field group and sham-exposure group. The NIH3T3 cells in the magnetic field group were stimulated by horse serum and then exposed to an extremely low-frequency magnetic field (50 Hz and 0.3 mT) for 48 hours, and those in the sham-exposure group were also stimulated by horse serum and then exposed to a coil for 48 hours. The NIH3T3 cells were collected, total RNA was extracted, and cDNA was obtained via reverse transcription. Real-time fluorescent quantitative RT-PCR was used to measure the changes in transcription cycles of Cry and Period genes in both groups. Results: There was no significant difference in the proliferation rate at 0, 12, 24, and 48 hours of exposure between the two groups ( P >0.05) . Both sham-exposure group and magnetic field group showed a rhythmic change in the expression of Cry gene, and compared with the sham-exposure group, the magnetic field group had a significantly shortened circadian rhythm of Cry gene in NIH3T3 cells ( t =2.57, P <0.05) . Both groups had rhythmic and periodical expression of Period gene and there was no significant difference between the two groups ( t =0.70, P >0.05) . Conclusion: Extremely low-frequency magnetic field can significantly shorten the circadian rhythm of Cry gene in mouse embryonic fibroblasts, while there is no significant change in the circadian rhythm of Period gene.

Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

PubMed

Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

1991-02-15

The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides.

PubMed

Hendrischk, Anne-Kathrin; Frühwirth, Sebastian Walter; Moldt, Julia; Pokorny, Richard; Metz, Sebastian; Kaiser, Gebhard; Jäger, Andreas; Batschauer, Alfred; Klug, Gabriele

2009-11-01

Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the alpha-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity (K(d) approximately 10(-8) M) but double-stranded DNA and single-stranded RNA with far lower affinity (K(d) approximately 10(-6) M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides. Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.

Cryptochrome 1 regulates growth and development in Brassica through alteration in the expression of genes involved in light, phytohormone and stress signalling.

PubMed

Sharma, Pooja; Chatterjee, Mithu; Burman, Naini; Khurana, Jitendra P

2014-04-01

The blue light photoreceptors cryptochromes are ubiquitous in higher plants and are vital for regulating plant growth and development. In spite of being involved in controlling agronomically important traits like plant height and flowering time, cryptochromes have not been extensively characterized from agriculturally important crops. Here we show that overexpression of CRY1 from Brassica napus (BnCRY1), an oilseed crop, results in short-statured Brassica transgenics, likely to be less prone to wind and water lodging. The overexpression of BnCRY1 accentuates the inhibition of cell elongation in hypocotyls of transgenic seedlings. The analysis of hypocotyl growth inhibition and anthocyanin accumulation responses in BnCRY1 overexpressors substantiates that regulation of seedling photomorphogenesis by cry1 is dependent on light intensity. This study highlights that the photoactivated cry1 acts through coordinated induction and suppression of specific downstream genes involved in phytohormone synthesis or signalling, and those involved in cell wall modification, during de-etiolation of Brassica seedlings. The microarray-based transcriptome profiling also suggests that the overexpression of BnCRY1 alters abiotic/biotic stress signalling pathways; the transgenic seedlings were apparently oversensitive to abscisic acid (ABA) and mannitol. © 2013 John Wiley & Sons Ltd.

Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae

USDA-ARS?s Scientific Manuscript database

Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and th...

Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

PubMed Central

Jakka, Siva R. K.; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J.; Narva, Kenneth; Blanco, Carlos A.

2015-01-01

Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae. PMID:26637593

cry1Aa lacks stability elements at its 5'-UTR but integrity of its transcription terminator is critical to prevent decay of its transcript.

PubMed

Ramírez-Prado, Jorge Humberto; Martínez-Márquez, Eva Isabel; Olmedo-Alvarez, Gabriela

2006-07-01

We analyzed the influence of the 5' and 3' untranslated regions of the Bacillus thuringiensis cry1Aa on its mRNA stability. Although the cry1Aa gene has a stable transcript (8 min), its 5' UTR did not provide stability to the reporter gene uidA. Stability of cry1Aa could be increased to 40 min by addition of an SP82 stability element at the 5' UTR, suggesting that once the 5' and 3' ends were protected initiation of decay could be effectively blocked. We generated mutations in the transcription terminator and found that changes that reduced the stability of the stem, a larger loop, or elimination of the U-trail sharply decreased the half-life of the transcript. Therefore, unlike some stable bacterial transcripts, cry1Aa lacks special features at the end 5' to prevent decay, but its terminator is the main determinant of its stability.

FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

PubMed

Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J

2017-04-11

The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cry1F Resistance in Fall Armyworm Spodoptera frugiperda: Single Gene versus Pyramided Bt Maize

PubMed Central

Huang, Fangneng; Qureshi, Jawwad A.; Meagher, Robert L.; Reisig, Dominic D.; Head, Graham P.; Andow, David A.; Ni, Xinzi; Kerns, David; Buntin, G. David; Niu, Ying; Yang, Fei; Dangal, Vikash

2014-01-01

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012–2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda. PMID:25401494

Cry1F resistance in fall armyworm Spodoptera frugiperda: single gene versus pyramided Bt maize.

PubMed

Huang, Fangneng; Qureshi, Jawwad A; Meagher, Robert L; Reisig, Dominic D; Head, Graham P; Andow, David A; Ni, Xinzi; Kerns, David; Buntin, G David; Niu, Ying; Yang, Fei; Dangal, Vikash

2014-01-01

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012-2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda.

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests, Ostrinia nubilalis and Spodoptera frugiperda

PubMed Central

Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

2013-01-01

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case. PMID:23861865

Unilateral incompatibility gene ui1.1 encodes an S-locus F-box protein expressed in pollen of Solanum species.

PubMed

Li, Wentao; Chetelat, Roger T

2015-04-07

Unilateral interspecific incompatibility (UI) is a postpollination, prezygotic reproductive barrier that prevents hybridization between related species when the female parent is self-incompatible (SI) and the male parent is self-compatible (SC). In tomato and related Solanum species, two genes, ui1.1 and ui6.1, are required for pollen compatibility on pistils of SI species or hybrids. We previously showed that ui6.1 encodes a Cullin1 (CUL1) protein. Here we report that ui1.1 encodes an S-locus F-box (SLF) protein. The ui1.1 gene was mapped to a 0.43-cM, 43.2-Mbp interval at the S-locus on chromosome 1, but positional cloning was hampered by low recombination frequency. We hypothesized that ui1.1 encodes an SLF protein(s) that interacts with CUL1 and Skp1 proteins to form an SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligase complex. We identified 23 SLF genes in the S. pennellii genome, of which 19 were also represented in cultivated tomato (S. lycopersicum). Data from recombination events, expression analysis, and sequence annotation highlighted 11 S. pennellii genes as candidates. Genetic transformations demonstrated that one of these, SpSLF-23, is sufficient for ui1.1 function. A survey of cultivated and wild tomato species identified SLF-23 orthologs in each of the SI species, but not in the SC species S. lycopersicum, S. cheesmaniae, and S. galapagense, pollen of which lacks ui1.1 function. These results demonstrate that pollen compatibility in UI is mediated by protein degradation through the ubiquitin-proteasome pathway, a mechanism related to that which controls pollen recognition in SI.

Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests

PubMed Central

Pinto, Laura M.N.; Dörr, Natália C.; Ribeiro, Ana Paula A.; de Salles, Silvia M.; de Oliveira, Jaime V.; Menezes, Valmir G.; Fiuza, Lidia M.

2012-01-01

The screening of Bacillus thuringiensis (Bt) Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively) as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil), which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64%) and Bt dendrolimus HD-37 (62%) strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82%) to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations. PMID:24031872

Variants of CEP68 Gene Are Associated with Acute Urticaria/Angioedema Induced by Multiple Non-Steroidal Anti-Inflammatory Drugs

PubMed Central

Cornejo-García, José Antonio; Flores, Carlos; Plaza-Serón, María C.; Acosta-Herrera, Marialbert; Blanca-López, Natalia; Doña, Inmaculada; Torres, María J.; Mayorga, Cristobalina; Guéant-Rodríguez, Rosa M.; Ayuso, Pedro; Fernández, Javier; Laguna, José J.; Agúndez, José A. G.; García-Martín, Elena; Guéant, Jean-Louis; Canto, Gabriela; Blanca, Miguel

2014-01-01

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most consumed drugs worldwide because of their efficacy and utility in the treatment of pain and inflammatory diseases. However, they are also responsible for an important number of adverse effects including hypersensitivity reactions. The most important group of these reactions is triggered by non-immunological, pharmacological mechanisms catalogued under the denomination of cross-intolerance (CRI), with acute urticaria/angioedema induced by multiple NSAIDs (MNSAID-UA) the most frequently associated clinical entity. A recent genome-wide association study identified the gene encoding the centrosomal protein of 68 KDa (CEP68) as the major locus associated with aspirin intolerance susceptibility in asthmatics. In this study, we aimed to assess the role of this locus in susceptibility to CRI to NSAIDs by examining 53 common gene variants in a total of 635 patients that were classified as MNSAID-UA (n = 399), airway exacerbations (n = 110) or blended pattern (n = 126), and 425 controls. We found in the MNSAID-UA group a number of variants (17) associated (lowest p-value = 1.13×10−6), including the non-synonymous Gly74Ser variant (rs7572857) previously associated with aspirin intolerance susceptibility in asthmatics. Although not being significant in the context of multiple testing, eight of these variants were also associated with exacerbated respiratory disease or blended reactions. Our results suggest that CEP68 gene variants may play an important role in MNSAID-UA susceptibility and, despite the different regulatory mechanisms involved depending on the specific affected organ, in the development of hypersensitivity reactions to NSAIDs. PMID:24618698

Cryptochrome and Phytochrome Cooperatively but Independently Reduce Active Gibberellin Content in Rice Seedlings under Light Irradiation

PubMed Central

Hirose, Fumiaki; Inagaki, Noritoshi; Hanada, Atsushi; Yamaguchi, Shinjiro; Kamiya, Yuji; Miyao, Akio; Hirochika, Hirohiko; Takano, Makoto

2012-01-01

In contrast to a wealth of knowledge about the photoregulation of gibberellin metabolism in dicots, that in monocots remains largely unclear. In this study, we found that a blue light signal triggers reduction of active gibberellin content in rice seedlings with simultaneous repression of two gibberellin 20-oxidase genes (OsGA20ox2 and OsGA20ox4) and acute induction of four gibberellin 2-oxidase genes (OsGA2ox4–OsGA2ox7). For further examination of the regulation of these genes, we established a series of cryptochrome-deficient lines through reverse genetic screening from a Tos17 mutant population and construction of knockdown lines based on an RNA interference technique. By using these lines and phytochrome mutants, we elucidated that cryptochrome 1 (cry1), consisting of two species in rice plants (cry1a and cry1b), is indispensable for robust induction of the GA2ox genes. On the other hand, repression of the GA20ox genes is mediated by phytochromes. In addition, we found that the phytochromes also mediate the repression of a gibberellin 3-oxidase gene (OsGA3ox2) in the light. These results imply that, in rice seedlings, phytochromes mediate the repression of gibberellin biosynthesis capacity, while cry1 mediates the induction of gibberellin inactivation capacity. The cry1 action was demonstrated to be dominant in the reduction of active gibberellin content, but, in rice seedlings, the cumulative effects of these independent actions reduced active gibberellin content in the light. This pathway design in which different types of photoreceptors independently but cooperatively regulate active gibberellin content is unique from the viewpoint of dicot research. This redundancy should provide robustness to the response in rice plants. PMID:22764280

Cryptochrome and phytochrome cooperatively but independently reduce active gibberellin content in rice seedlings under light irradiation.

PubMed

Hirose, Fumiaki; Inagaki, Noritoshi; Hanada, Atsushi; Yamaguchi, Shinjiro; Kamiya, Yuji; Miyao, Akio; Hirochika, Hirohiko; Takano, Makoto

2012-09-01

In contrast to a wealth of knowledge about the photoregulation of gibberellin metabolism in dicots, that in monocots remains largely unclear. In this study, we found that a blue light signal triggers reduction of active gibberellin content in rice seedlings with simultaneous repression of two gibberellin 20-oxidase genes (OsGA20ox2 and OsGA20ox4) and acute induction of four gibberellin 2-oxidase genes (OsGA2ox4-OsGA2ox7). For further examination of the regulation of these genes, we established a series of cryptochrome-deficient lines through reverse genetic screening from a Tos17 mutant population and construction of knockdown lines based on an RNA interference technique. By using these lines and phytochrome mutants, we elucidated that cryptochrome 1 (cry1), consisting of two species in rice plants (cry1a and cry1b), is indispensable for robust induction of the GA2ox genes. On the other hand, repression of the GA20ox genes is mediated by phytochromes. In addition, we found that the phytochromes also mediate the repression of a gibberellin 3-oxidase gene (OsGA3ox2) in the light. These results imply that, in rice seedlings, phytochromes mediate the repression of gibberellin biosynthesis capacity, while cry1 mediates the induction of gibberellin inactivation capacity. The cry1 action was demonstrated to be dominant in the reduction of active gibberellin content, but, in rice seedlings, the cumulative effects of these independent actions reduced active gibberellin content in the light. This pathway design in which different types of photoreceptors independently but cooperatively regulate active gibberellin content is unique from the viewpoint of dicot research. This redundancy should provide robustness to the response in rice plants.

Variants in glucose- and circadian rhythm-related genes affect the response of energy expenditure to weight-loss diets: the POUNDS LOST Trial.

PubMed

Mirzaei, Khadijeh; Xu, Min; Qi, Qibin; de Jonge, Lilian; Bray, George A; Sacks, Frank; Qi, Lu

2014-02-01

Circadian rhythm has been shown to be related to glucose metabolism and risk of diabetes, probably through effects on energy balance. Recent genome-wide association studies identified variants in circadian rhythm-related genes (CRY2 and MTNR1B) associated with glucose homeostasis. We tested whether CRY2 and MTNR1B genotypes affected changes in measures of energy expenditure in response to a weight-loss diet intervention in a 2-y randomized clinical trial, the POUNDS (Preventing Overweight Using Novel Dietary Strategies) LOST Trial. The variants CRY2 rs11605924 (n = 721) and MTNR1B rs10830963 (n = 722) were genotyped in overweight or obese adults who were randomly assigned to 1 of 4 weight-loss diets that differed in their proportions of macronutrients. Respiratory quotient (RQ) and resting metabolic rate (RMR) were measured. By 2 y of diet intervention, the A allele of CRY2 rs11605924 was significantly associated with a greater reduction in RQ (P = 0.03) and a greater increase in RMR and RMR/kg (both P = 0.04). The G allele of MTNR1B rs10830963 was significantly associated with a greater increase in RQ (P = 0.01) but was not related to changes in RMR and RMR/kg. In addition, we found significant gene-diet fat interactions for both CRY2 (P-interaction = 0.02) and MTNR1B (P-interaction < 0.001) in relation to 2-y changes in RQ. Our data indicate that variants in the circadian-related genes CRY2 and MTNR1B may affect long-term changes in energy expenditure, and dietary fat intake may modify the genetic effects. This trial was registered at www.clinicaltrials.gov as NCT00072995.

Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India.

PubMed

Patel, Ketan D; Chudasama, Chaitanyasinh J; Ingle, Sanjay S

2012-08-01

Bacillus thuringiensis (Bt) strains were isolated from 94 samples from different geographical regions. Novel types of crystalline inclusion bodies were observed from some of the isolates. Crystalline inclusions of bipyramidal, spherical and cuboidal morphology were found produced by most of the isolates. Isolate GS12 showed crystal on one side of spore while isolate GM108 formed crystals on both termini of spore. Isolate GN31 produced large sized bipyramidal crystals. SDS-PAGE analysis of the spore crystal suspension showed major protein bands in the range of 29 and 140 kDa. Two new serovars of Bt viz. GS4 and GN24 having H3abce and H3ab serotype respectively were isolated. Toxicity comparable to the reference strain Bacillus thuringiensis subs. kurstaki (Btk) HD-1 was observed for the isolates GM20, GM17 and MP3 against larvae of Helicoverpa armigera. Some of the isolates harboring cry genes like cry1Ac and cry2 did not show any toxicity towards H. armigera while most of the isolates were harboring cry1, cry1Ac and cry2 gene. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1.

PubMed

Ode, Koji L; Ukai, Hideki; Susaki, Etsuo A; Narumi, Ryohei; Matsumoto, Katsuhiko; Hara, Junko; Koide, Naoshi; Abe, Takaya; Kanemaki, Masato T; Kiyonari, Hiroshi; Ueda, Hiroki R

2017-01-05

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1 -/- :Cry2 -/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

PubMed

Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu

2003-01-01

The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

Knockout of a P-glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua.

PubMed

Zuo, Y-Y; Huang, J-L; Wang, J; Feng, Y; Han, T-T; Wu, Y-D; Yang, Y-H

2018-02-01

P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua. © 2017 The Royal Entomological Society.

Bacterial bioluminescence regulates expression of a host cryptochrome gene in the squid-Vibrio symbiosis.

PubMed

Heath-Heckman, Elizabeth A C; Peyer, Suzanne M; Whistler, Cheryl A; Apicella, Michael A; Goldman, William E; McFall-Ngai, Margaret J

2013-04-02

The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut.

Bacterial Bioluminescence Regulates Expression of a Host Cryptochrome Gene in the Squid-Vibrio Symbiosis

PubMed Central

Heath-Heckman, Elizabeth A. C.; Peyer, Suzanne M.; Whistler, Cheryl A.; Apicella, Michael A.; Goldman, William E.; McFall-Ngai, Margaret J.

2013-01-01

ABSTRACT The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut. PMID:23549919

Life-History Traits of Spodoptera frugiperda Populations Exposed to Low-Dose Bt Maize

PubMed Central

Sousa, Fernanda F.; Mendes, Simone M.; Santos-Amaya, Oscar F.; Araújo, Octávio G.; Oliveira, Eugenio E.

2016-01-01

Exposure to Bacillus thuringiensis (Bt) toxins in low- and moderate-dose transgenic crops may induce sublethal effects and increase the rate of Bt resistance evolution, potentially compromising control efficacy against target pests. We tested this hypothesis using the fall armyworm Spodoptera frugiperda, a major polyphagous lepidopteran pest relatively tolerant to Bt notorious for evolving field-relevant resistance to single-gene Bt maize. Late-instar larvae were collected from Bt Cry1Ab and non-Bt maize fields in five locations in Brazil, and their offspring was compared for survival, development, and population growth in rearing environment without and with Cry1Ab throughout larval development. Larval survival on Cry1Ab maize leaves varied from 20 to 80% among the populations. Larvae reared on Cry1Ab maize had seven-day delay in development time in relation to control larvae, and such delay was shorter in offspring of armyworms from Cry1Ab maize. Population growth rates were 50–70% lower for insects continuously exposed to Cry1Ab maize relative to controls, showing the population-level effect of Cry1Ab, which varied among the populations and prior exposure to Cry1Ab maize in the field. In three out of five populations, armyworms derived from Bt maize reared on Cry1Ab maize showed higher larval weight, faster larval development and better reproductive performance than the armyworms derived from non-Bt maize, and one of these populations showed better performance on both Cry1Ab and control diets, indicating no fitness cost of the resistance trait. Altogether, these results indicate that offspring of armyworms that developed on field-grown, single-gene Bt Cry1Ab maize had reduced performance on Cry1Ab maize foliage in two populations studied, but in other three populations, these offspring had better overall performance on the Bt maize foliage than that of the armyworms from non-Bt maize fields, possibly because of Cry1Ab resistance alleles in these populations. Implications of these findings for resistance management of S. frugiperda in Bt crops are discussed. PMID:27243977

Life-History Traits of Spodoptera frugiperda Populations Exposed to Low-Dose Bt Maize.

PubMed

Sousa, Fernanda F; Mendes, Simone M; Santos-Amaya, Oscar F; Araújo, Octávio G; Oliveira, Eugenio E; Pereira, Eliseu J G

2016-01-01

Exposure to Bacillus thuringiensis (Bt) toxins in low- and moderate-dose transgenic crops may induce sublethal effects and increase the rate of Bt resistance evolution, potentially compromising control efficacy against target pests. We tested this hypothesis using the fall armyworm Spodoptera frugiperda, a major polyphagous lepidopteran pest relatively tolerant to Bt notorious for evolving field-relevant resistance to single-gene Bt maize. Late-instar larvae were collected from Bt Cry1Ab and non-Bt maize fields in five locations in Brazil, and their offspring was compared for survival, development, and population growth in rearing environment without and with Cry1Ab throughout larval development. Larval survival on Cry1Ab maize leaves varied from 20 to 80% among the populations. Larvae reared on Cry1Ab maize had seven-day delay in development time in relation to control larvae, and such delay was shorter in offspring of armyworms from Cry1Ab maize. Population growth rates were 50-70% lower for insects continuously exposed to Cry1Ab maize relative to controls, showing the population-level effect of Cry1Ab, which varied among the populations and prior exposure to Cry1Ab maize in the field. In three out of five populations, armyworms derived from Bt maize reared on Cry1Ab maize showed higher larval weight, faster larval development and better reproductive performance than the armyworms derived from non-Bt maize, and one of these populations showed better performance on both Cry1Ab and control diets, indicating no fitness cost of the resistance trait. Altogether, these results indicate that offspring of armyworms that developed on field-grown, single-gene Bt Cry1Ab maize had reduced performance on Cry1Ab maize foliage in two populations studied, but in other three populations, these offspring had better overall performance on the Bt maize foliage than that of the armyworms from non-Bt maize fields, possibly because of Cry1Ab resistance alleles in these populations. Implications of these findings for resistance management of S. frugiperda in Bt crops are discussed.

Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie)▿

PubMed Central

Anilkumar, Konasale J.; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J.

2008-01-01

Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments. PMID:18024681

Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests.

PubMed

Boddupally, Dayakar; Tamirisa, Srinath; Gundra, Sivakrishna Rao; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2018-05-31

To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.

Introduction and expression of the cry1Ac gene of Bacillus thuringiensis in a cereal-associated bacterium, Bacillus polymyxa.

PubMed

Sudha, S N; Jayakumar, R; Sekar, V

1999-03-01

The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B. polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made to introduce the cry1Ac gene of B. thuringiensis subsp. kurstaki into B. polymyxa so that the application of such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic.

Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria

PubMed Central

Downing, Katrina J.; Leslie, Graeme; Thomson, Jennifer A.

2000-01-01

The cry1Ac7 gene of Bacillus thuringiensis strain 234, showing activity against the sugarcane borer Eldana saccharina, was cloned under the control of the tac promoter. The fusion was introduced into the broad-host-range plasmid pKT240 and the integration vector pJFF350 and without the tac promoter into the broad-host-range plasmids pML122 and pKmM0. These plasmids were introduced into a Pseudomonas fluorescens strain isolated from the phylloplane of sugarcane and the endophytic bacterium Herbaspirillum seropedicae found in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the tac promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nmr promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher mortality of larvae than did P. fluorescens 14::tox. An increased toxic effect was observed when P. fluorescens 14::ptac-tox was combined with P. fluorescens carrying the Serratia marcescens chitinase gene chiA, under the control of the tac promoter, integrated into the chromosome. PMID:10877771

Evaluation of Bt (Bacillus thuringiensis) rice varieties against stem borer (Chilo suppressalis).

PubMed

Kiani, Ghaffar; Nematzadeh, Ghorban Ali; Ghareyazie, Behzad; Sattari, Majid

2008-02-15

Three transgenic rice varieties namely Khazar, Neda and Nemat, all containing a cry1Ab gene, were evaluated through PCR analysis and field examinations for their resistance at natural infestation of insect pests during 2007. The results showed that all transgenic varieties produced 1.2 kb PCR product derived from application of cry1Ab gene. In field conditions, transgenic varieties exhibited high levels of resistance against natural infestation of stem borer and the damaged plants based on dead heart or white heat for them were less than 1%. Moreover, in stem-cut bioassay 100% of released larvae died within four days after infestation. These results demonstrate that expression of cry1Ab gene in the genome of transgenic varieties provided season-long protection from the natural infestation of lepidopteran insects.

Comparative analysis of the genetic basis of Cry1F resistance in two strains of Spodoptera frugiperda originated from Puerto Rico and Florida.

PubMed

Camargo, Ana M; Castañera, Pedro; Farinós, Gema P; Huang, Fangneng

2017-06-01

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target pest of Bacillus thuringiensis (Bt) maize and cotton in America. Since the commercialization of Cry1F maize (event TC1507) in 2003, resistance to Cry1F maize in field populations of S. frugiperda has occurred in Puerto Rico, Brazil and the southeast region of the United States. In this paper, we conducted a comparative analysis of the inheritance of two Cry1F-resistant colonies of S. frugiperda originated from Puerto Rico (PR) and Florida (FL), respectively. The objective of the analysis was to determine if the genetic basis of the resistance was similar in the two different originated colonies. To accomplish the objective, besides PR, FL, and a known Cry1F-susceptible colony, 14 additional colonies were developed by reciprocal crosses among the three parents, F 1 by F 1 crosses, backcrosses, and intercolony-crosses between PR and FL. Larval mortalities of the 17 colonies were assayed on both Cry1F maize leaf tissue and Cry1F-treated diet at the concentrations of 3.16, 10.00, and 31.60µg/g. Resistance to Cry1F in both PR and FL was autosomal and recessive or incompletely recessive. Segregations in F 2 and backcrossed generations associated with FL fitted the Mendelian monogenic model well, while with PR the segregations did not follow the single gene model in some bioassays. Further analyses with the intercolony complementation tests showed a similar level of resistance in the F 1 progeny as their parents FL and PR. Together with the data, it was likely that a single (or a few tightly-linked) gene was involved in FL; PR shared the same locus of the major resistance gene as FL, but the resistance in PR might also be associated with additional minor factors. Information generated from this study should be useful in understanding the origin of Cry1F resistance in the U.S. mainland and developing effective strategies for Bt resistance management in S. frugiperda. Copyright © 2017 Elsevier Inc. All rights reserved.

Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds.

PubMed

Wang, Qing; Zhu, Yi; Sun, Lin; Li, Lebin; Jin, Shuangxia; Zhang, Xianlong

2016-02-01

A promoter of the PNZIP (Pharbitis nil leucine zipper) gene (1.459 kb) was cloned from Pharbitis nil and fused to the GUS (β-glucuronidase) and Bacillus thuringiensis endotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 μg g(-1) fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 μg g(-1) fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 μg g(-1) fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.

Unraveling flp-11/flp-32 dichotomy in nematodes.

PubMed

Atkinson, Louise E; Miskelly, Iain R; Moffett, Christy L; McCoy, Ciaran J; Maule, Aaron G; Marks, Nikki J; Mousley, Angela

2016-10-01

FMRFamide-like peptide (FLP) signalling systems are core to nematode neuromuscular function. Novel drug discovery efforts associated with nematode FLP/FLP receptor biology are advanced through the accumulation of basic biological data that can reveal subtle complexities within the neuropeptidergic system. This study reports the characterisation of FMRFamide-like peptide encoding gene-11 (flp-11) and FMRFamide-like peptide encoding gene-32 (flp-32), two distinct flp genes which encode the analogous peptide, AMRN(A/S)LVRFamide, in multiple nematode species - the only known example of this phenomenon within the FLPergic system of nematodes. Using bioinformatics, in situ hybridisation, immunocytochemistry and behavioural assays we show that: (i) flp-11 and -32 are distinct flp genes expressed individually or in tandem across multiple nematode species, where they encode a highly similar peptide; (ii) flp-11 does not appear to be the most widely expressed flp in Caenorhabditis elegans; (iii) in species expressing both flp-11 and flp-32, flp-11 displays a conserved, restricted expression pattern across nematode clades and lifestyles; (iv) in species expressing both flp-11 and flp-32, flp-32 expression is more widespread and less conserved than flp-11; (v) in species expressing only flp-11, the flp-11 expression profile is more similar to the flp-32 profile observed in species expressing both; and (vi) FLP-11 peptides inhibit motor function in multiple nematode species. The biological significance and evolutionary origin of flp-11 and -32 peptide duplication remains unclear despite attempts to identify a common ancestor; this may become clearer as the availability of genomic data improves. This work provides insight into the complexity of the neuropeptidergic system in nematodes, and begins to examine how nematodes may compensate for structural neuronal simplicity. From a parasite control standpoint, this work underscores the importance of basic biological data, and has wider implications for the utility of C. elegans as a model for parasite neurobiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions.

PubMed

González-Ponce, Karen S; Casados-Vázquez, Luz E; Salcedo-Hernández, Rubén; Bideshi, Dennis K; Del Rincón-Castro, María C; Barboza-Corona, José E

2017-05-01

In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry - B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74∆sp produced amorphous inclusions in both Cry - B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74∆sp chimeric inclusions of recombinant HD73 strain showed LC 50 s of 396.86 and 290.25 ng/cm 2 , respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.

Stable integration and expression of a cry1Ia gene conferring resistance to fall armyworm and boll weevil in cotton plants.

PubMed

Silva, Carliane Rc; Monnerat, Rose; Lima, Liziane M; Martins, Érica S; Melo Filho, Péricles A; Pinheiro, Morganna Pn; Santos, Roseane C

2016-08-01

Boll weevil is a serious pest of cotton crop. Effective control involves applications of chemical insecticides, increasing the cost of production and environmental pollution. The current genetically modified Bt crops have allowed great benefits to farmers but show activity limited to lepidopteran pests. This work reports on procedures adopted for integration and expression of a cry transgene conferring resistance to boll weevil and fall armyworm by using molecular tools. Four Brazilian cotton cultivars were microinjected with a minimal linear cassette generating 1248 putative lines. Complete gene integration was found in only one line (T0-34) containing one copy of cry1Ia detected by Southern blot. Protein was expressed in high concentration at 45 days after emergence (dae), decreasing by approximately 50% at 90 dae. Toxicity of the cry protein was demonstrated in feeding bioassays revealing 56.7% mortality to boll weevil fed buds and 88.1% mortality to fall armyworm fed leaves. A binding of cry1Ia antibody was found in the midgut of boll weevils fed on T0-34 buds in an immunodetection assay. The gene introduced into plants confers resistance to boll weevil and fall armyworm. Transmission of the transgene occurred normally to T1 progeny. All plants showed phenotypically normal growth, with fertile flowers and abundant seeds. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

PubMed Central

de Souza Freire, Ingrid; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Martins, Erica Soares; Monnerat, Rose Gomes; Grisolia, Cesar Koppe

2014-01-01

The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually. PMID:25268978

Detection and characterization of cry1Ac transgene construct in Bt cotton: multiple polymerase chain reaction approach.

PubMed

Singh, Chandra K; Ojha, Abhishek; Kachru, Devendra N

2007-01-01

To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.

Dysfunction of SHANK2 and CHRNA7 in a patient with intellectual disability and language impairment supports genetic epistasis of the two loci.

PubMed

Chilian, B; Abdollahpour, H; Bierhals, T; Haltrich, I; Fekete, G; Nagel, I; Rosenberger, G; Kutsche, K

2013-12-01

Synaptopathies constitute a group of neurological diseases including autism spectrum disorders (ASD) and intellectual disability (ID). They have been associated with mutations in genes encoding proteins important for the formation and stabilization of synapses, such as SHANK1-3. Loss-of-function mutations in the SHANK genes have been identified in individuals with ASD and ID suggesting that other factors modify the neurological phenotype. We report a boy with severe ID, behavioral anomalies, and language impairment who carries a balanced de novo triple translocation 46,XY,t(11;17;19)(q13.3;q25.1;q13.42). The 11q13.3 breakpoint was found to disrupt the SHANK2 gene. The patient also carries copy number variations at 15q13.3 and 10q22.11 encompassing ARHGAP11B and two synaptic genes. The CHRNA7 gene encoding α7-nicotinic acetylcholine receptor subunit and the GPRIN2 gene encoding G-protein-regulated inducer of neurite growth 2 were duplicated. Co-occurrence of a de novo SHANK2 mutation and a CHRNA7 duplication in two reported patients with ASD and ID as well as in the patient with t(11;17;19), severe ID and behavior problems suggests convergence of these genes on a common synaptic pathway. Our results strengthen the oligogenic inheritance model and highlight the presence of a large effect mutation and modifier genes collectively determining phenotypic expression of the synaptopathy. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

CHARACTERIZATION OF H-Y AND CR-Y ZEOLITE CATALYSTS DURING THE OXIDATIVE DESTRUCTION OF CFC11 AND CFC12

EPA Science Inventory

The long term stability-deactivation characteristics of two Y zeolite catalysts, namely H-Y and cation exchanged Cr-Y, were studied during the oxidative destruction of CFC11 and CFC12 feeds. Experiments were carried out at 300 degrees C and 500 h-1 space velocity. Properties of...

Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

PubMed Central

Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

2012-01-01

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

Genomic Epidemiology of Hypervirulent Serogroup W, ST-11 Neisseria meningitidis

PubMed Central

Mustapha, Mustapha M.; Marsh, Jane W.; Krauland, Mary G.; Fernandez, Jorge O.; de Lemos, Ana Paula S.; Dunning Hotopp, Julie C.; Wang, Xin; Mayer, Leonard W.; Lawrence, Jeffrey G.; Hiller, N. Luisa; Harrison, Lee H.

2015-01-01

Neisseria meningitidis is a leading bacterial cause of sepsis and meningitis globally with dynamic strain distribution over time. Beginning with an epidemic among Hajj pilgrims in 2000, serogroup W (W) sequence type (ST) 11 emerged as a leading cause of epidemic meningitis in the African ‘meningitis belt’ and endemic cases in South America, Europe, Middle East and China. Previous genotyping studies were unable to reliably discriminate sporadic W ST-11 strains in circulation since 1970 from the Hajj outbreak strain (Hajj clone). It is also unclear what proportion of more recent W ST-11 disease clusters are caused by direct descendants of the Hajj clone. Whole genome sequences of 270 meningococcal strains isolated from patients with invasive meningococcal disease globally from 1970 to 2013 were compared using whole genome phylogenetic and major antigen-encoding gene sequence analyses. We found that all W ST-11 strains were descendants of an ancestral strain that had undergone unique capsular switching events. The Hajj clone and its descendants were distinct from other W ST-11 strains in that they shared a common antigen gene profile and had undergone recombination involving virulence genes encoding factor H binding protein, nitric oxide reductase, and nitrite reductase. These data demonstrate that recent acquisition of a distinct antigen-encoding gene profile and variations in meningococcal virulence genes was associated with the emergence of the Hajj clone. Importantly, W ST-11 strains unrelated to the Hajj outbreak contribute a significant proportion of W ST-11 cases globally. This study helps illuminate genomic factors associated with meningococcal strain emergence and evolution. PMID:26629539

Crystallin-αB Regulates Skeletal Muscle Homeostasis via Modulation of Argonaute2 Activity*

PubMed Central

Neppl, Ronald L.; Kataoka, Masaharu; Wang, Da-Zhi

2014-01-01

The core functional machinery of the RNAi pathway is the RNA-induced silencing complex (RISC), wherein Argonaute2 (Ago2) is essential for siRNA-directed endonuclease activity and RNAi/microRNA-mediated gene silencing. Crystallin-αB (CryAB) is a small heat shock protein involved in preventing protein aggregation. We demonstrate that CryAB interacts with the N and C termini of Ago2, not the catalytic site defined by the convergence of the PAZ, MID, and PIWI domains. We further demonstrate significantly reduced Ago2 activity in the absence of CryAB, highlighting a novel role of CryAB in the mammalian RNAi/microRNA pathway. In skeletal muscle of CryAB null mice, we observe a shift in the hypertrophy-atrophy signaling axis toward atrophy under basal conditions. Moreover, loss of CryAB altered the capability of satellite cells to regenerate skeletal muscle. These studies establish that CryAB is necessary for normal Ago2/RISC activity and cellular homeostasis in skeletal muscle. PMID:24782307

Extra-hypothalamic brain clocks in songbirds: Photoperiodic state dependent clock gene oscillations in night-migratory blackheaded buntings, Emberiza melanocephala.

PubMed

Singh, Devraj; Kumar, Vinod

2017-04-01

The avian circadian pacemaker system is comprised of independent clocks in the retina, pineal and hypothalamus, as shown by daily and circadian oscillations of core clock genes (Per2, Cry1, Bmal1 and Clock) in several birds including migratory blackheaded buntings (Emberiza melanocephala). This study investigated the extra-hypothalamic brain circadian clocks in blackheaded buntings, and measured Per2, Cry1, Cry2, Bmal1 and Clock mRNA expressions at 4h intervals over 24h beginning 1h after light-on in the left and right telencephalon, optic tectum and cerebellum, the brain regions involved in several physiological and cognitive functions. Because of seasonal alterations in the circadian clock dependent brain functions, we measured daily clock gene oscillations in buntings photoperiod-induced with the non-migratory state under short days (SDnM), and the pre-migratory (LDpM), migratory (LDM) and post-migratory (refractory, LDR) states under long days. Daily Per2 oscillations were not altered with changes in the photoperiodic states, except for about 2-3h phase difference in the optic tectum between the SDnM and LDpM states. However, there were about 3-5h differences in the phase and 2 to 4 fold change in the amplitude of daily Bmal1 and Cry1 mRNA oscillations between the photoperiod-induced states. Further, Cry2 and Clock genes lacked a significant oscillation, except in Cb (Cry2) and TeO and Rt (Clock) under LDR state. Overall, these results show the presence of circadian clocks in extra-hypothalamic brain regions of blackheaded buntings, and suggest tissue-dependent alterations in the waveforms of mRNA oscillations with transitions in the photoperiod-induced seasonal states in a long-day species. Copyright © 2017 Elsevier B.V. All rights reserved.

Variants in glucose- and circadian rhythm–related genes affect the response of energy expenditure to weight-loss diets: the POUNDS LOST Trial123

PubMed Central

Mirzaei, Khadijeh; Xu, Min; Qi, Qibin; de Jonge, Lilian; Bray, George A; Sacks, Frank; Qi, Lu

2014-01-01

Background: Circadian rhythm has been shown to be related to glucose metabolism and risk of diabetes, probably through effects on energy balance. Recent genome-wide association studies identified variants in circadian rhythm–related genes (CRY2 and MTNR1B) associated with glucose homeostasis. Objective: We tested whether CRY2 and MTNR1B genotypes affected changes in measures of energy expenditure in response to a weight-loss diet intervention in a 2-y randomized clinical trial, the POUNDS (Preventing Overweight Using Novel Dietary Strategies) LOST Trial. Design: The variants CRY2 rs11605924 (n = 721) and MTNR1B rs10830963 (n = 722) were genotyped in overweight or obese adults who were randomly assigned to 1 of 4 weight-loss diets that differed in their proportions of macronutrients. Respiratory quotient (RQ) and resting metabolic rate (RMR) were measured. Results: By 2 y of diet intervention, the A allele of CRY2 rs11605924 was significantly associated with a greater reduction in RQ (P = 0.03) and a greater increase in RMR and RMR/kg (both P = 0.04). The G allele of MTNR1B rs10830963 was significantly associated with a greater increase in RQ (P = 0.01) but was not related to changes in RMR and RMR/kg. In addition, we found significant gene-diet fat interactions for both CRY2 (P-interaction = 0.02) and MTNR1B (P-interaction < 0.001) in relation to 2-y changes in RQ. Conclusions: Our data indicate that variants in the circadian-related genes CRY2 and MTNR1B may affect long-term changes in energy expenditure, and dietary fat intake may modify the genetic effects. This trial was registered at www.clinicaltrials.gov as NCT00072995. PMID:24335056

Deletion of a Single-Copy Trna Affects Microtubule Function in Saccharomyces Cerevisiae

PubMed Central

Reijo, R. A.; Cho, D. S.; Huffaker, T. C.

1993-01-01

rts1-1 was identified as an extragenic suppressor of tub2-104, a cold-sensitive allele of the sole gene encoding β-tubulin in the yeast, Saccharomyces cerevisiae. In addition, rts1-1 cells are heat sensitive and resistant to the microtubule-destabilizing drug, benomyl. The rts1-1 mutation is a deletion of approximately 5 kb of genomic DNA on chromosome X that includes one open reading frame and three tRNA genes. Dissection of this region shows that heat sensitivity is due to deletion of the open reading frame (HIT1). Suppression and benomyl resistance are caused by deletion of the gene encoding a tRNA(AGG)(Arg) (HSX1). Northern analysis of rts1-1 cells indicates that HSX1 is the only gene encoding this tRNA. Deletion of HSX1 does not suppress the tub2-104 mutation by misreading at the AGG codons in TUB2. It also does not suppress by interfering with the protein arginylation that targets certain proteins for degradation. These results leave open the prospect that this tRNA(AGG)(Arg) plays a novel role in the cell. PMID:8307335

Safety assessment of lepidopteran insect-protected transgenic rice with cry2A* gene.

PubMed

Zou, Shiying; Huang, Kunlun; Xu, Wentao; Luo, Yunbo; He, Xiaoyun

2016-04-01

Numerous genetically modified (GM) crops expressing proteins for insect resistance have been commercialized following extensive testing demonstrating that the foods obtained from them are as safe as that obtained from their corresponding non-GM varieties. In this paper, we report the outcome of safety studies conducted on a newly developed insect-resistant GM rice expressing the cry2A* gene by a subchronic oral toxicity study on rats. GM rice and non-GM rice were incorporated into the diet at levels of 30, 50, and 70% (w/w), No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. These results demonstrate that the GM rice with cry2A* gene is as safe for food as conventional non-GM rice.

Regional brain responses in nulliparous women to emotional infant stimuli.

PubMed

Montoya, Jessica L; Landi, Nicole; Kober, Hedy; Worhunsky, Patrick D; Rutherford, Helena J V; Mencl, W Einar; Mayes, Linda C; Potenza, Marc N

2012-01-01

Infant cries and facial expressions influence social interactions and elicit caretaking behaviors from adults. Recent neuroimaging studies suggest that neural responses to infant stimuli involve brain regions that process rewards. However, these studies have yet to investigate individual differences in tendencies to engage or withdraw from motivationally relevant stimuli. To investigate this, we used event-related fMRI to scan 17 nulliparous women. Participants were presented with novel infant cries of two distress levels (low and high) and unknown infant faces of varying affect (happy, sad, and neutral) in a randomized, counter-balanced order. Brain activation was subsequently correlated with scores on the Behavioral Inhibition System/Behavioral Activation System scale. Infant cries activated bilateral superior and middle temporal gyri (STG and MTG) and precentral and postcentral gyri. Activation was greater in bilateral temporal cortices for low- relative to high-distress cries. Happy relative to neutral faces activated the ventral striatum, caudate, ventromedial prefrontal, and orbitofrontal cortices. Sad versus neutral faces activated the precuneus, cuneus, and posterior cingulate cortex, and behavioral activation drive correlated with occipital cortical activations in this contrast. Behavioral inhibition correlated with activation in the right STG for high- and low-distress cries relative to pink noise. Behavioral drive correlated inversely with putamen, caudate, and thalamic activations for the comparison of high-distress cries to pink noise. Reward-responsiveness correlated with activation in the left precentral gyrus during the perception of low-distress cries relative to pink noise. Our findings indicate that infant cry stimuli elicit activations in areas implicated in auditory processing and social cognition. Happy infant faces may be encoded as rewarding, whereas sad faces activate regions associated with empathic processing. Differences in motivational tendencies may modulate neural responses to infant cues.

Molecular identity and gene expression of aldosterone synthase cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamoto, Mitsuhiro; Nonaka, Yasuki; Takemori, Hiroshi

11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolatedmore » from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.« less

A cry in the dark: depressed mothers show reduced neural activation to their own infant’s cry

PubMed Central

Ablow, Jennifer C.

2012-01-01

This study investigated depression-related differences in primiparous mothers’ neural response to their own infant’s distress cues. Mothers diagnosed with major depressive disorder (n = 11) and comparison mothers with no diagnosable psychopathology (n = 11) were exposed to their own 18-months-old infant’s cry sound, as well as unfamiliar infant’s cry and control sound, during functional neuroimaging. Depressed mothers’ response to own infant cry greater than other sounds was compared to non-depressed mothers’ response in the whole brain [false discovery rate (FDR) corrected]. A continuous measure of self-reported depressive symptoms (CESD) was also tested as a predictor of maternal response. Non-depressed mothers activated to their own infant’s cry greater than control sound in a distributed network of para/limbic and prefrontal regions, whereas depressed mothers as a group failed to show activation. Non-depressed compared to depressed mothers showed significantly greater striatal (caudate, nucleus accumbens) and medial thalamic activation. Additionally, mothers with lower depressive symptoms activated more strongly in left orbitofrontal, dorsal anterior cingulate and medial superior frontal regions. Non-depressed compared to depressed mothers activated uniquely to own infant greater than other infant cry in occipital fusiform areas. Disturbance of these neural networks involved in emotional response and regulation may help to explain parenting deficits in depressed mothers. PMID:21208990

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light.

PubMed

Pepper, A E; Seong-Kim, M; Hebst, S M; Ivey, K N; Kwak, S J; Broyles, D E

2001-09-01

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).

The food and environmental safety of Bt crops.

PubMed

Koch, Michael S; Ward, Jason M; Levine, Steven L; Baum, James A; Vicini, John L; Hammond, Bruce G

2015-01-01

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms.

The food and environmental safety of Bt crops

PubMed Central

Koch, Michael S.; Ward, Jason M.; Levine, Steven L.; Baum, James A.; Vicini, John L.; Hammond, Bruce G.

2015-01-01

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms. PMID:25972882

Three cases of Creutzfeldt-Jakob disease with prion protein gene codon180 mutation presenting with pathological laughing and crying.

PubMed

Iwasaki, Yasushi

2012-08-15

Although there are no reports of pathological laughing and crying being observed in patients with Creutzfeldt-Jakob disease (CJD), the author experienced three patients with CJD with prion protein gene codon180 mutation (V180I CJD) who showed this characteristic clinical finding. This finding was observed from the early disease stage in all 3 patients and continued for several months. Startle reaction was also remarkable in all patients, although myoclonus was generally mild. The dissociation between the startle reaction and myoclonus was suspected to be another feature of V180I CJD. The pathological laughing and crying co-occured with the startle reaction and stopped right before the onset of akinetic mutism, and the degree of both symptoms was almost parallel during this period. On the basis of MRI and autopsy findings, pathological laughing and crying was suspected of being induced by the widespread cerebral cortical involvement that is characteristic of V180I CJD. From the present observations, the author speculated that pathological laughing and crying may be a comparatively frequent observation in V180I CJD patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Edible Safety Assessment of Genetically Modified Rice T1C-1 for Sprague Dawley Rats through Horizontal Gene Transfer, Allergenicity and Intestinal Microbiota.

PubMed

Zhao, Kai; Ren, Fangfang; Han, Fangting; Liu, Qiwen; Wu, Guogan; Xu, Yan; Zhang, Jian; Wu, Xiao; Wang, Jinbin; Li, Peng; Shi, Wei; Zhu, Hong; Lv, Jianjun; Zhao, Xiao; Tang, Xueming

2016-01-01

In this study, assessment of the safety of transgenic rice T1C-1 expressing Cry1C was carried out by: (1) studying horizontal gene transfer (HGT) in Sprague Dawley rats fed transgenic rice for 90 d; (2) examining the effect of Cry1C protein in vitro on digestibility and allergenicity; and (3) studying the changes of intestinal microbiota in rats fed with transgenic rice T1C-1 in acute and subchronic toxicity tests. Sprague Dawley rats were fed a diet containing either 60% GM Bacillus thuringiensis (Bt) rice T1C-1 expressing Cry1C protein, the parental rice Minghui 63, or a basic diet for 90 d. The GM Bt rice T1C-1 showed no evidence of HGT between rats and transgenic rice. Sequence searching of the Cry1C protein showed no homology with known allergens or toxins. Cry1C protein was rapidly degraded in vitro with simulated gastric and intestinal fluids. The expressed Cry1C protein did not induce high levels of specific IgG and IgE antibodies in rats. The intestinal microbiota of rats fed T1C-1 was also analyzed in acute and subchronic toxicity tests by DGGE. Cluster analysis of DGGE profiles revealed significant individual differences in the rats' intestinal microbiota.

Blue Light–Dependent Interaction between Cryptochrome2 and CIB1 Regulates Transcription and Leaf Senescence in Soybean[W

PubMed Central

Meng, Yingying; Li, Hongyu; Wang, Qin; Liu, Bin; Lin, Chentao

2013-01-01

Cryptochromes are blue light receptors that regulate light responses in plants, including various crops. The molecular mechanism of plant cryptochromes has been extensively investigated in Arabidopsis thaliana, but it has not been reported in any crop species. Here, we report a study of the mechanism of soybean (Glycine max) cryptochrome2 (CRY2a). We found that CRY2a regulates leaf senescence, which is a life history trait regulated by light and photoperiods via previously unknown mechanisms. We show that CRY2a undergoes blue light–dependent interaction with the soybean basic helix-loop-helix transcription activator CIB1 (for cryptochrome-interacting bHLH1) that specifically interacts with the E-box (CANNTG) DNA sequences. Analyses of transgenic soybean plants expressing an elevated or reduced level of the CRY2a or CIB1 demonstrate that CIB1 promotes leaf senescence, whereas CRY2a suppresses leaf senescence. Results of the gene expression and molecular interaction analyses support the hypothesis that CIB1 activates transcription of senescence-associated genes, such as WRKY DNA BINDING PROTEIN53b (WRKY53b), and leaf senescence. CIB1 interacts with the E-box–containing promoter sequences of the WRKY53b chromatin, whereas photoexcited CRY2a interacts with CIB1 to inhibit its DNA binding activity. These findings argue that CIB-dependent transcriptional regulation is an evolutionarily conserved CRY-signaling mechanism in plants, and this mechanism is opted in evolution to mediate light regulation of different aspects of plant development in different plant species. PMID:24272488

Lunar Phase-Dependent Expression of Cryptochrome and a Photoperiodic Mechanism for Lunar Phase-Recognition in a Reef Fish, Goldlined Spinefoot

PubMed Central

Fukushiro, Masato; Takeuchi, Takahiro; Takeuchi, Yuki; Hur, Sung-Pyo; Sugama, Nozomi; Takemura, Akihiro; Kubo, Yoko; Okano, Keiko; Okano, Toshiyuki

2011-01-01

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal. Their mRNA levels showed lunar cycle-dependent expression in the medial part of the brain (mesencephalon and diencephalon) peaking at the first quarter moon. Since this lunar phase coincided with the reproductive phase of the goldlined spinefoot, Cry gene expression was considered a state variable in the lunar phase recognition system. Based on the expression profiles of SgCrys together with the moonlight's pattern of timing and duration during its nightly lunar cycle, we have further speculated on a model of lunar phase recognition for reproductive control in the goldlined spinefoot, which integrates both moonlight and circadian signals in a manner similar to photoperiodic response. PMID:22163321

Lunar phase-dependent expression of cryptochrome and a photoperiodic mechanism for lunar phase-recognition in a reef fish, goldlined spinefoot.

PubMed

Fukushiro, Masato; Takeuchi, Takahiro; Takeuchi, Yuki; Hur, Sung-Pyo; Sugama, Nozomi; Takemura, Akihiro; Kubo, Yoko; Okano, Keiko; Okano, Toshiyuki

2011-01-01

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal. Their mRNA levels showed lunar cycle-dependent expression in the medial part of the brain (mesencephalon and diencephalon) peaking at the first quarter moon. Since this lunar phase coincided with the reproductive phase of the goldlined spinefoot, Cry gene expression was considered a state variable in the lunar phase recognition system. Based on the expression profiles of SgCrys together with the moonlight's pattern of timing and duration during its nightly lunar cycle, we have further speculated on a model of lunar phase recognition for reproductive control in the goldlined spinefoot, which integrates both moonlight and circadian signals in a manner similar to photoperiodic response.

A Spodoptera exigua Cadherin Serves as a Putative Receptor for Bacillus thuringiensis Cry1Ca Toxin and Shows Differential Enhancement of Cry1Ca and Cry1Ac Toxicity

PubMed Central

Ren, Xiang-Liang; Chen, Rui-Rui; Zhang, Ying; Ma, Yan; Cui, Jin-Jie; Han, Zhao-Jun; Mu, Li-Li

2013-01-01

Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca. PMID:23835184

Evaluation of cytotoxic and antimicrobial effects of two Bt Cry proteins on a GMO safety perspective.

PubMed

Farias, Davi Felipe; Viana, Martônio Ponte; de Oliveira, Gustavo Ramos; Beneventi, Magda Aparecida; Soares, Bruno Marques; Pessoa, Claudia; Pessoa, Igor Parra; Silva, Luciano Paulino; Vasconcelos, Ilka Maria; de Sá, Maria Fátima Grossi; Carvalho, Ana Fontenele Urano

2014-01-01

Studies have contested the innocuousness of Bacillus thuringiensis (Bt) Cry proteins to mammalian cells as well as to mammals microbiota. Thus, this study aimed to evaluate the cytotoxic and antimicrobial effects of two Cry proteins, Cry8Ka5 (a novel mutant protein) and Cry1Ac (a widely distributed protein in GM crops). Evaluation of cyto- and genotoxicity in human lymphocytes was performed as well as hemolytic activity coupled with cellular membrane topography analysis in mammal erythrocytes. Effects of Cry8Ka5 and Cry1Ac upon Artemia sp. nauplii and upon bacteria and yeast growth were assessed. The toxins caused no significant effects on the viability (IC50 > 1,000 µg/mL) or to the cellular DNA integrity of lymphocytes (no effects at 1,000 µg/mL). The Cry8Ka5 and Cry1Ac proteins did not cause severe damage to erythrocytes, neither with hemolysis (IC50 > 1,000 µg/mL) nor with alterations in the membrane. Likewise, the Cry8Ka5 and Cry1Ac proteins presented high LC50 (755.11 and >1,000 µg/mL, resp.) on the brine shrimp lethality assay and showed no growth inhibition of the microorganisms tested (MIC > 1,000 µg/mL). This study contributed with valuable information on the effects of Cry8Ka5 and Cry1Ac proteins on nontarget organisms, which reinforce their potential for safe biotechnological applications.

in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence, structure analysis of crystal proteins for stability.

PubMed

Ebenezer, King Solomon; Nachimuthu, Ramesh; Thiagarajan, Prabha; Velu, Rajesh Kannan

2013-01-01

Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125(th) amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.

Peptide selection and antibody generation for the prospective immunorecognition of Cry1Ab16 protein of transgenic maize.

PubMed

Costa, Joana; Marani, Mariela M; Grazina, Liliana; Villa, Caterina; Meira, Liliana; Oliveira, M Beatriz P P; Leite, José R S A; Mafra, Isabel

2017-09-15

The introduction of genes isolated from different Bacillus thuringiensis strains to express Cry-type toxins in transgenic crops is a common strategy to confer insect resistance traits. This work intended to extensively in silico analyse Cry1A(b)16 protein for the identification of peptide markers for the biorecognition of transgenic crops. By combining two different strategies based on several bioinformatic tools for linear epitope prediction, a set of seven peptides was successfully selected as potential Cry1A(b)16 immunogens. For the prediction of conformational epitopes, Cry1A(b)16 models were built on the basis of three independent templates of homologue proteins of Cry1A(a) and Cry1A(c) using an integrated approach. PcH_736-746 and PcH_876-886 peptides were selected as the best candidates, being synthesised and used for the production of polyclonal antibodies. To the best of our knowledge, this is the first attempt of selecting and defining linear peptides as immunogenic markers of Cry1A(b)-type toxins in transgenic maize. Copyright © 2017 Elsevier Ltd. All rights reserved.

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions*

PubMed Central

Yuan, Li; Seong, Eunju; Beuscher, James L.; Arikkath, Jyothi

2015-01-01

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. PMID:25724647

Down-regulation of aminopeptidase N and ABC transporter subfamily G transcripts in Cry1Ab and Cry1Ac resistant Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae)

PubMed Central

Zhang, Tiantao; Coates, Brad S.; Wang, Yueqin; Wang, Yidong; Bai, Shuxiong; Wang, Zhenying; He, Kanglai

2017-01-01

The Asian corn borer (ACB), Ostrinia furnacalis (Lepidoptera: Crambidae), is a highly destructive pest of cultivated maize throughout East Asia. Bacillus thuringiensis (Bt) crystalline protein (Cry) toxins cause mortality by a mechanism involving pore formation or signal transduction following toxin binding to receptors along the midgut lumen of susceptible insects, but this mechanism and mutations therein that lead to resistance are not fully understood. In the current study, quantitative comparisons were made among midgut expressed transcripts from O. furnacalis susceptible (ACB-BtS) and laboratory selected strains resistant to Cry1Ab (ACB-AbR) and Cry1Ac toxins (ACB-AcR) when feeding on non-Bt diet. From a combined de novo transcriptome assembly of 83,370 transcripts, ORFs of ≥ 100 amino acids were predicted and annotated for 28,940 unique isoforms derived from 12,288 transcripts. Transcriptome-wide expression estimated from RNA-seq read depths predicted significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as a putative ATP binding cassette transporter group G (abcg) gene in both ACB-AbR and -AcR (log2[fold-change] ≥ 1.36; P < 0.0001). The transcripts that were most highly differentially regulated in both ACB-AbR and -AcR compared to ACB-BtS (log2[fold-change] ≥ 2.0; P < 0.0001) included up- and down-regulation of serine proteases, storage proteins and cytochrome P450 monooxygenases, as well as up-regulation of genes with predicted transport function. This study predicted the significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as abccg gene in both ACB-AbR and -AcR. These data are important for the understanding of systemic differences between Bt resistant and susceptible genotypes. PMID:28808417

Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

PubMed

Martins, Erica Soares; Monnerat, Rose Gomes; Queiroz, Paulo Roberto; Dumas, Vinicius Fiuza; Braz, Shélida Vasconcelos; de Souza Aguiar, Raimundo Wagner; Gomes, Ana Cristina Menezes Mendes; Sánchez, Jorge; Bravo, Alejandra; Ribeiro, Bergmann Morais

2010-02-01

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis. 2009. Published by Elsevier Ltd.

Chilo suppressalis and Sesamia inferens display different susceptibility responses to Cry1A insecticidal proteins.

PubMed

Li, Bo; Xu, Yangyang; Han, Cao; Han, Lanzhi; Hou, Maolin; Peng, Yufa

2015-10-01

Chilo suppressalis and Sesamia inferens are important lepidopteran rice pests that occur concurrently in rice-growing areas of China. The development of transgenic rice expressing Cry1A insecticidal proteins has provided a useful strategy for controlling these pests. This study evaluated the baseline susceptibilities of C. suppressalis and S. inferens to Cry1A, as well as their responses to selection with Cry1A. Wide geographic variation in susceptibility was observed across all field populations. Within a given population, the LC50 of both Cry1Ab and Cry1Ac against S. inferens was drastically higher than that of C. suppressalis. Large LC50 differences (74.6-fold) were detected between the two species for Cry1Ab in the Poyang population, while small differences (3.6-fold) were detected for Cry1Ac in the Changsha population. The Cry1Ac LC50 of C. suppressalis and S. inferens increased 8.4- and 4.4-fold after 21 and eight selection generations respectively. Additionally, the estimated realised heritabilities (h(2) ) of Cry1Ac tolerance were 0.11 in C. suppressalis and 0.292 in S. inferens. S. inferens exhibited a significantly lower susceptibility and more rapidly evolved resistance to Cry1A compared with C. suppressalis. Therefore, S. inferens is more likely to evolve increased resistance, which threatens the sustainability of rice expressing Cry1A protein. © 2014 Society of Chemical Industry.

Mechanisms of Geomagnetic Field Influence on Gene Expression Using Influenza as a Model System: Basics of Physical Epidemiology

PubMed Central

Zaporozhan, Valeriy; Ponomarenko, Andriy

2010-01-01

Recent studies demonstrate distinct changes in gene expression in cells exposed to a weak magnetic field (MF). Mechanisms of this phenomenon are not understood yet. We propose that proteins of the Cryptochrome family (CRY) are “epigenetic sensors” of the MF fluctuations, i.e., magnetic field-sensitive part of the epigenetic controlling mechanism. It was shown that CRY represses activity of the major circadian transcriptional complex CLOCK/BMAL1. At the same time, function of CRY, is apparently highly responsive to weak MF because of radical pairs that periodically arise in the functionally active site of CRY and mediate the radical pair mechanism of magnetoreception. It is known that the circadian complex influences function of every organ and tissue, including modulation of both NF-κB- and glucocorticoids- dependent signaling pathways. Thus, MFs and solar cycles-dependent geomagnetic field fluctuations are capable of altering expression of genes related to function of NF-κB, hormones and other biological regulators. Notably, NF-κB, along with its significant role in immune response, also participates in differential regulation of influenza virus RNA synthesis. Presented data suggests that in the case of global application (example—geomagnetic field), MF-mediated regulation may have epidemiological and other consequences. PMID:20617011

Bacterial community structure in the rhizosphere of a Cry1Ac Bt-brinjal crop and comparison to its non-transgenic counterpart in the tropical soil.

PubMed

Singh, Amit Kishore; Rai, Govind Kumar; Singh, Major; Dubey, Suresh Kumar

2013-11-01

To elucidate whether the transgenic crop alters the rhizospheric bacterial community structure, a 2-year study was performed with Cry1Ac gene-inserted brinjal crop (Bt) and their near isogenic non-transformed trait (non-Bt). The event of Bt crop (VRBT-8) was screened using an insect bioassay and enzyme-linked immunosorbent assay. Soil moisture, NH4 (+)-N, NO3 (-)-N, and PO4 (-)-P level had non-significant variation. Quantitative polymerase chain reaction revealed that abundance of bacterial 16S rRNA gene copies were lower in soils associated with Bt brinjal. Microbial biomass carbon (MBC) showed slight reduction in Bt brinjal soils. Higher MBC values in the non-Bt crop soil may be attributed to increased root activity and availability of readily metabolizable carbon compounds. The restriction fragment length polymorphism of PCR-amplified rRNA gene fragments detected 13 different bacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fusobacteria in non-Bt, and Cyanobacteria and Bacteroidetes in Bt soils, respectively, reflecting minor changes in the community structure. Despite the detection of Cry1Ac protein in the rhizospheric soil, the overall impact of Cry1Ac expressing Bt brinjal was less compared to that due to seasonal changes.

One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins

PubMed Central

Tabashnik, Bruce E.; Liu, Yong-Biao; Finson, Naomi; Masson, Luke; Heckel, David G.

1997-01-01

Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected. PMID:9050831

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

PubMed

Tulinská, Jana; Adel-Patient, Karine; Bernard, Hervé; Líšková, Aurélia; Kuricová, Miroslava; Ilavská, Silvia; Horváthová, Mira; Kebis, Anton; Rollerová, Eva; Babincová, Júlia; Aláčová, Radka; Wal, Jean-Michel; Schmidt, Kerstin; Schmidtke, Jörg; Schmidt, Paul; Kohl, Christian; Wilhelm, Ralf; Schiemann, Joachim; Steinberg, Pablo

2018-07-01

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.

Mechanisms of Breast Cancer in Shift Workers: DNA Methylation in Five Core Circadian Genes in Nurses Working Night Shifts.

PubMed

Samulin Erdem, Johanna; Skare, Øivind; Petersen-Øverleir, Marte; Notø, Heidi Ødegaard; Lie, Jenny-Anne S; Reszka, Edyta; Pepłońska, Beata; Zienolddiny, Shanbeh

2017-01-01

Shift work has been suggested to be associated with breast cancer risk, and circadian disruption in shift workers is hypothesized as one of the mechanisms of increased cancer risk. There is, however, insufficient molecular evidence supporting this hypothesis. Using the quantitative methodology of pyrosequencing, epigenetic changes in 5-methyl cytosine (5mC) in five circadian genes CLOCK , BMAL1 , CRY1, PER1 and PER2 in female nurses working night shift work (278 breast cancer cases, 280 controls) were analyzed. In breast cancer cases, a medium exposure to night work was associated with increased methylation levels of the CLOCK (p=0.050), BMAL1 (p=0.001) and CRY1 (p=0.040) genes, compared with controls. Within the cases, analysis of the effects of shift work on the methylation patterns showed that methylation of CRY1 was lower in those who had worked night shift and had a high exposure (p=0.006) compared with cases that had worked only days. For cases with a medium exposure to night work, an increase in BMAL1 (p=0.003) and PER1 (p=0.035) methylation was observed compared with day working (unexposed) cases. The methylation levels of the five core circadian genes were also analyzed in relation to the estrogen and progesterone receptors status of the tumors in the cases, and no correlations were observed. Furthermore, nineteen polymorphisms in the five circadian genes were assessed for their effects on the methylation levels of the respective genes, but no associations were found. In summary, our data suggest that epigenetic regulation of CLOCK , BMAL1, CRY1 and PER1 may contribute to breast cancer in shift workers.

Mechanisms of Breast Cancer in Shift Workers: DNA Methylation in Five Core Circadian Genes in Nurses Working Night Shifts

PubMed Central

Samulin Erdem, Johanna; Skare, Øivind; Petersen-Øverleir, Marte; Notø, Heidi Ødegaard; Lie, Jenny-Anne S.; Reszka, Edyta; Pepłońska, Beata; Zienolddiny, Shanbeh

2017-01-01

Shift work has been suggested to be associated with breast cancer risk, and circadian disruption in shift workers is hypothesized as one of the mechanisms of increased cancer risk. There is, however, insufficient molecular evidence supporting this hypothesis. Using the quantitative methodology of pyrosequencing, epigenetic changes in 5-methyl cytosine (5mC) in five circadian genes CLOCK, BMAL1, CRY1, PER1 and PER2 in female nurses working night shift work (278 breast cancer cases, 280 controls) were analyzed. In breast cancer cases, a medium exposure to night work was associated with increased methylation levels of the CLOCK (p=0.050), BMAL1 (p=0.001) and CRY1 (p=0.040) genes, compared with controls. Within the cases, analysis of the effects of shift work on the methylation patterns showed that methylation of CRY1 was lower in those who had worked night shift and had a high exposure (p=0.006) compared with cases that had worked only days. For cases with a medium exposure to night work, an increase in BMAL1 (p=0.003) and PER1 (p=0.035) methylation was observed compared with day working (unexposed) cases. The methylation levels of the five core circadian genes were also analyzed in relation to the estrogen and progesterone receptors status of the tumors in the cases, and no correlations were observed. Furthermore, nineteen polymorphisms in the five circadian genes were assessed for their effects on the methylation levels of the respective genes, but no associations were found. In summary, our data suggest that epigenetic regulation of CLOCK, BMAL1, CRY1 and PER1 may contribute to breast cancer in shift workers. PMID:28928877

A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40).

PubMed

Li, Xiang; Wang, Xiuxiu; Yang, Jielin; Liu, Yueming; He, Yuping; Pan, Liangwen

2014-05-16

To date, over 150 genetically modified (GM) crops are widely cultivated. To comply with regulations developed for genetically modified organisms (GMOs), including labeling policies, many detection methods for GMO identification and quantification have been developed. To detect the entrance and exit of unauthorized GM crop events in China, we developed a novel quadruplex real-time PCR method for simultaneous detection and quantification of GM cotton events GHB119 and T304-40 in cotton-derived products (based on the 5'-flanking sequence) and the insect-resistance gene Cry2Ae. The limit of detection was 10 copies for GHB119 and Cry2Ae and 25 copies for T304-40. The limit of quantification was 25 copies for GHB119 and Cry2Ae and 50 copies for T304-40. Moreover, low bias and acceptable standard deviation and relative standard deviation values were obtained in quantification analysis of six blind samples containing different GHB119 and T304-40 ingredients. The developed quadruplex quantitative method could be used for quantitative detection of two GM cotton events (GHB119 and T304-40) and Cry2Ae gene ingredient in cotton derived products.

A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40)

PubMed Central

2014-01-01

Background To date, over 150 genetically modified (GM) crops are widely cultivated. To comply with regulations developed for genetically modified organisms (GMOs), including labeling policies, many detection methods for GMO identification and quantification have been developed. Results To detect the entrance and exit of unauthorized GM crop events in China, we developed a novel quadruplex real-time PCR method for simultaneous detection and quantification of GM cotton events GHB119 and T304-40 in cotton-derived products (based on the 5′-flanking sequence) and the insect-resistance gene Cry2Ae. The limit of detection was 10 copies for GHB119 and Cry2Ae and 25 copies for T304-40. The limit of quantification was 25 copies for GHB119 and Cry2Ae and 50 copies for T304-40. Moreover, low bias and acceptable standard deviation and relative standard deviation values were obtained in quantification analysis of six blind samples containing different GHB119 and T304-40 ingredients. Conclusions The developed quadruplex quantitative method could be used for quantitative detection of two GM cotton events (GHB119 and T304-40) and Cry2Ae gene ingredient in cotton derived products. PMID:24884946

Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

PubMed Central

Jiang, Jian; Huang, Ying; Shu, Changlong; Soberón, Mario; Bravo, Alejandra; Liu, Chunqing; Song, Fuping; Lai, Jinsheng

2017-01-01

ABSTRACT The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), containing two cry genes (cry8-like and cry8Ga), is toxic to Holotrichia oblita larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in H. oblita larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from H. oblita, and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from H. oblita larvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the H. oblita midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the H. oblita transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of Tribolium castaneum, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin. IMPORTANCE Holotrichia oblita is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses. H. oblita is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from Bacillus thuringiensis were shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control of H. oblita and for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins against H. oblita larvae. PMID:28389549

Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

PubMed

Obata, Fumiaki; Tanaka, Shiho; Kashio, Soshiro; Tsujimura, Hidenobu; Sato, Ryoichi; Miura, Masayuki

2015-07-08

Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery. Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation. With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

Time-related dynamics of variation in core clock gene expression levels in tissues relevant to the immune system.

PubMed

Mazzoccoli, G; Sothern, R B; Greco, A; Pazienza, V; Vinciguerra, M; Liu, S; Cai, Y

2011-01-01

Immune parameters show rhythmic changes with a 24-h periodicity driven by an internal circadian timing system that relies on clock genes (CGs). CGs form interlocked transcription-translation feedback loops to generate and maintain 24-h mRNA and protein oscillations. In this study we evaluate and compare the profiles and the dynamics of variation of CG expression in peripheral blood, and two lymphoid tissues of mice. Expression levels of seven recognized key CGs (mBmal1, mClock, mPer1, mPer2, mCry1, mCry2, and Rev-erbalpha) were evaluated by quantitative RT- PCR in spleen, thymus and peripheral blood of C57BL/6 male mice housed on a 12-h light (L)-dark (D) cycle and sacrificed every 4 h for 24 h (3-4 mice/time point). We found a statistically significant time-effect in spleen (S), thymus (T) and blood (B) for the original values of expression level of mBmal1 (S), mClock (T, B), mPer1 (S, B), mPer2 (S), mCry1 (S), mCry2 (B) and mRev-Erbalpha (S, T, B) and for the fractional variation calculated between single time-point expression value of mBmal1 (B), mPer2 (T), mCry2 (B) and mRev-Erbalpha (S). A significant 24-h rhythm was validated for five CGs in blood (mClock, mPer1, mPer2, mCry2, mRev-Erbalpha), for four CGs in the spleen (mBmal1, mPer1, mPer2, mRev-Erbalpha), and for three CGs in the thymus (mClock, mPer2, mRev-Erbalpha). The original values of acrophases for mBmal1, mClock, mPer1, mPer2, mCry1 and mCry2 were very similar for spleen and thymus and advanced by several hours for peripheral blood compared to the lymphoid tissues, whereas the phases of mRev-Erbalpha were coincident for all three tissues. In conclusion, central and peripheral lymphoid tissues in the mouse show different sequences of activation of clock gene expression compared to peripheral blood. These differences may underlie the compartmental pattern of web functioning in the immune system.

Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

PubMed

Bester, Michael C; Jacobson, Dan; Bauer, Florian F

2012-01-01

The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

Pyramids of QTLs enhance host-plant resistance and Bt-mediated resistance to leaf-chewing insects in soybean.

PubMed

Ortega, María A; All, John N; Boerma, H Roger; Parrott, Wayne A

2016-04-01

QTL-M and QTL-E enhance soybean resistance to insects. Pyramiding these QTLs with cry1Ac increases protection against Bt-tolerant pests, presenting an opportunity to effectively deploy Bt with host-plant resistance genes. Plant resistance to leaf-chewing insects minimizes the need for insecticide applications, reducing crop production costs and pesticide concerns. In soybean [Glycine max (L.) Merr.], resistance to a broad range of leaf-chewing insects is found in PI 229358 and PI 227687. PI 229358's resistance is conferred by three quantitative trait loci (QTLs): M, G, and H. PI 227687's resistance is conferred by QTL-E. The letters indicate the soybean Linkage groups (LGs) on which the QTLs are located. This study aimed to determine if pyramiding PI 229358 and PI 227687 QTLs would enhance soybean resistance to leaf-chewing insects, and if pyramiding these QTLs with Bt (cry1Ac) enhances resistance against Bt-tolerant pests. The near-isogenic lines (NILs): Benning(ME), Benning(MGHE), and Benning(ME+cry1Ac) were developed. Benning(ME) and Benning(MGHE) were evaluated in detached-leaf and greenhouse assays with soybean looper [SBL, Chrysodeixis includens (Walker)], corn earworm [CEW, Helicoverpa zea (Boddie)], fall armyworm [FAW, Spodoptera frugiperda (J.E. Smith)], and velvetbean caterpillar [VBC, Anticarsia gemmatalis (Hübner)]; and in field-cage assays with SBL. Benning(ME+cry1Ac) was tested in detached-leaf assays against SBL, VBC, and Southern armyworm [SAW, Spodoptera eridania (Cramer)]. In the detached-leaf assay, Benning(ME) showed the strongest antibiosis against CEW, FAW, and VBC. In field-cage conditions, Benning(ME) and Benning(MGHE) suffered 61 % less defoliation than Benning. Benning(ME+cry1Ac) was more resistant than Benning(ME) and Benning (cry1Ac) against SBL and SAW. Agriculturally relevant levels of resistance in soybean can be achieved with just two loci, QTL-M and QTL-E. ME+cry1Ac could present an opportunity to protect the durability of Bt genes in elite soybean cultivars. These results should assist the development of effective pest management strategies, and sustainable deployment of Bt genes in soybean.

Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

PubMed

Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

2003-10-10

The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

Effect of plant age, larval age, and fertilizer treatment on resistance of a cry1Ab-transformed aromatic rice to lepidopterous stem borers and foliage feeders.

PubMed

Alinia, F; Ghareyazie, B; Rubia, L; Bennett, J; Cohen, M B

2000-04-01

The resistance of vegetative, booting, and flowering stage plants of a variety of an aromatic rice, Oryza sativa L., transformed with a Bacillus thuringiensis Berliner cry1Ab gene under control of the maize phosphoenolpyruvate carboxylase (PEPC) promoter was evaluated against four lepidopterous rice pests--the stem borers Chilo suppressalis (Walker) (Lepidoptera: Crambidae) and Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae), and the foliage feeders Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae) and Naranga aenescens Moore (Lepidoptera: Noctuidae). Plants of the cry1Ab-transformed line (no. 827) were more resistant to young larvae of S. incertulas, C. suppressalis, and C. medinalis than control plants at the vegetative stage but not at the flowering stage. Survival of 10-d-old stem borer larvae did not differ on cry1Ab plants and control plants at either the vegetative or flowering stage, but the development of 10-d-old C. suppressalis larvae was retarded on the vegetative stage cry1Ab plants. Immunological analysis also showed an apparent decline in Cry1Ab titer in leaf blades and leaf sheaths at the reproductive stage. In experiments comparing three fertilizer treatments (NPK, PK, and none), there was a significant interaction between fertilizer treatment and variety on larval survival only in whole-plant assays at booting stage with C. suppressalis. On cry1Ab plants, larval survival did not differ significantly among the three fertilizer levels, whereas on control plants survival was highest with the NPK treatment. cry1Ab plants tested at the sixth and seventh generations after transformation were more resistant than control plants to N. aenescens and C. suppressalis, respectively, suggesting that gene silencing will not occur in line 827. The results of the experiments are discussed in terms of resistance management for B. thuringiensis toxins in rice.

Chromobacterium pathogenicity island 1 type III secretion system is a major virulence determinant for Chromobacterium violaceum-induced cell death in hepatocytes.

PubMed

Miki, Tsuyoshi; Iguchi, Mirei; Akiba, Kinari; Hosono, Masato; Sobue, Tomoyoshi; Danbara, Hirofumi; Okada, Nobuhiko

2010-08-01

Chromobacterium violaceum is a Gram-negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi-2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi-1/-1a-encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi-1/-1a or Cpi-2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi-1/-1a-dependent manner. Characterization of Chromobacterium-induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi-1/-1a effector, is implicated in translocator-mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium-induced cytotoxicity. These results strongly suggest that Cpi-1/-1a-encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes. © 2010 Blackwell Publishing Ltd.

Coevolution of Siglec-11 and Siglec-16 via gene conversion in primates.

PubMed

Hayakawa, Toshiyuki; Khedri, Zahra; Schwarz, Flavio; Landig, Corinna; Liang, Suh-Yuen; Yu, Hai; Chen, Xi; Fujito, Naoko T; Satta, Yoko; Varki, Ajit; Angata, Takashi

2017-11-23

Siglecs-11 and -16 are members of the sialic acid recognizing Ig-like lectin family, and expressed in same cells. Siglec-11 functions as an inhibitory receptor, whereas Siglec-16 exhibits activating properties. In humans, SIGLEC11 and SIGLEC16 gene sequences are extremely similar in the region encoding the extracellular domain due to gene conversions. Human SIGLEC11 was converted by the nonfunctional SIGLEC16P allele, and the converted SIGLEC11 allele became fixed in humans, possibly because it provides novel neuroprotective functions in brain microglia. However, the detailed evolutionary history of SIGLEC11 and SIGLEC16 in other primates remains unclear. We analyzed SIGLEC11 and SIGLEC16 gene sequences of multiple primate species, and examined glycan binding profiles of these Siglecs. The phylogenetic tree demonstrated that gene conversions between SIGLEC11 and SIGLEC16 occurred in the region including the exon encoding the sialic acid binding domain in every primate examined. Functional assays showed that glycan binding preference is similar between Siglec-11 and Siglec-16 in all analyzed hominid species. Taken together with the fact that Siglec-11 and Siglec-16 are expressed in the same cells, Siglec-11 and Siglec-16 are regarded as paired receptors that have maintained similar ligand binding preferences via gene conversions. Relaxed functional constraints were detected on the SIGLEC11 and SIGLEC16 exons that underwent gene conversions, possibly contributing to the evolutionary acceptance of repeated gene conversions. The frequency of nonfunctional SIGLEC16P alleles is much higher than that of SIGLEC16 alleles in every human population. Our findings indicate that Siglec-11 and Siglec-16 have been maintained as paired receptors by repeated gene conversions under relaxed functional constraints in the primate lineage. The high prevalence of the nonfunctional SIGLEC16P allele and the fixation of the converted SIGLEC11 imply that the loss of Siglec-16 and the gain of Siglec-11 in microglia might have been favored during the evolution of human lineage.

Geomagnetic field impacts on cryptochrome and phytochrome signaling.

PubMed

Agliassa, Chiara; Narayana, Ravishankar; Christie, John M; Maffei, Massimo E

2018-05-29

The geomagnetic field (GMF) is an environmental element whose instability affects plant growth and development. Despite known plant responses to GMF direction and intensity, the mechanism of magnetoreception in plants is still not known. Magnetic field variations affect many light-dependent plant processes, suggesting that the magnetoreception could require light. The objective of this work was to comprehensively investigate the influence of GMF on Arabidopsis thaliana (Col-0) photoreceptor signaling. Wild-type Arabidopsis seedlings and photoreceptor-deficient mutants (cry1cry2, phot1, phyA and phyAphyB) were exposed to near null magnetic field (NNMF, ≤40 nT) and GMF (~43 μT) under darkness and different light wavelengths. The GMF did not alter skotomorphogenic or photomorphogenic seedling development but had a significant impact on gene expression pathways downstream of cryptochrome and phytochrome photoactivation. GMF-induced changes in gene expression observed under blue light were partially associated with an alteration of cryptochrome activation. GMF impacts on phytochrome-regulated gene expression could be attributed to alterations in phytochrome protein abundance that were also dependent on the presence of cry1, cry2 and phot1. Moreover, the GMF was found to impact photomorphogenic-promoting gene expression in etiolated seedlings, indicating the existence of a light-independent magnetoreception mechanism. In conclusion, our data shows that magnetoreception alters photoreceptor signaling in Arabidopsis, but it does not necessarily depend on light. Copyright © 2018. Published by Elsevier B.V.

Performance of laying hens fed diets containing DAS-59122-7 maize grain compared with diets containing nontransgenic maize grain.

PubMed

Jacobs, C M; Utterback, P L; Parsons, C M; Rice, D; Smith, B; Hinds, M; Liebergesell, M; Sauber, T

2008-03-01

An experiment using 216 Hy-Line W-36 pullets was conducted to evaluate transgenic maize grain containing the cry34Ab1 and cry35Ab1 genes from a Bacillus thuringiensis (Bt) strain and the phosphinothricin ace-tyltransferase (pat) gene from Streptomyces viridochromogenes. Expression of the cry34Ab1 and cry35Ab1 genes confers resistance to corn rootworms, and the pat gene confers tolerance to herbicides containing glufosinate-ammonium. Pullets (20 wk of age) were placed in cage lots (3 hens/cage, 2 cages/lot) and were randomly assigned to 1 of 3 corn-soybean meal dietary treatments (12 lots/treatment) formulated with the following maize grains: near-isogenic control (control), conventional maize, and transgenic test corn line 59122 containing event DAS-59122-7. Differences between 59122 and control group means were evaluated with statistical significance at P < 0.05. Body weight and gain, egg production, egg mass, and feed efficiency for hens fed the 59122 corn were not significantly different from the respective values for hens fed diets formulated with control maize grain. Egg component weights, Haugh unit measures, and egg weight class distribution were similar regardless of the corn source. This research indicates that performance of hens fed diets containing 59122 maize grain, as measured by egg production and egg quality, was similar to that of hens fed diets formulated with near-isogenic corn grain.

Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.

PubMed

Pardo-López, Liliana; Soberón, Mario; Bravo, Alejandra

2013-01-01

Bacillus thuringiensis bacteria are insect pathogens that produce different Cry and Cyt toxins to kill their hosts. Here we review the group of three-domain Cry (3d-Cry) toxins. Expression of these 3d-Cry toxins in transgenic crops has contributed to efficient control of insect pests and a reduction in the use of chemical insecticides. The mode of action of 3d-Cry toxins involves sequential interactions with several insect midgut proteins that facilitate the formation of an oligomeric structure and induce its insertion into the membrane, forming a pore that kills midgut cells. We review recent progress in our understanding of the mechanism of action of these Cry toxins and focus our attention on the different mechanisms of resistance that insects have evolved to counter their action, such as mutations in cadherin, APN and ABC transporter genes. Activity of Cry1AMod toxins, which are able to form toxin oligomers in the absence of receptors, against different resistant populations, including those affected in the ABC transporter and the role of dominant negative mutants as antitoxins, supports the hypothesis that toxin oligomerization is a limiting step in the Cry insecticidal activity. Knowledge of the action of 3d-Cry toxin and the resistance mechanisms to these toxins will set the basis for a rational design of novel toxins to overcome insect resistance, extending the useful lifespan of Cry toxins in insect control programs. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Resistance to dual-gene Bt maize in Spodoptera frugiperda: selection, inheritance, and cross-resistance to other transgenic events

PubMed Central

Santos-Amaya, Oscar F.; Rodrigues, João V. C.; Souza, Thadeu C.; Tavares, Clébson S.; Campos, Silverio O.; Guedes, Raul N.C.; Pereira, Eliseu J.G.

2015-01-01

Transgenic crop “pyramids” producing two or more Bacillus thuringiensis (Bt) toxins active against the same pest are used to delay evolution of resistance in insect pest populations. Laboratory and greenhouse experiments were performed with fall armyworm, Spodoptera frugiperda, to characterize resistance to Bt maize producing Cry1A.105 and Cry2Ab and test some assumptions of the “pyramid” resistance management strategy. Selection of a field-derived strain of S. frugiperda already resistant to Cry1F maize with Cry1A.105 + Cry2Ab maize for ten generations produced resistance that allowed the larvae to colonize and complete the life cycle on these Bt maize plants. Greenhouse experiments revealed that the resistance was completely recessive (Dx = 0), incomplete, autosomal, and without maternal effects or cross-resistance to the Vip3Aa20 toxin produced in other Bt maize events. This profile of resistance supports some of the assumptions of the pyramid strategy for resistance management. However, laboratory experiments with purified Bt toxin and plant leaf tissue showed that resistance to Cry1A.105 + Cry2Ab2 maize further increased resistance to Cry1Fa, which indicates that populations of fall armyworm have high potential for developing resistance to some currently available pyramided maize used against this pest, especially where resistance to Cry1Fa was reported in the field. PMID:26675246

Resistance to dual-gene Bt maize in Spodoptera frugiperda: selection, inheritance, and cross-resistance to other transgenic events.

PubMed

Santos-Amaya, Oscar F; Rodrigues, João V C; Souza, Thadeu C; Tavares, Clébson S; Campos, Silverio O; Guedes, Raul N C; Pereira, Eliseu J G

2015-12-17

Transgenic crop "pyramids" producing two or more Bacillus thuringiensis (Bt) toxins active against the same pest are used to delay evolution of resistance in insect pest populations. Laboratory and greenhouse experiments were performed with fall armyworm, Spodoptera frugiperda, to characterize resistance to Bt maize producing Cry1A.105 and Cry2Ab and test some assumptions of the "pyramid" resistance management strategy. Selection of a field-derived strain of S. frugiperda already resistant to Cry1F maize with Cry1A.105 + Cry2Ab maize for ten generations produced resistance that allowed the larvae to colonize and complete the life cycle on these Bt maize plants. Greenhouse experiments revealed that the resistance was completely recessive (Dx = 0), incomplete, autosomal, and without maternal effects or cross-resistance to the Vip3Aa20 toxin produced in other Bt maize events. This profile of resistance supports some of the assumptions of the pyramid strategy for resistance management. However, laboratory experiments with purified Bt toxin and plant leaf tissue showed that resistance to Cry1A.105 + Cry2Ab2 maize further increased resistance to Cry1Fa, which indicates that populations of fall armyworm have high potential for developing resistance to some currently available pyramided maize used against this pest, especially where resistance to Cry1Fa was reported in the field.

Deregulation of Rab and Rab Effector Genes in Bladder Cancer

PubMed Central

Ho, Joel R.; Chapeaublanc, Elodie; Kirkwood, Lisa; Nicolle, Remy; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Southgate, Jennifer; Radvanyi, François; Goud, Bruno

2012-01-01

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer. PMID:22724020

Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions

PubMed Central

Chen, Tzu-Han; Shiau, Hsin-Chieh

2018-01-01

Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83–94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance. PMID:29920548

Evolution of Prdm Genes in Animals: Insights from Comparative Genomics

PubMed Central

Vervoort, Michel; Meulemeester, David; Béhague, Julien; Kerner, Pierre

2016-01-01

Prdm genes encode transcription factors with a subtype of SET domain known as the PRDF1-RIZ (PR) homology domain and a variable number of zinc finger motifs. These genes are involved in a wide variety of functions during animal development. As most Prdm genes have been studied in vertebrates, especially in mice, little is known about the evolution of this gene family. We searched for Prdm genes in the fully sequenced genomes of 93 different species representative of all the main metazoan lineages. A total of 976 Prdm genes were identified in these species. The number of Prdm genes per species ranges from 2 to 19. To better understand how the Prdm gene family has evolved in metazoans, we performed phylogenetic analyses using this large set of identified Prdm genes. These analyses allowed us to define 14 different subfamilies of Prdm genes and to establish, through ancestral state reconstruction, that 11 of them are ancestral to bilaterian animals. Three additional subfamilies were acquired during early vertebrate evolution (Prdm5, Prdm11, and Prdm17). Several gene duplication and gene loss events were identified and mapped onto the metazoan phylogenetic tree. By studying a large number of nonmetazoan genomes, we confirmed that Prdm genes likely constitute a metazoan-specific gene family. Our data also suggest that Prdm genes originated before the diversification of animals through the association of a single ancestral SET domain encoding gene with one or several zinc finger encoding genes. PMID:26560352

Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators.

PubMed

Klix, V; Nowrousian, M; Ringelberg, C; Loros, J J; Dunlap, J C; Pöggeler, S

2010-06-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.

Functional Characterization of MAT1-1-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators▿†

PubMed Central

Klix, V.; Nowrousian, M.; Ringelberg, C.; Loros, J. J.; Dunlap, J. C.; Pöggeler, S.

2010-01-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the α domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes. PMID:20435701

The Macrophage Activation Induced by Bacillus thuringiensis Cry1Ac Protoxin Involves ERK1/2 and p38 Pathways and the Interaction with Cell-Surface-HSP70.

PubMed

Rubio-Infante, Nestor; Ilhuicatzi-Alvarado, Damaris; Torres-Martínez, Marilu; Reyes-Grajeda, Juan Pablo; Nava-Acosta, Raúl; González-González, Edith; Moreno-Fierros, Leticia

2018-01-01

Here, we aimed to further characterize the mechanisms involved in protoxin (p) Cry1Ac-induced macrophage activation. We demonstrated that pCry1Ac induces MAPK ERK1/2, p38, and JNK phosphorylation in RAW264.7 macrophages. Because MAPK activation is mainly triggered via ligand-receptor interactions, we focused on the identification of potential pCry1Ac-receptor proteins. Flow cytometry and confocal analysis showed specific saturable pCry1Ac-binding to the macrophage surface and evidenced its internalization via the clathrin-pathway. We performed immunoprecipitation assays and identified by MALDI-TOF-TOF several possible pCry1Ac-binding proteins, such as heat shock proteins (HSPs), vimentin, α-enolase, and actin; whose interaction and presence was confirmed, respectively, by ligand blot and Western blot assays. We also detected cell-surface (cs) pCry1Ac-HSP70 colocalization, so HSP70 was chosen for further characterization. Co-immunoprecipitation with HSP70 antibodies followed by Western blot confirmed the pCry1Ac-HSP70 interaction. Furthermore, pretreatment of RAW264.7 cells with HSP70 antibodies reduced pCry1Ac-induced ERK1 phosphorylation and MCP-1 production; thus suggest the functional participation of csHSP70 in pCry1Ac-induced macrophage activation. csHSP70 also was evaluated in peritoneal-cavity (PerC) macrophages of untreated BALB/c mice, interestingly it was found that the predominant population namely large-peritoneal-macrophages (LPM) displayed csHSP70 + hi. Furthermore, the dynamics of PerC macrophage subsets, LPM, and small-peritoneal macrophages (SPM) were evaluated in response to in vivo pCry1Ac stimuli in presence or not of phenylethynesulfonamide (PES) a functional HSP70 inhibitor. It was found that pCry1Ac increased the proportion of SPM CD11b + F4/80 + lowMHCII + csHSP70 + low and markedly reduced the amount of LPM CD11b + F4/80 + hiMHCII-csHSP70 + hi; while PES, partially suppressed this pCry1Ac-induced effect, further suggesting the participation of HSP70 in macrophage activation process. J. Cell. Biochem. 119: 580-598, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

PubMed

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-06-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein

PubMed Central

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-01-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses. PMID:15890917

Degradation of endogenous and exogenous genes of genetically modified rice with Cry1Ab during food processing.

PubMed

Zhang, Wei; Xing, Fuguo; Selvaraj, Jonathan Nimal; Liu, Yang

2014-05-01

In order to assess the degradation of endogenous and exogenous genes during food processing, genetically modified rice with Cry1Ab was used as raw material to produce 4 processed foods: steamed rice, rice noodles, rice crackers, and sweet rice wine. The results showed various processing procedures caused different degrees of degradation of both endogenous and exogenous genes. During the processing of steamed rice and rice noodles, the procedures were so mild that only genes larger than 1500 bp were degraded, and no degradation of NOS terminator and Hpt gene was detected. For rice crackers, frying was the most severe procedure, followed by microwaving, baking, boiling, 1st drying, and 2nd drying. For sweet rice wine, fermentation had more impact on degradation of genes than the other processing procedures. All procedures in this study did not lead to degradation of genes to below 200 bp, except for NOS terminator. In the case of stability of the genes studied during processing of rice crackers and sweet rice wine, SPS gene was the most, followed by the Cry1Ab gene, Hpt gene, Pubi promoter, and NOS terminator. In our study, we gained some information about the degradation of endogenous and exogenous genes during 4 foods processing, compared the different stabilities between endogenous and exogenous genes, and analyzed different effects of procedure on degradation of genes. In addition, the fragments of endogenous and exogenous genes about 200 bp could be detected in final products, except NOS terminator. As a result, we provided some base information about risk assessment of genetically modified (GM) food and appropriate length of fragment to detect GM component in processed foods. © 2014 Institute of Food Technologists®

Enhanced toxicity of Bacillus thuringiensis Cry3A 8-endotoxin in coleopterans by mutagenesis in recetor binding loop

Treesearch

Sheng-Jiun Wu; Noah C. Koller; Deborah L. Miller; Leah S. Bauer; Donald H. Dean

2000-01-01

We used site-directed mutagenesis to modify the Bacillus cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R345A, Y350F, Y351F) and A2 (R345A,DeltaY350, DeltaY351, showed significantly improved...

A Genomic and Proteomic Approach to Identify and Quantify the Expressed Bacillus thuringiensis Proteins in the Supernatant and Parasporal Crystal.

PubMed

Gomis-Cebolla, Joaquín; Scaramal Ricietto, Ana Paula; Ferré, Juan

2018-05-10

The combined analysis of genomic and proteomic data allowed us to determine which cry and vip genes are present in a Bacillus thuringiensis ( Bt ) isolate and which ones are being expressed. Nine Bt isolates were selected from Spanish collections of Bt based on their vip1 and vip2 gene content. As a first step, nine isolates were analyzed by PCR to select those Bt isolates that contained genes with the lowest similarity to already described vip1 and vip2 genes (isolates E-SE10.2 and O-V84.2). Two selected isolates were subjected to a combined genomic and proteomic analysis. The results showed that the Bt isolate E-SE10.2 codifies for two new vegetative proteins, Vip2Ac-like_1 and Sip1Aa-like_1, that do not show expression differences at 24 h vs. 48 h and are expressed in a low amount. The Bt isolate O-V84.2 codifies for three new vegetative proteins, Vip4Aa-like_1, Vip4Aa-like_2, and Vip2Ac-like_2, that are marginally expressed. The Vip4Aa-like_1 protein was two-fold more abundant at 24 h vs. 48 h, while the Vip4Aa-like_2 was detected only at 24 h. For Vip2Ac-like_2, no differences in expression were found at 24 h vs. 48 h. Moreover, the parasporal crystal of the E-SE10.2 isolate contains a single type of crystal protein, Cry23Aa-like, while the parasporal crystal from O-V84.2 contains three kinds of crystal proteins: 7.0⁻9.8% weight of Cry45Aa-like proteins, 35⁻37% weight of Cry32-like proteins and 2.8⁻4.3% weight of Cry73-like protein.

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1988-10-31

00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...11. TITLE (include Security Classification) Cloning and Expression of Genes f or Dengue Virus Type 2 Fncoded Antigens for Rapid Diagnosis and Vaccine ...epidemics in Central and South Americas and the Caribbean is a cause of major concern. An effective vaccine is not available to protect individuals

Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

PubMed

Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

2015-01-01

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants

PubMed Central

Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

2015-01-01

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods. PMID:26513483

Circadian Clock-Regulated Expression of Phytochrome and Cryptochrome Genes in Arabidopsis1

PubMed Central

Tóth, Réka; Kevei, Éva; Hall, Anthony; Millar, Andrew J.; Nagy, Ferenc; Kozma-Bognár, László

2001-01-01

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC+) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC+ constructs, with the exception of PHYC::LUC+, display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock. PMID:11743105

Characterization of a highly toxic strain of Bacillus thuringiensis serovar kurstaki very similar to the HD-73 strain.

PubMed

Reinoso-Pozo, Yaritza; Del Rincón-Castro, Ma Cristina; Ibarra, Jorge E

2016-09-01

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed a 6.4 and 9.5 increase in toxicity, against Manduca sexta and Trichoplusia ni, respectively, compared to HD-73. However, LBIT-1200 was still highly similar to HD-73, including the production of bipyramidal crystals containing only one protein of ∼130 000 kDa, its flagellin gene sequence related to the kurstaki serotype, plasmid and RepPCR patterns similar to HD-73, no production of β-exotoxin and no presence of VIP genes. Sequencing of its cry gene showed the presence of a cry1Ac-type gene with four amino acid differences, including two amino acid replacements in domain III, compared to Cry1Ac1, which may explain its higher toxicity. In conclusion, the LBIT-1200 strain is a variant of the HD-73 strain but shows a much higher toxicity, which makes this new strain an important candidate to be developed as a bioinsecticide, once it passes other tests, throughout its biotechnological development. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Melting Lizards and Crying Mailboxes: Children's Preferential Recall of Minimally Counterintuitive Concepts

ERIC Educational Resources Information Center

Banerjee, Konika; Haque, Omar S.; Spelke, Elizabeth S.

2013-01-01

Previous research with adults suggests that a catalog of minimally counterintuitive concepts, which underlies supernatural or religious concepts, may constitute a cognitive optimum and is therefore cognitively encoded and culturally transmitted more successfully than either entirely intuitive concepts or maximally counterintuitive concepts. This…

Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice.

PubMed

Andreassen, Monica; Bøhn, Thomas; Wikmark, Odd-Gunnar; Bodin, Johanna; Traavik, Terje; Løvik, Martinus; Nygaard, Unni Cecilie

2016-05-04

In genetically modified (GM) crops there is a risk that the inserted genes may introduce new allergens and/or adjuvants into the food and feed chain. The MON810 maize, expressing the insecticidal Cry1Ab toxin, is grown in many countries worldwide. In animal models, intranasal and intraperitoneal immunisations with the purified Cry1Ab proteins have induced immune responses, and feeding trials with Cry1Ab-containing feed have revealed some altered immune responses. Previous investigations have primarily measured antibody responses to the protein, while investigations of clinical food allergy symptoms, or allergy promotion (adjuvant effect) associated with the Cry1Ab protein are largely missing. We aimed to investigate immunogenic, allergenic and adjuvant properties of purified Cry1Ab toxin (trypCry1Ab, i.e., trypsin activated Cry1Ab) in a mouse model of food allergy. Female C3H/HeJ mice were immunized by intragastric gavage of 10 μg purified, trypsin activated Cry1Ab toxin (trypCry1Ab) alone or together with the food allergen lupin. Cholera toxin was added as a positive control for adjuvant effect to break oral tolerance. Clinical symptoms (anaphylaxis) as well as humoral and cellular responses were assessed. In contrast to results from previous airway investigations, we observed no indication of immunogenic properties of trypCry1Ab protein after repeated intragastric exposures to one dose, with or without CT as adjuvant. Moreover, the results indicated that trypCry1Ab given by the intragastric route was not able to promote allergic responses or anaphylactic reactions against the co-administered allergen lupin at the given dose. The study suggests no immunogenic, allergenic or adjuvant capacity of the given dose of trypCry1Ab protein after intragastric exposure of prime aged mice.

Analysis of Homologs of Cry-toxin Receptor-Related Proteins in the Midgut of a Non-Bt Target, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

PubMed Central

Shao, Ensi; Lin, Li; Liu, Sijun; Zhang, Jiao; Chen, Xuelin; Sha, Li; Huang, Zhipeng; Huang, Biwang; Guan, Xiong

2018-01-01

Abstract The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests in the rice fields of Asia. Like other hemipteran insects, BPH is not susceptible to Cry toxins of Bacillus thuringiensis (Bt) or transgenic rice carrying Bt cry genes. Lack of Cry receptors in the midgut is one of the main reasons that BPH is not susceptible to the Cry toxins. The main Cry-binding proteins (CBPs) of the susceptible insects are cadherin, aminopeptidase N (APN), and alkaline phosphatase (ALP). In this study, we analyzed and validated de novo assembled transcripts from transcriptome sequencing data of BPH to identify and characterize homologs of cadherin, APN, and ALP. We then compared the cadherin-, APN-, and ALP-like proteins of BPH to previously reported CBPs to identify their homologs in BPH. The sequence analysis revealed that at least one cadherin, one APN, and two ALPs of BPH contained homologous functional domains identified from the Cry-binding cadherin, APN, and ALP, respectively. Quantitative real-time polymerase chain reaction used to verify the expression level of each putative Cry receptor homolog in the BPH midgut indicated that the CBPs homologous APN and ALP were expressed at high or medium-high levels while the cadherin was expressed at a low level. These results suggest that homologs of CBPs exist in the midgut of BPH. However, differences in key motifs of CBPs, which are functional in interacting with Cry toxins, may be responsible for insusceptibility of BPH to Cry toxins. PMID:29415259

Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs.

PubMed

Debode, Frederic; Janssen, Eric; Bragard, Claude; Berben, Gilbert

2017-08-01

The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

PubMed Central

Pigott, Craig R.; Ellar, David J.

2007-01-01

Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane

PubMed Central

2017-01-01

The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests. PMID:28123906

Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies.

PubMed

Xu, Chongxin; Zhang, Cunzheng; Zhong, Jianfeng; Hu, Hui; Luo, Shimin; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Liu, Xianjin

2017-07-26

In the present study, a Cry1F-immunized rabbit phage display library (6.96 × 10 8 cfu/mL) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFv's were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed in Escherichia coli HB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for Cry1F toxin. It indicated the 50% inhibition of the control (IC 50 ) was 11.56 ng/mL and the detection limit (IC 10 ) was 0.18 ng/mL and showed weak cross-reactivities for Cry1Ab (2.8%), Cry1Ac (1.3%), and Cry1B, Cry1C, Cry1Ie, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2-99.6%, and the coefficients of variation were in the range of 2.5-10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.

Molecular and functional characterization of cry1Ac transgenic pea lines.

PubMed

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-10-01

Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

Molecular and functional characterization of cry1Ac transgenic pea lines

PubMed Central

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-01-01

ABSTRACT Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses. PMID:27764552

Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis

PubMed Central

Espinasse, Sylvain; Gohar, Michel; Chaufaux, Josette; Buisson, Christophe; Perchat, Stéphane; Sanchis, Vincent

2002-01-01

Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 μg of β-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 μg/ml. However, these amounts of β-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of β-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of β-exotoxin I. PMID:12200263

New multiplex PCR methods for rapid screening of genetically modified organisms in foods

PubMed Central

Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

2015-01-01

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products. PMID:26257724

New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

PubMed

Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

2015-01-01

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

Genome-Wide Localization Study of Yeast Pex11 Identifies Peroxisome–Mitochondria Interactions through the ERMES Complex

PubMed Central

Mattiazzi Ušaj, M.; Brložnik, M.; Kaferle, P.; Žitnik, M.; Wolinski, H.; Leitner, F.; Kohlwein, S.D.; Zupan, B.; Petrovič, U.

2015-01-01

Pex11 is a peroxin that regulates the number of peroxisomes in eukaryotic cells. Recently, it was found that a mutation in one of the three mammalian paralogs, PEX11β, results in a neurological disorder. The molecular function of Pex11, however, is not known. Saccharomyces cerevisiae Pex11 has been shown to recruit to peroxisomes the mitochondrial fission machinery, thus enabling proliferation of peroxisomes. This process is essential for efficient fatty acid β-oxidation. In this study, we used high-content microscopy on a genome-wide scale to determine the subcellular localization pattern of yeast Pex11 in all non-essential gene deletion mutants, as well as in temperature-sensitive essential gene mutants. Pex11 localization and morphology of peroxisomes was profoundly affected by mutations in 104 different genes that were functionally classified. A group of genes encompassing MDM10, MDM12 and MDM34 that encode the mitochondrial and cytosolic components of the ERMES complex was analyzed in greater detail. Deletion of these genes caused a specifically altered Pex11 localization pattern, whereas deletion of MMM1, the gene encoding the fourth, endoplasmic-reticulum-associated component of the complex, did not result in an altered Pex11 localization or peroxisome morphology phenotype. Moreover, we found that Pex11 and Mdm34 physically interact and that Pex11 plays a role in establishing the contact sites between peroxisomes and mitochondria through the ERMES complex. Based on these results, we propose that the mitochondrial/cytosolic components of the ERMES complex establish a direct interaction between mitochondria and peroxisomes through Pex11. PMID:25769804

No impact of transgenic cry1C rice on the rove beetle Paederus fuscipes, a generalist predator of brown planthopper Nilaparvata lugens

NASA Astrophysics Data System (ADS)

Meng, Jiarong; Mabubu, Juma Ibrahim; Han, Yu; He, Yueping; Zhao, Jing; Hua, Hongxia; Feng, Yanni; Wu, Gang

2016-07-01

T1C-19 is newly developed transgenic rice active against lepidopteran pests, and expresses a synthesized cry1C gene driven by the maize ubiquitin promoter. The brown planthopper, Nilaparvata lugens, is a major non-target pest of rice, and the rove beetle (Paederus fuscipes) is a generalist predator of N. lugens nymphs. As P. fuscipes may be exposed to the Cry1C protein through preying on N. lugens, it is essential to assess the potential effects of transgenic cry1C rice on this predator. In this study, two experiments (a direct feeding experiment and a tritrophic experiment) were conducted to evaluate the ecological risk of cry1C rice to P. fuscipes. No significant negative effects were observed in the development, survival, female ratio and body weight of P. fuscipes in both treatments of direct exposure to elevated doses of Cry1C protein and prey-mediated exposure to realistic doses of the protein. This indicated that cry1C rice had no detrimental effects on P. fuscipes. This work represents the first study of an assessment continuum for the effects of transgenic cry1C rice on P. fuscipes. Use of the rove beetle as an indicator species to assess potential effects of genetically modified crops on non-target arthropods is feasible.

No impact of transgenic cry1C rice on the rove beetle Paederus fuscipes, a generalist predator of brown planthopper Nilaparvata lugens

PubMed Central

Meng, Jiarong; Mabubu, Juma Ibrahim; Han, Yu; He, Yueping; Zhao, Jing; Hua, Hongxia; Feng, Yanni; Wu, Gang

2016-01-01

T1C-19 is newly developed transgenic rice active against lepidopteran pests, and expresses a synthesized cry1C gene driven by the maize ubiquitin promoter. The brown planthopper, Nilaparvata lugens, is a major non-target pest of rice, and the rove beetle (Paederus fuscipes) is a generalist predator of N. lugens nymphs. As P. fuscipes may be exposed to the Cry1C protein through preying on N. lugens, it is essential to assess the potential effects of transgenic cry1C rice on this predator. In this study, two experiments (a direct feeding experiment and a tritrophic experiment) were conducted to evaluate the ecological risk of cry1C rice to P. fuscipes. No significant negative effects were observed in the development, survival, female ratio and body weight of P. fuscipes in both treatments of direct exposure to elevated doses of Cry1C protein and prey-mediated exposure to realistic doses of the protein. This indicated that cry1C rice had no detrimental effects on P. fuscipes. This work represents the first study of an assessment continuum for the effects of transgenic cry1C rice on P. fuscipes. Use of the rove beetle as an indicator species to assess potential effects of genetically modified crops on non-target arthropods is feasible. PMID:27444416

CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.

PubMed

Mandel, M A; Feldmann, K A; Herrera-Estrella, L; Rocha-Sosa, M; León, P

1996-05-01

An albino mutant designated cla1-1 (for "cloroplastos alterados', or "altered chloroplasts') has been isolated from a T-DNA-generated library of Arabidopsis thaliana. In cla1-1 plants, chloroplast development is arrested at an early stage. cla1-1 plants behave like wild-type in their capacity to etiolate and produce anthocyanins indicating that the light signal transduction pathway seems to be unaffected. Genetic and molecular analyses show that the disruption of a single gene, CLA1, by the T-DNA insertion is responsible for the mutant phenotype. RNA expression patterns indicate that CLA1 is positively regulated by light and that it has different effects on the steady-state RNA levels of some nuclear- and chloroplast-encoded photosynthetic genes. Although the specific function of the CLA1 gene is still unknown, it encodes a novel protein conserved in evolution between photosynthetic bacteria and plants which is essential for chloroplast development in Arabidopsis.

I thought that I heard you laughing: Contextual facial expressions modulate the perception of authentic laughter and crying.

PubMed

Lavan, Nadine; Lima, César F; Harvey, Hannah; Scott, Sophie K; McGettigan, Carolyn

2015-01-01

It is well established that categorising the emotional content of facial expressions may differ depending on contextual information. Whether this malleability is observed in the auditory domain and in genuine emotion expressions is poorly explored. We examined the perception of authentic laughter and crying in the context of happy, neutral and sad facial expressions. Participants rated the vocalisations on separate unipolar scales of happiness and sadness and on arousal. Although they were instructed to focus exclusively on the vocalisations, consistent context effects were found: For both laughter and crying, emotion judgements were shifted towards the information expressed by the face. These modulations were independent of response latencies and were larger for more emotionally ambiguous vocalisations. No effects of context were found for arousal ratings. These findings suggest that the automatic encoding of contextual information during emotion perception generalises across modalities, to purely non-verbal vocalisations, and is not confined to acted expressions.

Gq/11-Dependent Changes in the Murine Ovarian Transcriptome at the End of Gestation1

PubMed Central

Waite, Courtney; Mejia, Rachel; Ascoli, Mario

2016-01-01

Parturition in rodents is highly dependent on the engagement of the luteal prostaglandin F2 alpha receptor, which, through activation of the Gq/11 family of G proteins, increases the expression of Akr1c18, leading to an increase in progesterone catabolism. To further understand the involvement of Gq/11 on luteolysis and parturition, we used microarray analysis to compare the ovarian transcriptome of mice with a granulosa/luteal cell-specific deletion of Galphaq/11 with their control littermates on Day 18 of pregnancy, when mice from both genotypes are pregnant, and on Day 22, when mice with a granulosa/luteal cell-specific deletion of Galphaq/11 are still pregnant but their control littermates are 1–2 days postpartum. Ovarian genes up-regulated at the end of gestation in a Galphaq/11 -dependent fashion include genes involved in focal adhesion and extracellular matrix interactions. Genes down-regulated at the end of gestation in a Galphaq/11-dependent manner include Serpina6 (which encodes corticosteroid-binding globulin); Enpp2 (which encodes autotaxin, the enzyme responsible for the synthesis of lysophosphatidic acid); genes involved in protein processing and export; reproductive genes, such as Lhcgr; the three genes needed to convert progesterone to estradiol (Cyp17a1, Hsd17b7, and Cyp19a1); and Inha. Activation of ovarian Gq/11 by engagement of the prostaglandin F2 alpha receptor on Day 18 of pregnancy recapitulated the regulation of many but not all of these genes. Thus, although the ovarian transcriptome at the end of gestation is highly dependent on the activation of Gq/11, not all of these changes are dependent on the actions of prostaglandin F2 alpha. PMID:26843449

Isolation and characterization of cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits in Nitrosomonas sp. strain ENI-11.

PubMed

Hirota, Ryuichi; Kato, Junichi; Morita, Hiromu; Kuroda, Akio; Ikeda, Tsukasa; Takiguchi, Noboru; Ohtake, Hisao

2002-03-01

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87% identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions.

PubMed

Yuan, Li; Seong, Eunju; Beuscher, James L; Arikkath, Jyothi

2015-04-24

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A CRY-BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis.

PubMed

Wang, Xu; Wang, Qin; Han, Yun-Jeong; Liu, Qing; Gu, Lianfeng; Yang, Zhaohe; Su, Jun; Liu, Bobin; Zuo, Zecheng; He, Wenjin; Wang, Jian; Liu, Bin; Matsui, Minami; Kim, Jeong-Il; Oka, Yoshito; Lin, Chentao

2017-11-01

Cryptochromes are blue light receptors that regulate various light responses in plants. Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate blue light inhibition of hypocotyl elongation and long-day (LD) promotion of floral initiation. It has been reported recently that two negative regulators of Arabidopsis cryptochromes, Blue light Inhibitors of Cryptochromes 1 and 2 (BIC1 and BIC2), inhibit cryptochrome function by blocking blue light-dependent cryptochrome dimerization. However, it remained unclear how cryptochromes regulate the BIC gene activity. Here we show that cryptochromes mediate light activation of transcription of the BIC genes, by suppressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), resulting in activation of the transcription activator ELONGATED HYPOCOTYL 5 (HY5) that is associated with chromatins of the BIC promoters. These results demonstrate a CRY-BIC negative-feedback circuitry that regulates the activity of each other. Surprisingly, phytochromes also mediate light activation of BIC transcription, suggesting a novel photoreceptor co-action mechanism to sustain blue light sensitivity of plants under the broad spectra of solar radiation in nature. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

DOE PAGES

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; ...

2017-01-31

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

A Cryptochrome 2 mutation yields advanced sleep phase in humans.

PubMed

Hirano, Arisa; Shi, Guangsen; Jones, Christopher R; Lipzen, Anna; Pennacchio, Len A; Xu, Ying; Hallows, William C; McMahon, Thomas; Yamazaki, Maya; Ptáček, Louis J; Fu, Ying-Hui

2016-08-16

Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. We identified a missense mutation in the human Cryptochrome 2 (CRY2) gene that co-segregates with FASP in one family. The mutation leads to replacement of an alanine residue at position 260 with a threonine (A260T). In mice, the CRY2 mutation causes a shortened circadian period and reduced phase-shift to early-night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle. The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2, increasing its accessibility and affinity for FBXL3 (an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior.

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia.

PubMed

Rani, Lata; Mathur, Nitin; Gupta, Ritu; Gogia, Ajay; Kaur, Gurvinder; Dhanjal, Jaspreet Kaur; Sundar, Durai; Kumar, Lalit; Sharma, Atul

2017-01-01

In chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients. The integration of DNA methylation profile ( n  = 14) with the gene expression profile ( n  = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients ( n  = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1 , PMEPA1 , SOX7 , SPRY1 , CDK6 , TBX2 , and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup ( p  < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p  = 0.005) or PAX9 (RR = 1.87, p  = 0.001). High expression of CRY1 (HR: 3.53, p  < 0.001) or PAX9 (HR: 3.14, p  < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172-9.272, p  = 0.016) was also predictive of shorter overall survival in CLL. The DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.

Cryptochrome 2 expression level is critical for adrenocorticotropin stimulation of cortisol production in the capuchin monkey adrenal.

PubMed

Torres-Farfan, C; Abarzua-Catalan, L; Valenzuela, F J; Mendez, N; Richter, H G; Valenzuela, G J; Serón-Ferré, M

2009-06-01

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

PRKCDBP (CAVIN3) and CRY2 associate with major depressive disorder.

PubMed

Kovanen, Leena; Donner, Kati; Kaunisto, Mari; Partonen, Timo

2017-01-01

Dysfunctions in the intrinsic clocks are suggested in patients with depressive disorders. The cryptochrome circadian clocks 1 and 2 (CRY1 and CRY2) proteins modulate circadian rhythms in a cell and influence emotional reactions and mood in an individual. The protein kinase C delta binding protein (PRKCDBP, or CAVIN3), similar to the serum deprivation response protein (SDPR, or CAVIN2), reduces metabolic stability of the PER2-CRY2 transcription factor complex that plays a role in the circadian rhythm synchronization. Our aim was to study SDPR, PRKCDBP, CRY1 and CRY2 genetic variants in depressive disorders. The sample included 5910 Finnish individuals assessed with the Munich-Composite International Diagnostic Interview (M-CIDI) in year 2000. In year 2011, 3424 individuals were assessed again. After genotype quality control, there were 383 subjects with major depressive disorder, 166 with dysthymia, and 479 with depressive disorders (major depressive disorder, dysthymia or both), and 4154 healthy controls. A total of 48 single-nucleotide polymorphisms from SDPR, PRKCDBP, CRY1 and CRY2 genes were analyzed using logistic regression models controlling for age and gender. The earlier reported association of CRY2 variants with dysthymia was confirmed and extended to major depressive disorder (q<0.05). In addition, novel associations of PRKCDBP rs1488864 with depressive disorders (q=0.02) and with major depressive disorder in specific (q=0.007) were found. The number of cases was moderate and coverage of PRKCDB was limited. CRY2 and PRKCDBP variants may be risk factors of major depressive disorder and provide information for diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

PubMed

Dong, Sa; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Zhong, Jianfeng; Xu, Chongxin; Liu, Xianjin

2017-03-01

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D ) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10 -8  M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL -1 , respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL -1 ), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Comparison of compensatory reserve during lower-body negative pressure and hemorrhage in nonhuman primates.

PubMed

Hinojosa-Laborde, Carmen; Howard, Jeffrey T; Mulligan, Jane; Grudic, Greg Z; Convertino, Victor A

2016-06-01

Compensatory reserve was measured in baboons (n = 13) during hemorrhage (Hem) and lower-body negative pressure (LBNP) using a machine-learning algorithm developed to estimate compensatory reserve by detecting reductions in central blood volume during LBNP. The algorithm calculates compensatory reserve index (CRI) from normovolemia (CRI = 1) to cardiovascular decompensation (CRI = 0). The hypothesis was that Hem and LBNP will elicit similar CRI values and that CRI would have higher specificity than stroke volume (SV) in predicting decompensation. Blood was removed in four steps: 6.25%, 12.5%, 18.75%, and 25% of total blood volume. Four weeks after Hem, the same animals were subjected to four levels of LBNP that was matched on the basis of their central venous pressure. Data (mean ± 95% confidence interval) indicate that CRI decreased (P < 0.001) from baseline during Hem (0.69 ± 0.10, 0.57 ± 0.09, 0.36 ± 0.10, 0.16 ± 0.08, and 0.08 ± 0.03) and LBNP (0.76 ± 0.05, 0.66 ± 0.08, 0.36 ± 0.13, 0.23 ± 0.11, and 0.14 ± 0.09). CRI was not different between Hem and LBNP (P = 0.20). Linear regression analysis between Hem CRI and LBNP CRI revealed a slope of 1.03 and a correlation coefficient of 0.96. CRI exhibited greater specificity than SV in both Hem (92.3 vs. 82.1) and LBNP (94.8 vs. 83.1) and greater ROC AUC in Hem (0.94 vs. 0.84) and LBNP (0.94 vs. 0.92). These data support the hypothesis that Hem and LBNP elicited the same CRI response, suggesting that measurement of compensatory reserve is superior to SV as a predictor of cardiovascular decompensation.

Comparison of compensatory reserve during lower-body negative pressure and hemorrhage in nonhuman primates

PubMed Central

Howard, Jeffrey T.; Mulligan, Jane; Grudic, Greg Z.; Convertino, Victor A.

2016-01-01

Compensatory reserve was measured in baboons (n = 13) during hemorrhage (Hem) and lower-body negative pressure (LBNP) using a machine-learning algorithm developed to estimate compensatory reserve by detecting reductions in central blood volume during LBNP. The algorithm calculates compensatory reserve index (CRI) from normovolemia (CRI = 1) to cardiovascular decompensation (CRI = 0). The hypothesis was that Hem and LBNP will elicit similar CRI values and that CRI would have higher specificity than stroke volume (SV) in predicting decompensation. Blood was removed in four steps: 6.25%, 12.5%, 18.75%, and 25% of total blood volume. Four weeks after Hem, the same animals were subjected to four levels of LBNP that was matched on the basis of their central venous pressure. Data (mean ± 95% confidence interval) indicate that CRI decreased (P < 0.001) from baseline during Hem (0.69 ± 0.10, 0.57 ± 0.09, 0.36 ± 0.10, 0.16 ± 0.08, and 0.08 ± 0.03) and LBNP (0.76 ± 0.05, 0.66 ± 0.08, 0.36 ± 0.13, 0.23 ± 0.11, and 0.14 ± 0.09). CRI was not different between Hem and LBNP (P = 0.20). Linear regression analysis between Hem CRI and LBNP CRI revealed a slope of 1.03 and a correlation coefficient of 0.96. CRI exhibited greater specificity than SV in both Hem (92.3 vs. 82.1) and LBNP (94.8 vs. 83.1) and greater ROC AUC in Hem (0.94 vs. 0.84) and LBNP (0.94 vs. 0.92). These data support the hypothesis that Hem and LBNP elicited the same CRI response, suggesting that measurement of compensatory reserve is superior to SV as a predictor of cardiovascular decompensation PMID:27030667

Cry1A(b)16 toxin from Bacillus thuringiensis: Theoretical refinement of three-dimensional structure and prediction of peptides as molecular markers for detection of genetically modified organisms.

PubMed

Plácido, Alexandra; Coelho, Andreia; Abreu Nascimento, Lucas; Gomes Vasconcelos, Andreanne; Fátima Barroso, Maria; Ramos-Jesus, Joilson; Costa, Vladimir; das Chagas Alves Lima, Francisco; Delerue-Matos, Cristina; Martins Ramos, Ricardo; Marani, Mariela M; Roberto de Souza de Almeida Leite, José

2017-07-01

Transgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody-peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248-1257. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Identification of differentially expressed microRNAs between Bacillus thuringiensis Cry1Ab-resistant and -susceptible strains of Ostrinia furnacalis

PubMed Central

Xu, Li-Na; Ling, Ying-Hui; Wang, Yue-Qin; Wang, Zhen-Ying; Hu, Ben-Jin; Zhou, Zi-Yan; Hu, Fei; He, Kang-Lai

2015-01-01

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée), can develop strong resistance to Cry1Ab, the most widely commercialized Cry toxin for Bt maize worldwide. It is essential to understand the mechanism of resistance for management of this species, but information on the post-transcriptional regulation of Bt resistance in this target insect is limited. In the present study, RNA was extracted from the ACB in various larval stages (1–5 instar) from Cry1Ab-sensitive (ACB-BtS) and -resistant (ACB-AbR) strains, each of which included two biological replicates. Using Illumina sequencing, a total of 23,809,890 high-quality reads were collected from the four ACB libraries. The numbers of known microRNAs (miRNAs) were 302 and 395 for ACB-BtS and 268 and 287 for ACB-AbR. Using Mireap software, we identified 32 and 16 potential novel miRNAs for ACB-BtS and 18 and 22 for ACB-AbR. Among them, 21 known and 1 novel miRNAs had significantly different expression between ACB-BtS and ACB-AbR. Several miRNAs were observed to target potential Bt receptor genes, such as aminopeptidase N and cadherin-like protein. The glycosylphosphatidylinositol-anchor biosynthetic process and ABC transporters pathway were identified through Gene Ontology and KEGG pathway analysis of target genes of the differentially expressed miRNAs. PMID:26486179

Continuous evolution of B. thuringiensis toxins overcomes insect resistance

PubMed Central

Badran, Ahmed H.; Guzov, Victor M.; Huai, Qing; Kemp, Melissa M.; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M.; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H.; Wang, Ping; Malvar, Thomas; Liu, David R.

2016-01-01

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. PMID:27120167

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

USDA-ARS?s Scientific Manuscript database

European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn, and reduces yield of food, feed, and biofuel materials. Transgenic hybrid corn expressing the Bacillus thuringiensis (Bt) toxin Cry1F is highly lethal to O...

Genomics and relative expression analysis identifies key genes associated with high female to male flower ratio in Jatropha curcas L.

PubMed

Gangwar, Manali; Sood, Hemant; Chauhan, Rajinder Singh

2016-04-01

Jatropha curcas, has been projected as a major source of biodiesel due to high seed oil content (42 %). A major roadblock for commercialization of Jatropha-based biodiesel is low seed yield per inflorescence, which is affected by low female to male flower ratio (1:25-30). Molecular dissection of female flower development by analyzing genes involved in phase transitions and floral organ development is, therefore, crucial for increasing seed yield. Expression analysis of 42 genes implicated in floral organ development and sex determination was done at six floral developmental stages of a J. curcas genotype (IC561235) with inherently higher female to male flower ratio (1:8-10). Relative expression analysis of these genes was done on low ratio genotype. Genes TFL1, SUP, AP1, CRY2, CUC2, CKX1, TAA1 and PIN1 were associated with reproductive phase transition. Further, genes CUC2, TAA1, CKX1 and PIN1 were associated with female flowering while SUP and CRY2 in female flower transition. Relative expression of these genes with respect to low female flower ratio genotype showed up to ~7 folds increase in transcript abundance of SUP, TAA1, CRY2 and CKX1 genes in intermediate buds but not a significant increase (~1.25 folds) in female flowers, thereby suggesting that these genes possibly play a significant role in increased transition towards female flowering by promoting abortion of male flower primordia. The outcome of study has implications in feedstock improvement of J. curcas through functional validation and eventual utilization of key genes associated with female flowering.

A conceptual contribution to battles in the brain

PubMed Central

2010-01-01

Badcock and Crespi have advanced the hypothesis that autism and schizophrenia are caused by imbalanced imprinting in the brain. They argue that an imbalance between the effects of paternally and maternally expressed genes on brain development results in either an extreme paternal (autism) or maternal brain (schizophrenia). In this paper their conceptual model is discussed and criticized since it presupposes an incoherent distinction between observable physical and hidden mental phenomena. An alternative model is discussed that may be more fruitful for investigating the possible role of imprinted genes in the development of social behaviour. The development of crying and reactive crying and behaviours necessary for collaborative action are discussed as a promising research area for understanding the effects of imprinted genes. PMID:21212823

Immunotoxicological Evaluation of Corn Genetically Modified with Bacillus thuringiensis Cry1Ah Gene by a 30-Day Feeding Study in BALB/c Mice

PubMed Central

Song, Yan; Liang, Chunlai; Wang, Wei; Fang, Jin; Sun, Nana; Jia, Xudong; Li, Ning

2014-01-01

This study was to investigate the immunotoxicological potential of corn genetically modified (GM) with Bacillus thuringiensis (Bt) Cry1Ah gene in BALB/c mice. Female BALB/c mice were randomly assigned to one of the four groups: the negative control group, the parental corn group, the GM corn group and the positive control group with 10 mice per group. Mice in the GM corn group and the parental corn group were fed with diets containing 70% corresponding corn for 30 days. Mice in the negative control group and the positive control group were fed with AIN93G diet, administered with saline or 200 mg/kg of cyclophosphamide (CY) via intraperitoneal injection 24 h before the termination of the study, respectively. At the end of the study, the immunotoxicological effects of the GM corn were evaluated through immunopathology parameters including body and organ weights, hematology and clinical chemistry parameters, histological examination, peripheral blood lymphocytes phenotype; humoral immunity including antibody plaque-forming cell, serum immunoglobulin, cytokine and half hemolysis value; cellular immunity such as mitogen-induced splenocyte proliferation, cytotoxic T-lymphocyte reaction, delayed-type hypersensitivity reaction; non-specific immunity including phagocytic activities of phagocytes, natural killer cell activity. A single dose of cyclophosphamide (200 mg/kg bw) was found to have significant adverse effects on immunopathology, cellular immunity, and humoral immunity in mice. The corn genetically modified with Bt Cry1Ah gene is considered consistent with the parental corn in terms of immunopathology, humoral immunity, cellular immunity and non-specific immunity. No adverse immunotoxicological effects of GM corn with Bt Cry1Ah gene were found when feeding mice for 30 days. PMID:24520311

Associations of polymorphisms in circadian genes with abdominal obesity in Chinese adult population.

PubMed

Ye, Ding; Cai, Shaofang; Jiang, Xiyi; Ding, Ye; Chen, Kun; Fan, Chunhong; Jin, Mingjuan

2016-09-01

Circadian rhythm, which is controlled by circadian genes, regulates metabolic balance including the circulating levels of glucose, fatty acids, triglycerides, various hormones and so on. The study aimed to investigate the impact of potential polymorphisms in circadian genes on abdominal obesity among Chinese Han adults. A total of 260 cases with abdominal obesity and 260 controls were recruited by individual matching. Demographic characteristics and lifestyle information were collected by a validated questionnaire, and anthropometric parameters was measured by physical examination. Twenty-three single nucleotide polymorphisms (SNPs) in three circadian genes, CLOCK, CRY1 and CRY2, were genotyped by MassArray technique. Five SNPs significantly deviated from Hardy-Weinberg equilibrium (HWE) among controls, so eighteen SNPs were taken into logistic regression analysis. Independently, CLOCK rs10002541 (CC genotype vs. TT genotype: OR: 0.45, 95% CI: 0.23-0.86), CLOCK rs6850524 (CC genotype vs. GG genotype: OR: 0.50, 95% CI: 0.25-0.99) and CRY1 rs10861688 (TT genotype vs. CC genotype: OR: 0.50, 95% CI: 0.25-0.97) were negatively associated with the risk of abdominal obesity. Haplotype analysis showed that the haplotypes of CG and TG for CLOCK rs10002541 and rs4864546 had significant associations with abdominal obesity. Compared with the carriers of TA, those of CG were observed to have a lower risk (OR: 0.74, 95% CI: 0.56-0.99) of abdominal obesity, and those of TG presented a higher risk (OR: 1.70, 95% CI: 1.03-2.81). Our findings suggest that CLOCK and CRY1 polymorphisms might be involved in individual susceptibility to abdominal obesity in Chinese Han population. Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper.

PubMed

Wan, Gui-Jun; Wang, Wen-Jing; Xu, Jing-Jing; Yang, Quan-Feng; Dai, Ming-Jiang; Zhang, Feng-Jiao; Sword, Gregory A; Pan, Wei-Dong; Chen, Fa-Jun

2015-01-01

Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE.

Construction of a standard reference plasmid containing seven target genes for the detection of transgenic cotton.

PubMed

Wang, Xujing; Tang, Qiaoling; Dong, Lei; Dong, Yufeng; Su, Yueyan; Jia, Shirong; Wang, Zhixing

2014-07-01

Insect resistance and herbicide tolerance are the dominant traits of commercialized transgenic cotton. In this study, we constructed a general standard reference plasmid for transgenic cotton detection. Target genes, including the cowpea trypsin gene cptI, the insect resistance gene cry1Ab/1Ac, the herbicide tolerance gene cp4-epsps, the Agrobacterium tumefaciens nopaline synthase (Nos) terminator that exists in transgenic cotton and part of the endogenous cotton SadI gene were amplified from plasmids pCPT1, pBT, pCP4 and pBI121 and from DNA of the nontransgenic cotton line K312, respectively. The genes cry1Ab/1Ac and cptI, as well as cp4-epsps and the Nos terminator gene, were ligated together to form the fusion genes cptI-Bt and cp4-Nos, respectively, by overlapping PCR. We checked the validity of genes Sad1, cptI-Bt and cp4-Nos by DNA sequencing. Then, positive clones of cptI-Bt, cp4-Nos and Sad1 were digested with the corresponding restriction enzymes and ligated sequentially into vector pCamBIA2300, which contains the CAMV 35S promoter and nptII gene, to form the reference plasmid pMCS. Qualitative detection showed that pMCS is a good positive control for transgenic cotton detection. Real-time PCR detection efficiencies with pMCS as a calibrator ranged from 94.35% to 98.67% for the standard curves of the target genes (R(2)⩾0.998). The relative standard deviation of the mean value for the known sample was 11.95%. These results indicate that the strategy of using the pMCS plasmid as a reference material is feasible and reliable for the detection of transgenic cotton. Therefore, this plasmid can serve as a useful reference tool for qualitative and quantitative detection of single or stacked trait transgenic cotton, thus paving the way for the identification of various products containing components of transgenic cotton. Copyright © 2014 Elsevier Inc. All rights reserved.

Occurrence, leaching, and degradation of Cry1Ab protein from transgenic maize detritus in agricultural streams

DOE PAGES

Griffiths, Natalie A.; Tank, Jennifer L.; Royer, Todd V.; ...

2017-03-15

The insecticidal Cry1Ab protein expressed by transgenic (Bt) maize can enter adjacent water bodies via multiple pathways, but its fate in stream ecosystems is not as well studied as in terrestrial systems. In this study, we used a combination of field sampling and laboratory experiments to examine the occurrence, leaching, and degradation of soluble Cry1Ab protein derived from Bt maize in agricultural streams. We surveyed 11 agricultural streams in northwestern Indiana, USA, on 6 dates that encompassed the growing season, crop harvest, and snowmelt/spring flooding, and detected Cry1Ab protein in the water column and in flowing subsurface tile drains atmore » concentrations of 3–60 ng/L. In a series of laboratory experiments, submerged Bt maize leaves leached Cry1Ab into stream water with 1% of the protein remaining in leaves after 70 d. Laboratory experiments suggested that dissolved Cry1Ab protein degraded rapidly in microcosms containing water-column microorganisms, and light did not enhance breakdown by stimulating assimilatory uptake of the protein by autotrophs. Here, the common detection of Cry1Ab protein in streams sampled across an agricultural landscape, combined with laboratory studies showing rapid leaching and degradation, suggests that Cry1Ab may be pseudo-persistent at the watershed scale due to the multiple input pathways from the surrounding terrestrial environment.« less

Occurrence, leaching, and degradation of Cry1Ab protein from transgenic maize detritus in agricultural streams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffiths, Natalie A.; Tank, Jennifer L.; Royer, Todd V.

The insecticidal Cry1Ab protein expressed by transgenic (Bt) maize can enter adjacent water bodies via multiple pathways, but its fate in stream ecosystems is not as well studied as in terrestrial systems. In this study, we used a combination of field sampling and laboratory experiments to examine the occurrence, leaching, and degradation of soluble Cry1Ab protein derived from Bt maize in agricultural streams. We surveyed 11 agricultural streams in northwestern Indiana, USA, on 6 dates that encompassed the growing season, crop harvest, and snowmelt/spring flooding, and detected Cry1Ab protein in the water column and in flowing subsurface tile drains atmore » concentrations of 3–60 ng/L. In a series of laboratory experiments, submerged Bt maize leaves leached Cry1Ab into stream water with 1% of the protein remaining in leaves after 70 d. Laboratory experiments suggested that dissolved Cry1Ab protein degraded rapidly in microcosms containing water-column microorganisms, and light did not enhance breakdown by stimulating assimilatory uptake of the protein by autotrophs. Here, the common detection of Cry1Ab protein in streams sampled across an agricultural landscape, combined with laboratory studies showing rapid leaching and degradation, suggests that Cry1Ab may be pseudo-persistent at the watershed scale due to the multiple input pathways from the surrounding terrestrial environment.« less

Gene cloning, overexpression, and characterization of a xylanase from Penicillium sp. CGMCC 1669.

PubMed

Liu, Wanli; Shi, Pengjun; Chen, Qiang; Yang, Peilong; Wang, Guozeng; Wang, Yaru; Luo, Huiying; Yao, Bin

2010-09-01

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml(-1). After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40 degrees C, was stable at acidic buffers of pH 4.5-9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and alpha-chymotrypsin). The specific activity, K (m), and V (max) for oat spelt xylan substrate was 7,988 U mg(-1), 22.2 mg ml(-1), and 15,105.7 micromol min(-1) mg(-1), respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.

Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

PubMed Central

Wu, S; Kriz, A L; Widholm, J M

1994-01-01

The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions. PMID:7972490

The Arabidopsis WRINKLED1 transcription factor affects auxin homeostasis in roots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kong, Que; Ma, Wei; Yang, Haibing

WRINKLED1 (WRI1) is a key transcriptional regulator of fatty acid biosynthesis genes in diverse oil-containing tissues. Loss of function of Arabidopsis WRI1 leads to a reduction in the expression of genes for fatty acid biosynthesis and glycolysis, and concomitant strong reduction of seed oil content. The wri1-1 loss-of-function mutant shows reduced primary root growth and decreased acidification of the growth medium. The content of a conjugated form of the plant growth hormone auxin, indole-3-acetic acid (IAA)-Asp, was higher in wri1-1 plants compared with the wild-type. GH3.3, a gene encoding an enzyme involved in auxin degradation, displayed higher expression in themore » wri1-1 mutant. EMSAs demonstrated that AtWRI1 bound to the promoter of GH3.3. Specific AtWRI1-binding motifs were identified in the promoter of GH3.3. In addition, wri1-1 displayed decreased auxin transport. Expression of some PIN genes, which encode IAA carrier proteins, was reduced in wri1-1 plants as well. Correspondingly, AtWRI1 bound to the promoter regions of some PIN genes. It is well known that auxin exerts its maximum effects at a specific, optimal concentration in roots requiring a finely balanced auxin homeostasis. This process appears to be disrupted when the expression of WRI1 and in turn a subset of its target genes are misregulated, highlighting a role for WRI1 in root auxin homeostasis.« less

The Arabidopsis WRINKLED1 transcription factor affects auxin homeostasis in roots

DOE PAGES

Kong, Que; Ma, Wei; Yang, Haibing; ...

2017-08-26

WRINKLED1 (WRI1) is a key transcriptional regulator of fatty acid biosynthesis genes in diverse oil-containing tissues. Loss of function of Arabidopsis WRI1 leads to a reduction in the expression of genes for fatty acid biosynthesis and glycolysis, and concomitant strong reduction of seed oil content. The wri1-1 loss-of-function mutant shows reduced primary root growth and decreased acidification of the growth medium. The content of a conjugated form of the plant growth hormone auxin, indole-3-acetic acid (IAA)-Asp, was higher in wri1-1 plants compared with the wild-type. GH3.3, a gene encoding an enzyme involved in auxin degradation, displayed higher expression in themore » wri1-1 mutant. EMSAs demonstrated that AtWRI1 bound to the promoter of GH3.3. Specific AtWRI1-binding motifs were identified in the promoter of GH3.3. In addition, wri1-1 displayed decreased auxin transport. Expression of some PIN genes, which encode IAA carrier proteins, was reduced in wri1-1 plants as well. Correspondingly, AtWRI1 bound to the promoter regions of some PIN genes. It is well known that auxin exerts its maximum effects at a specific, optimal concentration in roots requiring a finely balanced auxin homeostasis. This process appears to be disrupted when the expression of WRI1 and in turn a subset of its target genes are misregulated, highlighting a role for WRI1 in root auxin homeostasis.« less

A novel gene encoding a TIG multiple domain protein is a positional candidate for autosomal recessive polycystic kidney disease.

PubMed

Xiong, Huaqi; Chen, Yongxiong; Yi, Yajun; Tsuchiya, Karen; Moeckel, Gilbert; Cheung, Joseph; Liang, Dan; Tham, Kyi; Xu, Xiaohu; Chen, Xing-Zhen; Pei, York; Zhao, Zhizhuang Jeo; Wu, Guanqing

2002-07-01

Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-p12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. We recently identified a novel gene in this genetic interval from kidney cDNA, using cloning strategies. The gene PKHD1 (PKHD1-tentative) encodes a novel 3396-amino-acid protein with no apparent homology with any known proteins. We named its gene product "tigmin" because it contains multiple TIG domains, which usually are seen in proteins containing immunoglobulin-like folds. PKHD1 encodes an 11.6-kb transcript and is composed of 61 exons spanning an approximately 365-kb genomic region on chromosome 6p12-p11.2 adjacent to the marker D6S1714. Northern blot analyses demonstrated that the gene has discrete bands with one peak signal at approximately 11 kb, indicating that PKHD1 is likely to have multiple alternative transcripts. PKHD1 is highly expressed in adult and infant kidneys and weakly expressed in liver in northern blot analysis. This expression pattern parallels the tissue involvement observed in ARPKD. In situ hybridization analysis further revealed that the expression of PKHD1 in the kidney is mainly localized to the epithelial cells of the collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features of PKHD1 make it a strong positional candidate gene for ARPKD.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

PubMed

Reid, S D; Green, N M; Buss, J K; Lei, B; Musser, J M

2001-06-19

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Effects on intestinal microbiota and immune genes of Solea senegalensis after suspension of the administration of Shewanella putrefaciens Pdp11.

PubMed

Vidal, Sara; Tapia-Paniagua, Silvana Teresa; Moriñigo, Jesús Miguel; Lobo, Carmen; García de la Banda, Inés; Balebona, María Del Carmen; Moriñigo, Miguel Ángel

2016-11-01

The interaction host-intestinal microbiota is essential for the immunological homeostasis of the host. Probiotics, prebiotics and synbiotics are promising tools for the manipulation of the intestinal microbiota towards beneficial effects to the host. The objective of this study was to evaluate the modulation effect on the intestinal microbiota and the transcription of genes involved in the immune response in head kidney of Solea senegalensis after administration of diet supplemented with the prebiotic alginate and the probiotic Shewanella putrefaciens Pdp11 CECT 7627 (SpPdp11). The results showed higher adaptability to dietary changes in the intestinal microbiota of fish fed diet with alginate and SpPdp11 together compared to those fish that received an alginate-supplemented diet. The alginate-supplemented diet produced up-regulation of genes encoding proteins involved in immunological responses, such as complement, lysozyme G and transferrin, and oxidative stress, such as NADPH oxidase and glutation peroxidase. On the other hand, the administration of alginate combined with SpPdp11 resulted in a significant increase of the transcription of genes encoding for glutation peroxidase and HSP70, indicating a potential protective effect of SpPdp11 against oxidative stress. In addition, these effects were maintained after the suspension of the probiotic treatment. The relationship between the modulation of the intestinal microbiota and the expression of genes with protective effect against the oxidative stress was demonstrated by the Principal Components Analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mapping and Sequencing of the Canine NRAMP1 Gene and Identification of Mutations in Leishmaniasis-Susceptible Dogs

PubMed Central

Altet, Laura; Francino, Olga; Solano-Gallego, Laia; Renier, Corinne; Sánchez, Armand

2002-01-01

The NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control of intraphagosomal replication of parasites and in macrophage activation. It has been described in mice as the determinant of natural resistance or susceptibility to infection with antigenically unrelated pathogens, including Leishmania. Our aims were to sequence and map the canine Slc11a1 gene and to identify mutations that may be associated with resistance or susceptibility to Leishmania infection. The canine Slc11a1 gene has been mapped to dog chromosome CFA37 and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellite in intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1 proteins of different mammalian species. A case-control study with 33 resistant and 84 susceptible dogs showed an association between allele 145 of the microsatellite and susceptible dogs. Sequence variant analysis was performed by direct sequencing of the cDNA and the promoter region of four unrelated beagles experimentally infected with Leishmania infantum to search for possible functional mutations. Two of the dogs were classified as susceptible and the other two were classified as resistant based on their immune responses. Two important mutations were found in susceptible dogs: a G-rich region in the promoter that was common to both animals and a complete deletion of exon 11, which encodes the consensus transport motif of the protein, in the unique susceptible dog that needed an additional and prolonged treatment to avoid continuous relapses. A study with a larger dog population would be required to prove the association of these sequence variants with disease susceptibility. PMID:12010961

Viral repression of fungal pheromone precursor gene expression.

PubMed

Zhang, L; Baasiri, R A; Van Alfen, N K

1998-02-01

Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus. This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture. At the molecular level, the virus is known to affect the transcription of a number of fungal genes. Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains. We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2. To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains. The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus. Although published reports suggest that a G alpha(i) subunit may be involved in this regulation, our results do not support this hypothesis. The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe. This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete.

Compatibility of garlic (Allium sativum L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding.

PubMed

Upadhyay, Santosh Kumar; Singh, Seema; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna Kumar

2012-03-01

δ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.

DDB1-Mediated CRY1 Degradation Promotes FOXO1-Driven Gluconeogenesis in Liver.

PubMed

Tong, Xin; Zhang, Deqiang; Charney, Nicholas; Jin, Ethan; VanDommelen, Kyle; Stamper, Kenneth; Gupta, Neil; Saldate, Johnny; Yin, Lei

2017-10-01

Targeted protein degradation through ubiquitination is an important step in the regulation of glucose metabolism. Here, we present evidence that the DDB1-CUL4A ubiquitin E3 ligase functions as a novel metabolic regulator that promotes FOXO1-driven hepatic gluconeogenesis. In vivo, hepatocyte-specific Ddb1 deletion leads to impaired hepatic gluconeogenesis in the mouse liver but protects mice from high-fat diet-induced hyperglycemia. Lack of Ddb1 downregulates FOXO1 protein expression and impairs FOXO1-driven gluconeogenic response. Mechanistically, we discovered that DDB1 enhances FOXO1 protein stability via degrading the circadian protein cryptochrome 1 (CRY1), a known target of DDB1 E3 ligase. In the Cry1 depletion condition, insulin fails to reduce the nuclear FOXO1 abundance and suppress gluconeogenic gene expression. Chronic depletion of Cry1 in the mouse liver not only increases FOXO1 protein but also enhances hepatic gluconeogenesis. Thus, we have identified the DDB1-mediated CRY1 degradation as an important target of insulin action on glucose homeostasis. © 2017 by the American Diabetes Association.

Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

PubMed Central

2013-01-01

Background Hydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins. Results We have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1–40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1–11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species’ ecological strategy, with the non-pathogenic fungi having increased numbers of hydrophobins compared with their pathogenic counterparts. However, there was no significant relationship between the number of hydrophobin-encoding genes and genome size. Furthermore, our results revealed significant differences in the expression levels of the 16 H. annosum s.s. hydrophobin-encoding genes which suggest possible differences in their regulatory patterns. Conclusions A considerable expansion of the hydrophobin-encoding genes in basidiomycetes has been observed. The distribution and number of hydrophobin-encoding genes in the analyzed species may be connected to their ecological preferences. Results of our analysis also have shown that H. annosum s.l. hydrophobin-encoding genes may be under positive selection. Our gene expression analysis revealed differential expression of H. annosum s.s. hydrophobin genes under different growth conditions, indicating their possible functional diversification. PMID:24188142

Bioinformatics analysis and characteristics of VP23 encoded by the newly identified UL18 gene of duck enteritis virus

NASA Astrophysics Data System (ADS)

Chen, Xiwen; Cheng, Anchun; Wang, Mingshu; Xiang, Jun

2011-10-01

In this study, the predicted information about structures and functions of VP23 encoded by the newly identified DEV UL18 gene through bioinformatics softwares and tools. The DEV UL18 was predicted to encode a polypeptide with 322 amino acids, termed VP23, with a putative molecular mass of 35.250 kDa and a predicted isoelectric point (PI) of 8.37, no signal peptide and transmembrane domain in the polypeptide. The prediction of subcellular localization showed that the DEV-VP23 located at endoplasmic reticulum with 33.3%, mitochondrial with 22.2%, extracellular, including cell wall with 11.1%, vesicles of secretory system with 11.1%, Golgi with 11.1%, and plasma membrane with 11.1%. The acid sequence of analysis showed that the potential antigenic epitopes are situated in 45-47, 53-60, 102-105, 173-180, 185-189, 260-265, 267-271, and 292-299 amino acids. All the consequences inevitably provide some insights for further research about the DEV-VP23 and also provide a fundament for further study on the the new type clinical diagnosis of DEV and can be used for the development of new DEV vaccine.

Partial genome assembly for a candidate division OP11 single cell from an anoxic spring (Zodletone Spring, Oklahoma).

PubMed

Youssef, Noha H; Blainey, Paul C; Quake, Stephen R; Elshahed, Mostafa S

2011-11-01

Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ∼270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (β-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.

Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

PubMed

Karimi, Ashkan; Milewicz, Dianna M

2016-01-01

The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Gene-environment interactions of circadian-related genes for cardiometabolic traits

USDA-ARS?s Scientific Manuscript database

Objective: Common circadian-related gene variants associate with increased risk for metabolic alterations including type 2 diabetes. However, little is known about whether diet and sleep could modify associations between circadian-related variants (CLOCK-rs1801260, CRY2-rs11605924, MTNR1B-rs1387153,...

Chronobiology in mammalian health.

PubMed

Liu, Zhihua; Chu, Guiyan

2013-03-01

Circadian rhythms are daily cycles of physiology and behavior that are driven by an endogenous oscillator with a period of approximately one day. In mammals, the hypothalamic suprachiasmatic nuclei are our principal circadian oscillators which influences peripheral tissue clocks via endocrine, autonomic and behavioral cues, and other brain regions and most peripheral tissues contain circadian clocks as well. The circadian molecular machinery comprises a group of circadian genes, namely Clock, Bmal1, Per1, Per2, Per3, Cry1 and Cry2. These circadian genes drive endogenous oscillations which promote rhythmically expression of downstream genes and thereby physiological and behavioral processes. Disruptions in circadian homeostasis have pronounced impact on physiological functioning, overall health and disease susceptibility. This review introduces the general profile of circadian gene expression and tissue-specific circadian regulation, highlights the connection between the circadian rhythms and physiological processes, and discusses the role of circadian rhythms in human disease.

Neurobiology of culturally common maternal responses to infant cry

PubMed Central

Bornstein, Marc H.; Rigo, Paola; Esposito, Gianluca; Swain, James E.; Suwalsky, Joan T. D.; Su, Xueyun; Du, Xiaoxia; Zhang, Kaihua; Cote, Linda R.; De Pisapia, Nicola; Venuti, Paola

2017-01-01

This report coordinates assessments of five types of behavioral responses in new mothers to their own infants’ cries with neurobiological responses in new mothers to their own infants’ cries and in experienced mothers and inexperienced nonmothers to infant cries and other emotional and control sounds. We found that 684 new primipara mothers in 11 countries (Argentina, Belgium, Brazil, Cameroon, France, Kenya, Israel, Italy, Japan, South Korea, and the United States) preferentially responded to their infants’ vocalizing distress by picking up and holding and by talking to their infants, as opposed to displaying affection, distracting, or nurturing. Complementary functional magnetic resonance imaging (fMRI) analyses of brain responses to their own infants’ cries in 43 new primipara US mothers revealed enhanced activity in concordant brain territories linked to the intention to move and to speak, to process auditory stimulation, and to caregive [supplementary motor area (SMA), inferior frontal regions, superior temporal regions, midbrain, and striatum]. Further, fMRI brain responses to infant cries in 50 Chinese and Italian mothers replicated, extended, and, through parcellation, refined the results. Brains of inexperienced nonmothers activated differently. Culturally common responses to own infant cry coupled with corresponding fMRI findings to own infant and to generic infant cries identified specific, common, and automatic caregiving reactions in mothers to infant vocal expressions of distress and point to their putative neurobiological bases. Candidate behaviors embedded in the nervous systems of human caregivers lie at the intersection of evolutionary biology and developmental cultural psychology. PMID:29078366

Herbicide and insect resistant Bt cotton pollen assessment finds no detrimental effects on adult honey bees.

PubMed

Niu, Lin; Ma, Weihua; Lei, Chaoliang; Jurat-Fuentes, Juan Luis; Chen, Lizhen

2017-11-01

One important concern regarding the use of transgenic cotton expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) is its potential detrimental effect on non-target organisms. The honey bee (Apis mellifera) is the most important pollinator species worldwide and it is directly exposed to transgenic crops by the consumption of genetically modified (GM) pollen. However, the potential effects of Bt cotton on A. mellifera remain unclear. In the present study, we assessed the effects of two Bt cotton varieties; ZMSJ expressing the Cry1Ac and Cry2Ab insecticidal proteins, and ZMKCKC producing Cry1Ac and EPSPS, on A. mellifera. Feeding on pollen from two Bt cotton varieties led to detection of low levels of Cry toxins (<10 ng/g fresh weight) in the midgut of A. mellifera adults, yet expression of detoxification genes did not change significantly compared to feeding on non-Bt cotton. Binding assays showed no Cry1Ac or Cry2Ab binding to midgut brush border membrane proteins from A. mellifera adults. Taken together, these results support minimal risk for potential negative effects on A. mellifera by exposure to Bt cotton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Two Functional Copies of the DGCR6 Gene Are Present on Human Chromosome 22q11 Due to a Duplication of an Ancestral Locus

PubMed Central

Edelmann, Lisa; Stankiewicz, Pavel; Spiteri, Elizabeth; Pandita, Raj K.; Shaffer, Lisa; Lupski, James; Morrow, Bernice E.

2001-01-01

The DGCR6 (DiGeorge critical region) gene encodes a putative protein with sequence similarity to gonadal (gdl), a Drosophila melanogaster gene of unknown function. We mapped the DGCR6 gene to chromosome 22q11 within a low copy repeat, termed sc11.1a, and identified a second copy of the gene, DGCR6L, within the duplicate locus, termed sc11.1b. Both sc11.1 repeats are deleted in most persons with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), and they map immediately adjacent and internal to the low copy repeats, termed LCR22, that mediate the deletions associated with VCFS/DGS. We sequenced genomic clones from both loci and determined that the putative initiator methionine is located further upstream than originally described, but in a position similar to the mouse and chicken orthologs. DGCR6L encodes a highly homologous, functional copy of DGCR6, with some base changes rendering amino acid differences. Expression studies of the two genes indicate that both genes are widely expressed in fetal and adult tissues. Evolutionary studies using FISH mapping in several different species of ape combined with sequence analysis of DGCR6 in a number of different primate species indicate that the duplication is at least 12 million years old and may date back to before the divergence of Catarrhines from Platyrrhines, 35 mya. These data suggest that there has been selective evolutionary pressure toward the functional maintenance of both paralogs. Interestingly, a full-length HERV-K provirus integrated into the sc11.1a locus after the divergence of chimpanzees and humans. PMID:11157784

Emergency Department Triage of the "Incessantly Crying" Baby.

PubMed

Chua, Caroline; Setlik, Jennifer; Niklas, Victoria

2016-11-01

Incessant crying is one of the most common caregiver complaints during emergency department (ED) visits in the first few months of the child's life. Although the majority of cases are attributed to normal infant behavior, the differential diagnosis remains broad. Moreover, the potential for the negative impact of incessant crying on the mental well-being of caregivers as well as the infants necessitates that complaints be taken seriously and that "red flags" for underlying organic causes be ruled out and caregiver anxiety quelled. In addition, the apparent triviality of incessant crying in the face of the life-threatening illnesses or injuries that confront practitioners in the ED necessitates a high level of due diligence in the evaluation of these infants and their families. Ensuring the availability of family support is essential in the discharge planning. Families should also perceive the empathy of the physician and feel reassured about their safe discharge home. Although it is a challenge to examine an incessantly crying infant in all care settings, the failure to recognize the small percentage of infants that present with incessant crying as a manifestation of an underlying organic illness may have grave consequences. [Pediatr Ann. 2016;45(11):e394-e398.]. Copyright 2016, SLACK Incorporated.

Estimating Foodborne Gastroenteritis, Australia

PubMed Central

Kirk, Martyn D.; Becker, Niels; Gregory, Joy E.; Unicomb, Leanne; Millard, Geoffrey; Stafford, Russell; Lalor, Karin

2005-01-01

We estimated for Australia the number of cases, hospitalizations, and deaths due to foodborne gastroenteritis in a typical year, circa 2000. The total amount of infectious gastroenteritis was measured by using a national telephone survey. The foodborne proportion was estimated from Australian data on each of 16 pathogens. To account for uncertainty, we used simulation techniques to calculate 95% credibility intervals (CrI). The estimate of incidence of gastroenteritis in Australia is 17.2 million (95% confidence interval 14.5–19.9 million) cases per year. We estimate that 32% (95% CrI 24%–40%) are foodborne, which equals 0.3 (95% CrI 0.2–0.4) episodes per person, or 5.4 million (95% CrI 4.0–6.9 million) cases annually in Australia. Norovirus, enteropathogenic Escherichia coli, Campylobacter spp., and Salmonella spp. cause the most illnesses. In addition, foodborne gastroenteritis causes ≈15,000 (95% CrI 11,000–18,000) hospitalizations and 80 (95% CrI 40–120) deaths annually. This study highlights global public health concerns about foodborne diseases and the need for standardized methods, including assessment of uncertainty, for international comparison. PMID:16102316

Defective canalicular transport and toxicity of dietary ursodeoxycholic acid in the abcb11-/- mouse: transport and gene expression studies.

PubMed

Wang, Renxue; Liu, Lin; Sheps, Jonathan A; Forrest, Dana; Hofmann, Alan F; Hagey, Lee R; Ling, Victor

2013-08-15

The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

PubMed

Masson, L; Moar, W J; van Frankenhuyzen, K; Bossé, M; Brousseau, R

1992-02-01

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNAmore » is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.« less

GmHs1-1, encoding a calcineurin-like protein, controls hard-seededness in soybean

USDA-ARS?s Scientific Manuscript database

Loss of seed-coat impermeability was an essential step towards domestication of many leguminous crops for production of their highly nutritious seeds. Here we show that seed-coat impermeability in wild soybean is controlled by a single gene, Hard seededness 1 (Hs1), which encodes a calcineurin-like ...

Effects of 90-day feeding of transgenic Bt rice TT51 on the reproductive system in male rats.

PubMed

Wang, Er Hui; Yu, Zhou; Hu, Jing; Xu, Hai Bin

2013-12-01

Rice is a staple food crop; however, the threat of pests leads to a serious decline in its output and quality. The CryAb/CryAc gene, encodes a synthetic fusion Bacillus thuringiensis (Bt) crystal protein, was introduced into rice MingHui63 to produce insect-resistant rice TT51. This study was undertaken to investigate potential unintended effects of TT51 on the reproductive system in male rats. Male rats were treated with diets containing 60% of either TT51 or MingHui63 by weight, nutritionally balanced to an AIN93G diet, for 90days. An additional negative control group of rats were fed with a rice-based AIN93G diet. Body weights, food intake, hematology, serum chemistry, serum hormone levels, sperm parameters and relative organ/body weights were measured, and gross as well as microscopic pathology were examined. No diet-related significant differences in the values of response variables were observed between rats that were fed with diet containing transgenic TT51, MingHui63 and the control in this 90-day feeding study. In addition, necropsy and histopathology examination indicated no treatment-related changes. The results from the present study indicated that TT51 does not appear to exert any effect on the reproductive system in male rats compared with MingHui63 or the control. Copyright © 2013 Elsevier Ltd. All rights reserved.

Correlated expression of gfp and Bt cry1Ac gene facilitates quantification of transgenic hybridization between Brassicas.

PubMed

Shen, B-C; Stewart, C N; Zhang, M-Q; Le, Y-T; Tang, Z-X; Mi, X-C; Wei, W; Ma, K-P

2006-09-01

Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.

New alleles of mgm1: a gene encoding a protein with a GTP-binding domain related to dynamin.

PubMed

Backer, J S

1995-10-01

Three previously described genes that affect baker's yeast (Saccharomyces cerevisiae) mitochondrial DNA (mtDNA) or mitochondrial RNA, tpm2-1, mna1-1, and mgm-1-1, are shown to be alleles of the same gene. This report demonstrates that tpm2-1 does not affect recombination of mtDNA. Therefore, there is no evidence that this dynamin-like protein is involved in movement of mtDNA within a cell.

Gene-environment interactions of circadian-related genes for cardiometabolic traits

USDA-ARS?s Scientific Manuscript database

Common circadian-related gene variants associate with increased risk for metabolic alterations including type 2 diabetes. However, little is known about whether diet and sleep could modify associations between circadian-related variants (CLOCK-rs1801260, CRY2-rs11605924, MTNR1B-rs1387153, MTNR1B-rs1...

Toxin stability improvement and toxicity increase against dipteran and lepidopteran larvae of Bacillus thuringiensis crystal protein Cry2Aa.

PubMed

Elleuch, Jihen; Jaoua, Samir; Ginibre, Carole; Chandre, Fabrice; Tounsi, Slim; Zghal, Raida Z

2016-12-01

Bacillus thuringiensis δ-endotoxins are the most widely used biopesticides for controlling economically important crop pests and disease vectors. Improving their efficacy is of great benefit. Here, an improvement in Cry2Aa δ-endotoxin toxicity was attempted via a cry gene over expression system using P20 from B. thuringiensis israelensis. The coexpression of Cry2Aa with P20 resulted in a seven fold increase in its production yield in B. thuringiensis. Generated crystals proved to be significantly more toxic (505.207 µg g -1 , 1.99 mg L -1 and 1.49 mg L -1 ) than the P20-lacking control (720.78 µg g -1 , 705.69 mg L -1 and 508.51 mg L -1 ) against Ephestia kuehniella, Aedes aegypti and Culex pipiens larvae respectively. In vitro, processing experiments revealed a P20-mediated protection of Cry2Aa against degradation under larval gut conditions. Thus, P20 could promote the maintenance of a tightly packaged conformation of Cry2Aa toxins in the larval midgut upon correct activation and binding to its membrane receptors. Based on their resistance against excessive proteolysis, Cry2Aa δ-endotoxins, produced in the presence of P20, could be considered as a successful control agent for E. kuehniella and an effective alternative for mosquito control, implying its possible exploitation in pest management programmes. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Bacillus thuringiensis Cry1Ia10 and Vip3Aa protein interactions and their toxicity in Spodoptera spp. (Lepidoptera).

PubMed

Bergamasco, V B; Mendes, D R P; Fernandes, O A; Desidério, J A; Lemos, M V F

2013-02-01

The polyphagous pests belonging to the genus Spodoptera are considered to be among the most important causes of damage and are widely distributed throughout the Americas'. Due to the extensive use of genetically modified plants containing Bacillus thuringiensis genes that code for insecticidal proteins, resistant insects may arise. To prevent the development of resistance, pyramided plants, which express multiple insecticidal proteins that act through distinct mode of actions, can be used. This study analyzed the mechanisms of action for the proteins Cry1Ia10 and Vip3Aa on neonatal Spodoptera frugiperda, Spodoptera albula, Spodoptera eridania and Spodoptera cosmioides larvae. The interactions of these toxins with receptors on the intestinal epithelial membrane were also analyzed by binding biotinylated toxins to brush border membrane vesicles (BBMVs) from the intestines of these insects. A putative receptor of approximately 65 kDa was found by ligand blotting in all of these species. In vitro competition assays using biotinylated proteins have indicated that Vip3Aa and Cry1Ia10 do not compete for the same receptor for S. frugiperda, S. albula and S. cosmioides and that Vip3Aa was more efficient than Cry1Ia10 when tested individually, by bioassays. A synergistic effect of the toxins in S. frugiperda, S. albula and S. cosmioides was observed when they were combined. However, in S. eridania, Cry1Ia10 and Vip3Aa might compete for the same receptor and through bioassays Cry1Ia10 was more efficient than Vip3Aa and showed an antagonistic effect when the proteins were combined. These results suggest that using these genes to develop pyramided plants may not prove effective in preventing the development of resistance in S. eridiana. Copyright © 2012 Elsevier Inc. All rights reserved.

Trojan Horse Strategy for Non-invasive Interference of Clock Gene in the Oyster Crassostrea gigas.

PubMed

Payton, Laura; Perrigault, Mickael; Bourdineaud, Jean-Paul; Marcel, Anjara; Massabuau, Jean-Charles; Tran, Damien

2017-08-01

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

75 FR 51043 - Pesticide Experimental Use Permit; Receipt of Application; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-18

... (vector pCOT1) in event COT102 cotton, Bt Cry1Ac protein and the genetic material necessary for its production (vector PV-GHBK04) in event MON 15985 cotton, and Bt Cry2Ab2 protein and the genetic material necessary for its production (vector PV-GHBK11) in event MON 15985 cotton. The Agency has determined that...

Photoperiod regulates multiple gene expression in the suprachiasmatic nuclei and pars tuberalis of the Siberian hamster (Phodopus sungorus).

PubMed

Johnston, Jonathan D; Ebling, Francis J P; Hazlerigg, David G

2005-06-01

Photoperiod regulates the seasonal physiology of many mammals living in temperate latitudes. Photoperiodic information is decoded by the master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus and then transduced via pineal melatonin secretion. This neurochemical signal is interpreted by tissues expressing melatonin receptors (e.g. the pituitary pars tuberalis, PT) to drive physiological changes. In this study we analysed the photoperiodic regulation of the circadian clockwork in the SCN and PT of the Siberian hamster. Female hamsters were exposed to either long or short photoperiod for 8 weeks and sampled at 2-h intervals across the 24-h cycle. In the SCN, rhythmic expression of the clock genes Per1, Per2, Cry1, Rev-erbalpha, and the clock-controlled genes arginine vasopressin (AVP) and d-element binding protein (DBP) was modulated by photoperiod. All of these E-box-containing genes tracked dawn, with earlier peak mRNA expression in long, compared to short, photoperiod. This response occurred irrespective of the presence of additional regulatory cis-elements, suggesting photoperiodic regulation of SCN gene expression through a common E-box-related mechanism. In long photoperiod, expression of Cry1 and Per1 in the PT tracked the onset and offset of melatonin secretion, respectively. However, whereas Cry1 tracked melatonin onset in short period, Per1 expression was not detectably rhythmic. We therefore propose that, in the SCN, photoperiodic regulation of clock gene expression primarily occurs via E-boxes, whereas melatonin-driven signal transduction drives the phasing of a subset of clock genes in the PT, independently of the E-box.

Acute Sleep Loss Induces Tissue-Specific Epigenetic and Transcriptional Alterations to Circadian Clock Genes in Men.

PubMed

Cedernaes, Jonathan; Osler, Megan E; Voisin, Sarah; Broman, Jan-Erik; Vogel, Heike; Dickson, Suzanne L; Zierath, Juleen R; Schiöth, Helgi B; Benedict, Christian

2015-09-01

Shift workers are at increased risk of metabolic morbidities. Clock genes are known to regulate metabolic processes in peripheral tissues, eg, glucose oxidation. This study aimed to investigate how clock genes are affected at the epigenetic and transcriptional level in peripheral human tissues following acute total sleep deprivation (TSD), mimicking shift work with extended wakefulness. In a randomized, two-period, two-condition, crossover clinical study, 15 healthy men underwent two experimental sessions: x sleep (2230-0700 h) and overnight wakefulness. On the subsequent morning, serum cortisol was measured, followed by skeletal muscle and subcutaneous adipose tissue biopsies for DNA methylation and gene expression analyses of core clock genes (BMAL1, CLOCK, CRY1, PER1). Finally, baseline and 2-h post-oral glucose load plasma glucose concentrations were determined. In adipose tissue, acute sleep deprivation vs sleep increased methylation in the promoter of CRY1 (+4%; P = .026) and in two promoter-interacting enhancer regions of PER1 (+15%; P = .036; +9%; P = .026). In skeletal muscle, TSD vs sleep decreased gene expression of BMAL1 (-18%; P = .033) and CRY1 (-22%; P = .047). Concentrations of serum cortisol, which can reset peripheral tissue clocks, were decreased (2449 ± 932 vs 3178 ± 723 nmol/L; P = .039), whereas postprandial plasma glucose concentrations were elevated after TSD (7.77 ± 1.63 vs 6.59 ± 1.32 mmol/L; P = .011). Our findings demonstrate that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in key metabolic tissues. Tissue-specific clock alterations could explain why shift work may disrupt metabolic integrity as observed herein.

Cloning and characterization of a novel zinc finger gene in Xp11.2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Derry, J.M.J.; Jess, U.; Francke, U.

1995-11-20

During a systematic search for open reading frames in chromosome band Xp11.2, a novel gene (ZNF157) that encodes a putative 506-amino-acid protein with the sequence characteristics of a zinc-finger-containing transcription factor was isolated. ZNF157 is encoded by four exons distributed over >20 kb of genomic DNA. The second and third exons contain sequences similar to those of the previously described KRAB-A and KRAB-B domains, motifs that have been shown to mediate transcriptional repression in other members of the protein family. A fourth exon contains 12 zinc finger DNA binding motifs and finger linking regions characteristic of ZNF proteins of themore » Krueppel family. ZNF157 maps to the telomeric end of a cluster of ZNF genes that includes ZNF21, ZNF41, and ZNF81. 19 refs., 2 figs.« less

Agronomic performance of insect-protected and herbicide-tolerant MON 89034 × TC1507 × NK603 × DAS-40278-9 corn is equivalent to that of conventional corn.

PubMed

de Cerqueira, Denise T Rezende; Schafer, Ariane C; Fast, Brandon J; Herman, Rod A

2017-07-03

Agronomic characteristics of genetically modified (GM) MON 89034 × TC1507 × NK603 × DAS-40278-9 (PowerCore™ Enlist™), MON 89034 × TC1507 × NK603 (PowerCore™), and DAS-40278-9 (Enlist™) corn, a non-GM near-isogenic hybrid, and 2 commercial non-GM hybrids were assessed in a field study to determine if the agronomic performance of the GM corn hybrids is equivalent to that of non-transgenic hybrid corn. The MON 89034 × TC1507 × NK603 × DAS-40278-9 hybrid corn was developed through stacking of 4 individual transgenic events, MON 89034, TC1507, NK603, and DAS-40278-9 by traditional breeding and contains the cry1A.105 and cry2Ab2 (MON 89034), cry1F and pat (TC1507), cp4 epsps (NK603) and aad-1 (DAS-40278-9) transgenes. These transgenes encode the proteins Cry1A.105, Cry2Ab2, and Cry1F, which confer insect resistance, PAT, CP4 EPSPS, and AAD-1, which confer herbicide tolerance. The following agronomic characteristics were assessed in the study: initial and final stand count, seedling vigor, time to silk, time to pollen shed, pollen viability, plant height, ear height, stalk lodging, root lodging, days to maturity, stay green, disease incidence, insect damage, herbicide injury, and yield. The agronomic assessment was conducted in 2 regions of Brazil (Indianopolis-MG; Cravinhos-SP). The agronomic attributes for all GM entries were statistically indistinguishable from the non-GM near-isogenic hybrid. In addition, most of the agronomic assessments fell within the range of the commercial varieties included in the study. Taken together, MON 89034 × TC1507 × NK603 × DAS-40278, MON 89034 × TC1507 × NK603, and DAS-40278-9 were found to be agronomically equivalent to non-GM corn.

Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

PubMed

Chen, Wen-Bo; Lu, Guo-Qing; Cheng, Hong-Mei; Liu, Chen-Xi; Xiao, Yu-Tao; Xu, Chao; Shen, Zhi-Cheng; Wu, Kong-Ming

2017-10-01

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China. Copyright © 2017 Elsevier Inc. All rights reserved.

Vaccinia-related kinase 3 (VRK3) sets the circadian period and amplitude by affecting the subcellular localization of clock proteins in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Nayoung; Department of Brain Science, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon, Kyunggi-do, 16499; Song, Jieun

In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in themore » subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells. - Highlights: • Screening was performed to identify kinases that regulate CRY1 subcellular localization. • VRK3 alters the subcellular localization of CRY1, CRY2, PER2, and BMAL1. • VRK3 knock-down alters the circadian bioluminescence rhythm in mammalian cells.« less

Identification of a fourth locus (EVR4) for familial exudative vitreoretinopathy (FEVR).

PubMed

Toomes, Carmel; Downey, Louise M; Bottomley, Helen M; Scott, Sheila; Woodruff, Geoffrey; Trembath, Richard C; Inglehearn, Chris F

2004-01-15

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous inherited blinding disorder of the retinal vascular system. To date three loci have been mapped: EVR1 on chromosome 11q, EVR2 on chromosome Xp, and EVR3 on chromosome 11p. The gene underlying EVR3 remains unidentified whilst the EVR2 gene, which encodes the Norrie disease protein (NDP), was identified over a decade ago. More recently, FZD4, the gene that encodes the Wnt receptor Frizzled-4, was identified as the mutated gene at the EVR1 locus. The purpose of this study was to screen FZD4 in a large family previously proven to be linked to the EVR1 locus. PCR products were generated using genomic DNA from affected family members with primers designed to amplify the coding sequence of FZD4. The PCR products were screened for mutations by direct sequencing. Genotyping was performed in all available family members using fluorescently labeled microsatellite markers from chromosome 11q. Sequencing of the EVR1 gene, FZD4, in this family identified no mutation. To investigate this family further we performed high-resolution genotyping with markers spanning chromosome 11q. Haplotype analysis excluded FZD4 as the mutated gene in this family and identified a candidate region approximately 10 cM centromeric to EVR1. This new FEVR locus is flanked by markers D11S1368 (centromeric) and D11S937 (telomeric) and spans approximately 15 cM. High-resolution genotyping and haplotype analysis excluded FZD4 as the defective gene in a family previously linked to the EVR1 locus. The results indicate that the gene mutated in this family lies centromeric to the EVR1 gene, FZD4, and is also genetically distinct from the EVR3 locus. This new locus has been designated EVR4 and is the fourth FEVR locus to be described.

Biallelic missense variants in ZBTB11 can cause intellectual disability in human.

PubMed

Fattahi, Zohreh; Sheikh, Taimoor I; Musante, Luciana; Rasheed, Memoona; Taskiran, Ibrahim Ihsan; Harripaul, Ricardo; Hu, Hao; Kazeminasab, Somayeh; Alam, Muhammad Rizwan; Hosseini, Masoumeh; Larti, Farzaneh; Ghaderi, Zhila; Celik, Arzu; Ayub, Muhammad; Ansar, Muhammad; Haddadi, Mohammad; Wienker, Thomas F; Ropers, Hans Hilger; Kahrizi, Kimia; Vincent, John B; Najmabadi, H

2018-06-08

Exploring genes and pathways underlying Intellectual Disability (ID) provides insight into brain development and function, clarifying the complex puzzle of how cognition develops. As part of ongoing systematic studies to identify candidate ID genes, linkage analysis and next generation sequencing revealed ZBTB11, as a novel candidate ID gene. ZBTB11 encodes a less-studied transcription regulator and the two identified missense variants in this study may disrupt canonical Zn2+-binding residues of its C2H2 zinc finger domain, leading to possible altered DNA binding. Using HEK293T cells transfected with wild type and mutant GFP-ZBTB11 constructs, we found the ZBTB11 mutants being excluded from the nucleolus, where the wild-type recombinant protein is predominantly localized. Pathway analysis applied to ChIP-seq data deposited in the ENCODE database supports the localization of ZBTB11 in nucleoli, highlighting associated pathways such as rRNA synthesis, ribosomal assembly, RNA modification, stress sensing and provides a direct link between subcellular ZBTB11 location and its function. Furthermore, considering the report of prominent brain and spinal cord degeneration in a zebrafish Zbtb11 mutant, we investigated ZBTB11-ortholog knockdown in Drosophila melanogaster brain by targeting RNAi using the UAS/Gal4 system. The observed approximate reduction to a third of the mushroom body size - possibly through neuronal reduction or degeneration - may affect neuronal circuits in the brain that are required for adaptive behavior, specifying the role of this gene in nervous system. In conclusion, we report two ID families segregating ZBTB11 biallelic mutations disrupting Zn2+-binding motifs, and provide functional evidence linking ZBTB11 dysfunction to this phenotype.

Role of monochromatic light on daily variation of clock gene expression in the pineal gland of chick.

PubMed

Jiang, Nan; Wang, Zixu; Cao, Jing; Dong, Yulan; Chen, Yaoxing

2016-11-01

The avian pineal gland is a master clock that can receive external photic cues and translate them into output rhythms. To clarify whether a shift in light wavelength can influence the circadian expression in chick pineal gland, a total of 240 Arbor Acre male broilers were exposed to white light (WL), red light (RL), green light (GL) or blue light (BL). After 2weeks light illumination, circadian expressions of seven core clock genes in pineal gland and the level of melatonin in plasma were examined. The results showed after illumination with monochromatic light, 24h profiles of all clock gene mRNAs retained circadian oscillation, except that RL tended to disrupt the rhythm of cCry2. Compared to WL, BL advanced the acrophases of the negative elements (cCry1, cCry2, cPer2 and cPer3) by 0.1-1.5h and delayed those of positive elements (cClock, cBmal1 and cBmal2) by 0.2-0.8h. And, RL advanced all clock genes except cClock and cPer2 by 0.3-2.1h, while GL delayed all clock genes by 0.5-1.5h except cBmal2. Meanwhile, GL increased the amplitude and mesor of positive and reduced both parameters of negative clock genes, but RL showed the opposite pattern. Although the acrophase of plasma melatonin was advanced by both GL and RL, the melatonin level was significantly increased in GL and decreased in RL. This tendency was consistent with the variations in the positive clock gene mRNA levels under monochromatic light and contrasted with those of negative clock genes. Therefore, we speculate that GL may enhance positive clock genes expression, leading to melatonin synthesis, whereas RL may enhance negative genes expression, suppressing melatonin synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Viral Repression of Fungal Pheromone Precursor Gene Expression

PubMed Central

Zhang, Lei; Baasiri, Rudeina A.; Van Alfen, Neal K.

1998-01-01

Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus. This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture. At the molecular level, the virus is known to affect the transcription of a number of fungal genes. Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains. We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2. To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains. The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus. Although published reports suggest that a Gαi subunit may be involved in this regulation, our results do not support this hypothesis. The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe. This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete. PMID:9447992

Light-Responsive Cryptochromes from a Simple Multicellular Animal, the Coral Acropora millepora

NASA Astrophysics Data System (ADS)

Levy, O.; Appelbaum, L.; Leggat, W.; Gothlif, Y.; Hayward, D. C.; Miller, D. J.; Hoegh-Guldberg, O.

2007-10-01

Hundreds of species of reef-building corals spawn synchronously over a few nights each year, and moonlight regulates this spawning event. However, the molecular elements underpinning the detection of moonlight remain unknown. Here we report the presence of an ancient family of blue-light-sensing photoreceptors, cryptochromes, in the reef-building coral Acropora millepora. In addition to being cryptochrome genes from one of the earliest-diverging eumetazoan phyla, cry1 and cry2 were expressed preferentially in light. Consistent with potential roles in the synchronization of fundamentally important behaviors such as mass spawning, cry2 expression increased on full moon nights versus new moon nights. Our results demonstrate phylogenetically broad roles of these ancient circadian clock-related molecules in the animal kingdom.

Identification of Loci and Functional Characterization of Trichothecene Biosynthesis Genes in Filamentous Fungi of the Genus Trichoderma▿†

PubMed Central

Cardoza, R. E.; Malmierca, M. G.; Hermosa, M. R.; Alexander, N. J.; McCormick, S. P.; Proctor, R. H.; Tijerino, A. M.; Rumbero, A.; Monte, E.; Gutiérrez, S.

2011-01-01

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function. PMID:21642405

Geographical and Temporal Variability in Susceptibility to Cry1F Toxin From Bacillus thuringiensis in Spodoptera frugiperda (Lepidoptera: Noctuidae) Populations in Brazil.

PubMed

Farias, Juliano R; Horikoshi, Renato J; Santos, Antonio C; Omoto, Celso

2014-12-01

The genetically modified maize TC1507 event with the cry1F gene (Cry1F maize) has been used to control Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Brazil since the 2009-2010 cropping season. As part of the insect resistance management program, we conducted studies to determine the baseline susceptibility to Cry1F before the widespread planting of Cry1F maize. Subsequently, we evaluated the geographical and temporal variability of susceptibility to this toxin in populations of S. frugiperda collected from major maize-growing regions in Brazil. The baseline susceptibility to Cry1F was determined using a diet-overlay bioassay for a susceptible reference population and four field populations of S. frugiperda. We then monitored the susceptibility to Cry1F in 43 populations of S. frugiperda sampled in nine States of Brazil between 2011 and 2013. In the baseline study, the MIC50 (the concentration that inhibits molting to second instars in 50% of individuals) ranged from 3.59 to 72.47 ng Cry1F toxin per centimeter square. Based on the upper limit of the MIC99 value of the joint analysis from the baseline susceptibility data, the concentrations of 200 and 2,000 ng of Cry1F toxin per centimeter square were defined as diagnostic concentrations for potentially resistant individuals, and these were used to monitor the susceptibility of S. frugiperda to Cry1F. Survival at 2,000 ng Cry1F toxin per centimeter square increased significantly throughout the cropping seasons in S. frugiperda populations from São Paulo, Santa Catarina, Rio Grande do Sul, Bahia, Mato Grosso, Goiás, Mato Grosso do Sul, and Paraná. The highest survival (>50%) was reached in populations collected from Bahia, Mato Grosso, Goiás, Mato Grosso do Sul, and Paraná during the 2012-2013 cropping season. Therefore, a significant decrease in susceptibility to Cry1F was detected in S. frugiperda throughout cropping seasons, especially in regions with intensive maize production in Brazil, according to field-evolved resistance that has been demonstrated in Brazilian populations. © 2014 Entomological Society of America.

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

PubMed Central

Marshall, Kathryn E.; Robinson, Errol W.; Hengel, Shawna M.; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation. PMID:22470473

FRET imaging of diatoms expressing a biosilica-localized ribose sensor.

PubMed

Marshall, Kathryn E; Robinson, Errol W; Hengel, Shawna M; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.

Serotonin Transporter Gene Polymorphisms and Early Parent-Infant Interactions Are Related to Adult Male Heart Rate Response to Female Crying

PubMed Central

Truzzi, Anna; Bornstein, Marc H.; Senese, Vincenzo P.; Shinohara, Kazuyuki; Setoh, Peipei; Esposito, Gianluca

2017-01-01

Adults' adaptive interactions with intimate partners enhance well-being. Here we hypothesized that adult males' physiological responses to opposite-sex conspecifics' distress result from an interaction between an environmental factor (early social interaction with caregivers) and a genetic factor (a polymorphism within the promoter region of the serotonin transporter gene, 5-HTTLPR). We assessed heart rate changes in 42 non-married male adults to distress vocalizations (female, infant, and bonobo cries). Males' early interaction with parents was assessed using the Parental Bonding Instrument. Buccal mucosa cell samples were collected to assess their 5-HTTLPR genotype. A significant interaction emerged between early experience and genetic predisposition. Males with a genetic predisposition for higher sensitivity to environmental factors showed atypical physiological responses to adult female cries according to their experienced early maternal parenting. Environmental experiences and genetic characteristics are associated with adult males' physiological responses to socially meaningfully stimuli. Understanding the mechanisms that modulate responses to opposite-sex conspecifics may improve personal well-being and social adaptiveness. PMID:28293197

The characterization of electroplex generated from the interface between 2-(4-trifluoromethyl-2-hydroxyphenyl)benzothiazole] zinc and N,N'-diphenyl-N,N'- bis(1-naphthyl)-(1,1'-biphenyl)-4,4'-diamine

NASA Astrophysics Data System (ADS)

Zhang, Ye; Hao, Yuying; Meng, Weixin; Xu, Huixia; Wang, Hua; Xu, Bingshe

2012-03-01

The electroplex between (2-(4-trifluoromethyl-2-hydroxyphenyl)benzothiazole) zinc [Zn(4-TfmBTZ)2] as an electron-acceptor and N,N'-diphenyl-N,N'-bis(1-naphthyl)-(1,1'-biphenyl)-4,4'-diamine (NPB) as an electron-donor was characterized by bilayer, blend, and multilayer quantum-well (MQW) device, respectively. The blend composition and quantum-well number are effective parameters for tuning electroluminescence color. White light with high color purity and color rendering index (CRI) was observed from these devices based on Zn(4-TfmBTZ)2/NPB. Moreover, the blend and MQW devices all exhibit high operation stability, hence excellent color stability. For the device with 5 mol% NPB in blend layer, its Commission International Del'Eclairage (CIE) coordinate region is x=0.28-0.31, y=0.33-0.35 and CRI is 83.3-91.2 at 5-9 V. For MQW structure device with NPB of 60 nm thickness, its CIE coordinate region is x=0.29-0.32, y=0.31-0.34 and CRI=87.9-92.5 at 10-15 V. Such high color stability and purity and CRI, being close to ideal white light, are of current important for white OLED.

Cri du Chat syndrome

PubMed Central

Cerruti Mainardi, Paola

2006-01-01

The Cri du Chat syndrome (CdCS) is a genetic disease resulting from a deletion of variable size occurring on the short arm of chromosome 5 (5p-). The incidence ranges from 1:15,000 to 1:50,000 live-born infants. The main clinical features are a high-pitched monochromatic cry, microcephaly, broad nasal bridge, epicanthal folds, micrognathia, abnormal dermatoglyphics, and severe psychomotor and mental retardation. Malformations, although not very frequent, may be present: cardiac, neurological and renal abnormalities, preauricular tags, syndactyly, hypospadias, and cryptorchidism. Molecular cytogenetic analysis has allowed a cytogenetic and phenotypic map of 5p to be defined, even if results from the studies reported up to now are not completely in agreement. Genotype-phenotype correlation studies showed a clinical and cytogenetic variability. The identification of phenotypic subsets associated with a specific size and type of deletion is of diagnostic and prognostic relevance. Specific growth and psychomotor development charts have been established. Two genes, Semaphorin F (SEMAF) and δ-catenin (CTNND2), which have been mapped to the "critical regions", are potentially involved in cerebral development and their deletion may be associated with mental retardation in CdCS patients. Deletion of the telomerase reverse transcriptase (hTERT) gene, localised to 5p15.33, could contribute to the phenotypic changes in CdCS. The critical regions were recently refined by using array comparative genomic hybridisation. The cat-like cry critical region was further narrowed using quantitative polymerase chain reaction (PCR) and three candidate genes were characterised in this region. The diagnosis is based on typical clinical manifestations. Karyotype analysis and, in doubtful cases, FISH analysis will confirm the diagnosis. There is no specific therapy for CdCS but early rehabilitative and educational interventions improve the prognosis and considerable progress has been made in the social adjustment of CdCS patients. PMID:16953888

A male-specific odorant receptor conserved through the evolution of sex pheromones in Ostrinia moth species

PubMed Central

Miura, Nami; Nakagawa, Tatsuro; Tatsuki, Sadahiro; Touhara, Kazushige; Ishikawa, Yukio

2009-01-01

In many moths, mate-finding communication is mediated by the female sex pheromones. Since differentiation of sex pheromones is often associated with speciation, it is intriguing to know how the changes in female sex pheromone have been tracked by the pheromone recognition system of the males. A male-specific odorant receptor was found to have been conserved through the evolution of sex pheromone communication systems in the genus Ostrinia (Lepidoptera: Crambidae). In an effort to characterize pheromone receptors of O. scapulalis, which uses a mixture of (E)-11- and (Z)-11-tetradecenyl acetates as a sex pheromone, we cloned a gene (OscaOR1) encoding a male-specific odorant receptor. In addition, we cloned a gene of the Or83b family (OscaOR2). Functional assays using Xenopus oocytes co-expressing OscaOR1 and OscaOR2 have shown that OscaOR1 is, unexpectedly, a receptor of (E)-11-tetradecenol (E11-14:OH), a single pheromone component of a congener O. latipennis. Subsequent studies on O. latipennis showed that this species indeed has a gene orthologous to OscaOR1 (OlatOR1), a functional assay of which confirmed it to be a gene encoding the receptor of E11-14:OH. Furthermore, investigations of six other Ostrinia species have revealed that all of them have a gene orthologous to OscaOR1, although none of these species, except O. ovalipennis, a species most closely related to O. latipennis, uses E11-14:OH as the pheromone component. The present findings suggest that the male-specific receptor of E11-14:OH was acquired before the divergence of the genus Ostrinia, and functionally retained through the evolution of this genus. PMID:19421342

Capturing novel mouse genes encoding chromosomal and other nuclear proteins.

PubMed

Tate, P; Lee, M; Tweedie, S; Skarnes, W C; Bickmore, W A

1998-09-01

The burgeoning wealth of gene sequences contrasts with our ignorance of gene function. One route to assigning function is by determining the sub-cellular location of proteins. We describe the identification of mouse genes encoding proteins that are confined to nuclear compartments by splicing endogeneous gene sequences to a promoterless betageo reporter, using a gene trap approach. Mouse ES (embryonic stem) cell lines were identified that express betageo fusions located within sub-nuclear compartments, including chromosomes, the nucleolus and foci containing splicing factors. The sequences of 11 trapped genes were ascertained, and characterisation of endogenous protein distribution in two cases confirmed the validity of the approach. Three novel proteins concentrated within distinct chromosomal domains were identified, one of which appears to be a serine/threonine kinase. The sequence of a gene whose product co-localises with splicesome components suggests that this protein may be an E3 ubiquitin-protein ligase. The majority of the other genes isolated represent novel genes. This approach is shown to be a powerful tool for identifying genes encoding novel proteins with specific sub-nuclear localisations and exposes our ignorance of the protein composition of the nucleus. Motifs in two of the isolated genes suggest new links between cellular regulatory mechanisms (ubiquitination and phosphorylation) and mRNA splicing and chromosome structure/function.

Effects of bright light exposure during daytime on peripheral clock gene expression in humans.

PubMed

Sato, Maki; Wakamura, Tomoko; Morita, Takeshi; Okamoto, Akihiko; Akashi, Makoto; Matsui, Takuya; Sato, Motohiko

2017-06-01

Light is the strongest synchronizer controlling circadian rhythms. The intensity and duration of light change throughout the year, thereby influencing body weight, food preferences, and melatonin secretion in humans and animals. Although the expression of clock genes has been examined using human samples, it currently remains unknown whether bright light during the daytime affects the expression of these genes in humans. Therefore, we herein investigated the effects of bright light exposure during the daytime on clock gene expression in the hair follicular and root cells of the human scalp. Seven healthy men (20.4 ± 2.2 years old; 172.3 ± 5.8 cm; 64.3 ± 8.5 kg; BMI 21.7 ± 3.1 kg/m 2 , mean ± SD) participated in this study. Subjects completed 3-day experimental sessions twice in 1 month during which they were exposed to bright and dim light conditions. The mRNA expression of Per1-3, Cry1-2, Rev-erb-α (Nr1d1), Rev-erb-β (Nr1d2), and Dec1 was analyzed using branched DNA probes. No significant changes were observed in the expression of Per1, Per2, Per3, Cry1, Cry2, Rev-erb-α (Nr1d1), or Dec1 following exposure to bright light conditions. However, the expression of Rev-erb-β (Nr1d2) tended to be stronger under bright light than dim light conditions. These results suggest that the bright light stimulus did not influence the expression of clock genes in humans. Long-lasting bright light exposure during the daytime may be required to change the expression of clock genes in humans.

Mode of action of the qcr9 and cat3 mutations in restoring the ability of Saccharomyces cerevisiae tps1 mutants to grow on glucose.

PubMed

Blázquez, M A; Gancedo, C

1995-12-20

Mutations in the TPS1 gene, which encodes trehalose-6-P synthase, cause a glucose-negative phenotype in Saccharomyces cerevisiae. Antimycin A or disruption of the QCR9 gene, which encodes one subunit of the cytochrome bc1 complex, restore the ability to grow in glucose-containing media. Under these conditions the cell excreted a large amount of glycerol, corresponding to about 20% of the glucose taken up. Suppression appears to be achieved by diversion of accumulated glycolytic intermediates to the production of glycerol, thereby providing NAD+ and phosphate for the glyceraldehyde-3-P dehydrogenase reaction. Analysis of the mutation sci1-1, which also suppresses the glucose-negative phenotype of tps1 mutants, showed that glucose transport was decreased in sci1-1 mutants. The gene SCI1 was cloned and its nucleotide sequence revealed it to be identical to CAT3/SNF4. The suppression mediated by sci1-1 is attributable to a decrease in glycolytic flux.

Do educational materials change knowledge and behaviour about crying and shaken baby syndrome? A randomized controlled trial.

PubMed

Barr, Ronald G; Barr, Marilyn; Fujiwara, Takeo; Conway, Jocelyn; Catherine, Nicole; Brant, Rollin

2009-03-31

Shaken baby syndrome often occurs after shaking in response to crying bouts. We questioned whether the use of the educational materials from the Period of PURPLE Crying program would change maternal knowledge and behaviour related to shaking. We performed a randomized controlled trial in which 1279 mothers received materials from the Period of PURPLE Crying program or control materials during a home visit by a nurse by 2 weeks after the birth of their child. At 5 weeks, the mothers completed a diary to record their behaviour and their infants' behaviour. Two months after giving birth, the mothers completed a telephone survey to assess their knowledge and behaviour. The mean score (range 0-100 points) for knowledge about infant crying was greater among mothers who received the PURPLE materials (63.8 points) than among mothers who received the control materials (58.4 points) (difference 5.4 points, 95% confidence interval [CI] 4.1 to 6.5 points). The mean scores were similar for both groups for shaking knowledge and reported maternal responses to crying, inconsolable crying and self-talk responses. Compared with mothers who received control materials, mothers who received the PURPLE materials reported sharing information about walking away if frustrated more often (51.5% v. 38.5%, difference 13.0%, 95% CI 6.9% to 19.2%), the dangers of shaking (49.3% v. 36.4%, difference 12.9%, 95% CI 6.8% to 19.0%), and infant crying (67.6% v. 60.0%, difference 7.6%, 95% CI 1.7% to 13.5%). Walking away during inconsolable crying was significantly higher among mothers who received the PURPLE materials than among those who received control materials (0.067 v. 0.039 events per day, rate ratio 1.7, 95% CI 1.1 to 2.6). The receipt of the Period of PURPLE Crying materials led to higher maternal scores for knowledge about infant crying and for some behaviours considered to be important for the prevention of shaking.

Do educational materials change knowledge and behaviour about crying and shaken baby syndrome? A randomized controlled trial

PubMed Central

Barr, Ronald G.; Barr, Marilyn; Fujiwara, Takeo; Conway, Jocelyn; Catherine, Nicole; Brant, Rollin

2009-01-01

Background Shaken baby syndrome often occurs after shaking in response to crying bouts. We questioned whether the use of the educational materials from the Period of PURPLE Crying program would change maternal knowledge and behaviour related to shaking. Methods We performed a randomized controlled trial in which 1279 mothers received materials from the Period of PURPLE Crying program or control materials during a home visit by a nurse by 2 weeks after the birth of their child. At 5 weeks, the mothers completed a diary to record their behaviour and their infants' behaviour. Two months after giving birth, the mothers completed a telephone survey to assess their knowledge and behaviour. Results The mean score (range 0–100 points) for knowledge about infant crying was greater among mothers who received the PURPLE materials (63.8 points) than among mothers who received the control materials (58.4 points) (difference 5.4 points, 95% confidence interval [CI] 4.1 to 6.5 points). The mean scores were similar for both groups for shaking knowledge and reported maternal responses to crying, inconsolable crying and self-talk responses. Compared with mothers who received control materials, mothers who received the PURPLE materials reported sharing information about walking away if frustrated more often (51.5% v. 38.5%, difference 13.0%, 95% CI 6.9% to 19.2%), the dangers of shaking (49.3% v. 36.4%, difference 12.9%, 95% CI 6.8% to 19.0%), and infant crying (67.6% v. 60.0%, difference 7.6%, 95% CI 1.7% to 13.5%). Walking away during inconsolable crying was significantly higher among mothers who received the PURPLE materials than among those who received control materials (0.067 v. 0.039 events per day, rate ratio 1.7, 95% CI 1.1 to 2.6). Interpretation The receipt of the Period of PURPLE Crying materials led to higher maternal scores for knowledge about infant crying and for some behaviours considered to be important for the prevention of shaking. (ClinicalTrials.gov trial register no. NCT00175422.) PMID:19255065

Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers.

PubMed

Ye, G Y; Shu, Q Y; Yao, H W; Cui, H R; Cheng, X Y; Hu, C; Xia, Y W; Gao, M W; Altosaar, I

2001-02-01

Two transgenic rice (Oryza sativa L.) lines, KMD1 and KMD2 at the R4 generation, transformed with a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were first evaluated for stem borer resistance in the field during the rice growing season of 1998 in two areas of Zhejiang Province, China. Both KMD1 and KMD2 were highly resistant to the stem borers Chilo suppressalis (Walker) and Scirpophaga incertulas (Walker), and were completely undamaged during the whole rice growing season. In contrast, damage to the plants of the untransformed parental control (Xiushui 11) was in the form of deadhearts or whiteheads. Under natural infestation by the C. suppressalis, the damage to control plants reached a peak of 88.7% of plants and 20.1% of tillers encountered with deadhearts. Under artificial and natural infestation of neonate striped stem borers at the vegetative stage and booting stage, 100% of plants and 25.6% of tillers, 78.9% of plants and 15.6% of productive tillers among artificially infested control plants were observed with the symptom of deadhearts and whiteheads, respectively. Damage to the control plants from artificial infestation by the S. incertulas reached a peak of 97.0% of plants and 22.9% of tillers damaged. The field research indicated that both KMD1 and KMD2 show great potential for protecting rice from attack by these two stem borers.

SEMI-ROLLED LEAF1 Encodes a Putative Glycosylphosphatidylinositol-Anchored Protein and Modulates Rice Leaf Rolling by Regulating the Formation of Bulliform Cells1[W][OA

PubMed Central

Xiang, Jing-Jing; Zhang, Guang-Heng; Qian, Qian; Xue, Hong-Wei

2012-01-01

Leaf rolling is an important agronomic trait in rice (Oryza sativa) breeding and moderate leaf rolling maintains the erectness of leaves and minimizes shadowing between leaves, leading to improved photosynthetic efficiency and grain yields. Although a few rolled-leaf mutants have been identified and some genes controlling leaf rolling have been isolated, the molecular mechanisms of leaf rolling still need to be elucidated. Here we report the isolation and characterization of SEMI-ROLLED LEAF1 (SRL1), a gene involved in the regulation of leaf rolling. Mutants srl1-1 (point mutation) and srl1-2 (transferred DNA insertion) exhibit adaxially rolled leaves due to the increased numbers of bulliform cells at the adaxial cell layers, which could be rescued by complementary expression of SRL1. SRL1 is expressed in various tissues and is expressed at low levels in bulliform cells. SRL1 protein is located at the plasma membrane and predicted to be a putative glycosylphosphatidylinositol-anchored protein. Moreover, analysis of the gene expression profile of cells that will become epidermal cells in wild type but probably bulliform cells in srl1-1 by laser-captured microdissection revealed that the expression of genes encoding vacuolar H+-ATPase (subunits A, B, C, and D) and H+-pyrophosphatase, which are increased during the formation of bulliform cells, were up-regulated in srl1-1. These results provide the transcript profile of rice leaf cells that will become bulliform cells and demonstrate that SRL1 regulates leaf rolling through inhibiting the formation of bulliform cells by negatively regulating the expression of genes encoding vacuolar H+-ATPase subunits and H+-pyrophosphatase, which will help to understand the mechanism regulating leaf rolling. PMID:22715111

Genetic diversity of genes encoding OKP and LEN beta-lactamases produced by clinical Klebsiella pneumoniae strains in Portugal.

PubMed

Mendonça, Nuno; Ferreira, Eugénia; Caniça, Manuela

2009-03-01

Of the 308 clinical Klebsiella pneumoniae strains collected in 21 Portuguese health institutions, 11 encoded for LEN and 9 for OKP enzymes; of these, 15 were new enzymes. Ninety-one percent of LEN and all OKP producer strains were resistant to amoxicillin. We demonstrate that these beta-lactamase were highly diverse.

Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review.

PubMed

Parmar, Nehanjali; Singh, Kunwar Harendra; Sharma, Deepika; Singh, Lal; Kumar, Pankaj; Nanjundan, J; Khan, Yasin Jeshima; Chauhan, Devendra Kumar; Thakur, Ajay Kumar

2017-08-01

Genetic engineering technique offers myriads of applications in improvement of horticultural crops for biotic and abiotic stress tolerance, and produce quality enhancement. During last two decades, a large number of transgenic horticultural crops has been developed and more are underway. A number of genes including natural and synthetic Cry genes, protease inhibitors, trypsin inhibitors and cystatin genes have been used to incorporate insect and nematode resistance. For providing protection against fungal and bacterial diseases, various genes like chitinase, glucanase, osmotin, defensin and pathogenesis-related genes are being transferred to many horticultural crops world over. RNAi technique has been found quite successful in inducing virus resistance in horticultural crops in addition to coat protein genes. Abiotic stresses such as drought, heat and salinity adversely affect production and productivity of horticultural crops and a number of genes encoding for biosynthesis of stress protecting compounds including mannitol, glycine betaine and heat shock proteins have been employed for abiotic stress tolerance besides various transcription factors like DREB1, MAPK, WRKY, etc. Antisense gene and RNAi technologies have revolutionized the pace of improvement of horticultural crops, particularly ornamentals for color modification, increasing shelf-life and reducing post-harvest losses. Precise genome editing tools, particularly CRISPR/Cas9, have been efficiently applied in tomato, petunia, citrus, grape, potato and apple for gene mutation, repression, activation and epigenome editing. This review provides comprehensive overview to draw the attention of researchers for better understanding of genetic engineering advancements in imparting biotic and abiotic stress tolerance as well as on improving various traits related to quality, texture, plant architecture modification, increasing shelf-life, etc. in different horticultural crops.

The clp1 gene of the mushroom Coprinus cinereus is essential for A-regulated sexual development.

PubMed Central

Inada, K; Morimoto, Y; Arima, T; Murata, Y; Kamada, T

2001-01-01

Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development. PMID:11139497

Conventional and high-dose daunorubicin and idarubicin in acute myeloid leukaemia remission induction treatment: a mixed treatment comparison meta-analysis of 7258 patients.

PubMed

Sekine, Leo; Morais, Vinícius Daudt; Lima, Karine Margarites; Onsten, Tor Gunnar Hugo; Ziegelmann, Patrícia Klarmann; Ribeiro, Rodrigo Antonini

2015-12-01

Previous meta-analyses suggested that acute myeloid leukaemia induction regimens containing idarubicin (IDA) or high-dose daunorubicin (HDD) induce higher rates of complete remission (CR) than conventional-dose daunorubicin (CDD), with a possible benefit in overall survival. However, robust comparisons between these regimens are still lacking. We conducted a mixed treatment comparison meta-analysis regarding these three regimens. Mixed treatment comparison is a statistical method of data summarization that aggregates data from both direct and indirect effect estimates. Literature search strategy included MEDLINE, EMBASE, Cochrane, Scielo and LILACS, from inception until August 2013 and resulted in the inclusion of 17 trials enrolling 7258 adult patients. HDD [relative risk (RR) 1.13; 95% credible interval (CrI) 1.02-1.26] and IDA (RR 1.13; 95% CrI 1.05-1.23) showed higher CR rates than CDD. IDA also led to lower long-term overall mortality rates when compared with CDD (RR 0.93, 95% CrI 0.86-0.99), whereas HDD and CDD were no different (RR 0.94, 95% CrI 0.85-1.02). HDD and IDA comparison did not reach statistically significant differences in CR (RR 1.00; 95% CrI 0.89-1.11) and in long-term mortality (RR 1.01, 95% CrI 0.91-1.11). IDA and HDD are consistently superior to CDD in inducing CR, and IDA was associated with lower long-term mortality. On the basis of these findings, we recommend incorporation of IDA and HDD instead of the traditional CDD as standard treatments for acute myeloid leukaemia induction. The lack of HDD benefit on mortality, when compared with CDD in this study, should be cautiously addressed, because it may have been susceptible to underestimation because of statistical power limitations. Copyright © 2014 John Wiley & Sons, Ltd.

The complete genome sequence of Eubacterium limosum SA11, a metabolically versatile rumen acetogen.

PubMed

Kelly, William J; Henderson, Gemma; Pacheco, Diana M; Li, Dong; Reilly, Kerri; Naylor, Graham E; Janssen, Peter H; Attwood, Graeme T; Altermann, Eric; Leahy, Sinead C

2016-01-01

Acetogens are a specialized group of anaerobic bacteria able to produce acetate from CO2 and H2 via the Wood-Ljungdahl pathway. In some gut environments acetogens can compete with methanogens for H2, and as a result rumen acetogens are of interest in the development of microbial approaches for methane mitigation. The acetogen Eubacterium limosum SA11 was isolated from the rumen of a New Zealand sheep and its genome has been sequenced to examine its potential application in methane mitigation strategies, particularly in situations where hydrogenotrophic methanogens are inhibited resulting in increased H2 levels in the rumen. The 4.15 Mb chromosome of SA11 has an average G + C content of 47 %, and encodes 3805 protein-coding genes. There is a single prophage inserted in the chromosome, and several other gene clusters appear to have been acquired by horizontal transfer. These include genes for cell wall glycopolymers, a type VII secretion system, cell surface proteins and chemotaxis. SA11 is able to use a variety of organic substrates in addition to H2/CO2, with acetate and butyrate as the principal fermentation end-products, and genes involved in these metabolic pathways have been identified. An unusual feature is the presence of 39 genes encoding trimethylamine methyltransferase family proteins, more than any other bacterial genome. Overall, SA11 is a metabolically versatile organism, but its ability to grow on such a wide range of substrates suggests it may not be a suitable candidate to take the place of hydrogen-utilizing methanogens in the rumen.

An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

PubMed Central

Bearden, Scott W.; Staggs, Teanna M.; Perry, Robert D.

1998-01-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media. PMID:9495751

An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

PubMed

Bearden, S W; Staggs, T M; Perry, R D

1998-03-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

Structures of Preferred Human IgV Genes-Based Protective Antibodies Identify How Conserved Residues Contact Diverse Antigens and Assign Source of Specificity to CDR3 Loop Variation.

PubMed

Bryson, Steve; Thomson, Christy A; Risnes, Louise F; Dasgupta, Somnath; Smith, Kenneth; Schrader, John W; Pai, Emil F

2016-06-01

The human Ab response to certain pathogens is oligoclonal, with preferred IgV genes being used more frequently than others. A pair of such preferred genes, IGVK3-11 and IGVH3-30, contributes to the generation of protective Abs directed against the 23F serotype of the pneumonococcal capsular polysaccharide of Streptococcus pneumoniae and against the AD-2S1 peptide of the gB membrane protein of human CMV. Structural analyses of Fab fragments of mAbs 023.102 and pn132p2C05 in complex with portions of the 23F polysaccharide revealed five germline-encoded residues in contact with the key component, l-rhamnose. In the case of the AD-2S1 peptide, the KE5 Fab fragment complex identified nine germline-encoded contact residues. Two of these germline-encoded residues, Arg91L and Trp94L, contact both the l-rhamnose and the AD-2S1 peptide. Comparison of the respective paratopes that bind to carbohydrate and protein reveals that stochastic diversity in both CDR3 loops alone almost exclusively accounts for their divergent specificity. Combined evolutionary pressure by human CMV and the 23F serotype of S. pneumoniae acted on the IGVK3-11 and IGVH3-30 genes as demonstrated by the multiple germline-encoded amino acids that contact both l-rhamnose and AD-2S1 peptide. Copyright © 2016 by The American Association of Immunologists, Inc.

Only two mutations detected in 15 Tunisian patients with 11β-hydroxylase deficiency: the p.Q356X and the novel p.G379V.

PubMed

Kharrat, M; Trabelsi, S; Chaabouni, M; Maazoul, F; Kraoua, L; Ben Jemaa, L; Gandoura, N; Barsaoui, S; Morel, Y; M'rad, R; Chaabouni, H

2010-10-01

Steroid 11β-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11β-hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11β-hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population. © 2010 John Wiley & Sons A/S.

ENHANCED BIOACCUMULATION OF HEAVY METAL BY BACTERIA CELLS DISPLAYING SYNTHETIC PHYTOCHELATINS. (R827227)

EPA Science Inventory

A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)_nGly (EC8 (n = 8), EC11 (n = 11...

Effects of a diet containing genetically modified rice expressing the Cry1Ab/1Ac protein (Bacillus thuringiensis toxin) on broiler chickens.

PubMed

Li, Zeyang; Gao, Yang; Zhang, Minhong; Feng, Jinghai; Xiong, Yandan

2015-01-01

The aim of this study was to evaluate the effect of feeding Bacillus thuringiensis (Bt) rice expressing the Cry1Ab/1Ac protein on broiler chicken. The genetically modified (GM) Bt rice was compared with the corresponding non-GM rice regarding performance of feeding groups, their health status, relative organ weights, biochemical serum parameters and occurrence of Cry1Ab/1Ac gene fragments. One hundred and eighty day-old Arbor Acres female broilers with the same health condition were randomly allocated to the two treatments (6 replicate cages with 15 broilers in each cage per treatment). They received diets containing GM rice (GM group) or its parental non-GM rice (non-GM group) at 52-57% of the air-dried diet for 42 days. The results show that the transgenic rice had a similar nutrient composition as the non-GM rice and had no adverse effects on chicken growth, biochemical serum parameters and necropsy during the 42-day feeding period. In birds fed the GM rice, no transgenic gene fragments were detected in the samples of blood, liver, kidneys, spleen, jejunum, ileum, duodenum and muscle tissue. In conclusion, the results suggest that Bt rice expressing Cry1Ab/1Ac protein has no adverse effects on broiler chicken. Therefore, it can be considered as safe and used as feed source for broiler chicken.

Alternations in neuroendocrine and endocrine regulation of reproduction in male goldfish (Carassius auratus) following an acute and chronic exposure to vinclozolin, in vivo.

PubMed

Golshan, Mahdi; Hatef, Azadeh; Zare, Ava; Socha, Magdalena; Milla, Sylvain; Gosiewski, Grzegorz; Fontaine, Pascal; Sokołowska-Mikołajczyk, Mirosława; Habibi, Hamid R; Alavi, Sayyed Mohammad Hadi

2014-10-01

The fungicide vinclozolin (VZ) is in use globally and known to disrupt reproductive function in male. The present study tested the hypothesis that VZ disrupts testicular function in goldfish (Carassius auratus) by affecting brain-pituitary-testis axis. Goldfish were exposed to 100, 400 and 800 μg/L VZ and 5 μg/L 17β-estradiol (E2) for comparison. In VZ treated goldfish, 11-ketotesteosterone (11-KT) secretion was changed depending on dose and duration period of treatment. Following 7 days of exposure, 11-KT was decreased in goldfish exposed to 800 μg/L VZ, while it was increased in goldfish exposed to 100 μg/L VZ after 30 days of exposure. Circulating E2 level was unchanged in VZ treated goldfish, however the E2/11-KT ratio was increased in a concentration-related manner. In E2 treated goldfish, circulatory 11-KT and E2 levels were decreased and increased, respectively, which resulted in an increase in the E2/11-KT ratio. Exposure to VZ at 100 μg/L caused a significant increase in the circulatory luteinizing hormone (LH) after 30 days. In E2 treated fish circulatory LH was decreased, significantly. Transcripts of genes encoding gonadotropin-releasing hormone and androgen receptor in the brain, and those of genes encoding LH and follicle-stimulating hormone receptors, StAR, CYP17, and 3β-HSD in the testis changed in VZ-treated goldfish depending on concentration and period of treatment. mRNA of genes encoding vitellogenin and estrogen receptor in the liver and cytochrome P450 aromatase in the brain were increased in E2-treated goldfish. The results suggest that VZ-induced changes in 11-KT were due to disruption in brain-pituitary-testis axis and provide integrated characterization of VZ-related reproductive disorders in male fish. Copyright © 2014 Elsevier B.V. All rights reserved.

Light signaling to the zebrafish circadian clock by Cryptochrome 1a

PubMed Central

Tamai, T. Katherine; Young, Lucy C.; Whitmore, David

2007-01-01

Zebrafish tissues and cells have the unusual feature of not only containing a circadian clock, but also being directly light-responsive. Several zebrafish genes are induced by light, but little is known about their role in clock resetting or the mechanism by which this might occur. Here we show that Cryptochrome 1a (Cry1a) plays a key role in light entrainment of the zebrafish clock. Intensity and phase response curves reveal a strong correlation between light induction of Cry1a and clock resetting. Overexpression studies show that Cry1a acts as a potent repressor of clock function and mimics the effect of constant light to “stop” the circadian oscillator. Yeast two-hybrid analysis demonstrates that the Cry1a protein interacts directly with specific regions of core clock components, CLOCK and BMAL, blocking their ability to fully dimerize and transactivate downstream targets, providing a likely mechanism for clock resetting. A comparison of entrainment of zebrafish cells to complete versus skeleton photoperiods reveals that clock phase is identical under these two conditions. However, the amplitude of the core clock oscillation is much higher on a complete photoperiod, as are the levels of light-induced Cry1a. We believe that Cry1a acts on the core clock machinery in both a continuous and discrete fashion, leading not only to entrainment, but also to the establishment of a high-amplitude rhythm and even stopping of the clock under long photoperiods. PMID:17785416

The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management.

PubMed

Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

2015-09-03

Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management.

Evolutionary Diversifaction of Aminopeptidase N in Lepidoptera by Conserved Clade-specific Amino Acid Residues

PubMed Central

Hughes, Austin L.

2015-01-01

Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family. PMID:24675701

The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management

PubMed Central

Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

2015-01-01

Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management. PMID:26333918

Organization and transient expression of the gene for human U11 snRNA

PubMed Central

Clemens, Suter-Crazzolara; Walter, Keller

1991-01-01

The nucleotide sequence of U11 small nuclear RNA, a minor U RNA from HeLa cells, was determined. Computer analysis of the sequence (135 residues) predicts two strong hairpin loops which are separated by seventeen nucleotides containing an Sm binding site (AAUUUUUUGG). A synthetic gene was constructed in which the coding region of U11 RNA is under the control of a T7 promoter. This vector can be used to produce U11 RNA in vitro. Southern hybridization and PCR analysis of HeLa genomic DNA suggest that U11 RNA is encoded by a single copy gene, and that at least three genomic regions could be U11 RNA pseudogenes. A HeLa genomic copy of a U11 gene was isolated by inverted PCR. This gene contains the U11 RNA coding sequence and several sequence elements unique for the U RNA genes. These include a Distal Sequence Element (DSE, ATTTGCATA) present between positions −215 and −223 relative to the start of transcription; a Proximal Sequence Element (PSE, TTCACCTTTACCAAAAATG) located between positions −43 and −63 ; and a 3′box (GTTAGGCGAAATATTA) between positions +150 and +166. Transfection of HeLa cells with this gene revealed that it is functioning in vivo and can produce U11 RNA. PMID:1820214

A new yeast gene with a myosin-like heptad repeat structure.

PubMed

Kölling, R; Nguyen, T; Chen, E Y; Botstein, D

1993-03-01

We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.

Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoid liposarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schoenmakers, E.F.P.M.; Kools, P.F.J.; Mols, R.

1994-03-15

The authors report here the physical mapping of recurrent chromosome 12q13-q15 breakpoints in cell lines derived from primary myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands. In fluorescence in situ hybridization (FISH) experiments, they first mapped the position of the chromosome 12 translocation breakpoint in uterine leiomyoma cell line LM-30.1/SV40 relative to loci COL2A1, D12S4, D12S17, D12S6, D12S19, D12S8, and D12S7. It mapped between linkage probes CRI-C86 (D12S19) and p7G11 (D12S8). They then isolated YAC clones using CRI-C86- and p7G11-derived sequence-tagged sites, constructed corresponding YAC contigs of 310 and 800 kb, respectively, and a mixture ofmore » them was used to routinely study the various tumor cell lines by FISH analysis. The chromosome 12 breakpoints of all tumor cell lines tested mapped between cosmids LLNL12NCO1-98C10 and LLNL12NCO1-113D12. None of the breakpoints appeared to map within any of the isolated YAC clones. Furthermore, FISH analysis using cosmid LLNL12-NCO1-144G3, which maps at the CHOP locus, revealed that the chromosome 12 breakpoints in all cell lines of the three benign solid tumors that were tested were located distal to the chromosome 12 translocation breakpoint with the CHOP gene in myxoid liposarcoma cells with t(12;16). In conclusion, the studies seem to indicate that the chromosome 12 breakpoints of myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands are all clustered within the 7-cM interval between D12S19 and D12S8, with those of the benign solid tumors distal to CHOP. Finally, the MYF5 gene mapped telomeric to LLNL12NCO1-113D12, and the MIP gene mapped centromeric to the chromosome 12 translocation breakpoint in myxoid liposarcoma cells. 56 refs., 5 figs., 3 tabs.« less

Housing conditions associated with recurrent gastrointestinal infection in urban Aboriginal children in NSW, Australia: findings from SEARCH.

PubMed

Andersen, Melanie J; Skinner, Adam; Williamson, Anna B; Fernando, Peter; Wright, Darryl

2018-06-01

To examine the associations between housing and gastrointestinal infection in Aboriginal children in urban New South Wales. A total of 1,398 Aboriginal children were recruited through four Aboriginal Community Controlled Health Services. Multilevel regression modelling of survey data estimated associations between housing conditions and recurrent gastrointestinal infection, adjusting for sociodemographic and health factors. Of the sample, 157 children (11%) had recurrent gastrointestinal infection ever and 37 (2.7%) required treatment for recurrent gastrointestinal infection in the past month. Children in homes with 3+ housing problems were 2.51 (95% CrI 1.10, 2.49) times as likely to have recurrent gastrointestinal infection ever and 6.79 (95% CrI 2.11, 30.17) times as likely to have received recent treatment for it (versus 0-2 problems). For every additional housing problem, the prevalence of recurrent gastrointestinal infection ever increased by a factor of 1.28 (95% CrI 1.14, 1.47) and the prevalence of receiving treatment for gastrointestinal infection in the past month increased by a factor of 1.64 (95% CrI 1.20, 2.48). Housing problems were independently associated with recurrent gastrointestinal infection in a dose-dependent manner. Implications for public health: The role of housing as a potential determinant of health in urban Aboriginal children merits further attention in research and policy settings. © 2018 The Authors.

Molecular and genomic basis of volatile-mediated indirect defense against insects in rice.

PubMed

Yuan, Joshua S; Köllner, Tobias G; Wiggins, Greg; Grant, Jerome; Degenhardt, Jörg; Chen, Feng

2008-08-01

Rice plants fed on by fall armyworm (Spodoptera frugiperda, FAW) caterpillars emit a blend of volatiles dominated by terpenoids. These volatiles were highly attractive to females of the parasitoid Cotesia marginiventris. Microarray analysis identified 196 rice genes whose expression was significantly upregulated by FAW feeding, 18 of which encode metabolic enzymes potentially involved in volatile biosynthesis. Significant induction of expression of seven of the 11 terpene synthase (TPS) genes identified through the microarray experiments was confirmd using real-time RT-PCR. Enzymes encoded by three TPS genes, Os02g02930, Os08g07100 and Os08g04500, were biochemically characterized. Os02g02930 was found to encode a monoterpene synthase producing the single product S-linalool, which is the most abundant volatile emitted from FAW-damaged rice plants. Both Os08g07100 and Os08g04500 were found to encode sesquiterpene synthases, each producing multiple products. These three enzymes are responsible for production of the majority of the terpenes released from FAW-damaged rice plants. In addition to TPS genes, several key genes in the upstream terpenoid pathways were also found to be upregulated by FAW feeding. This paper provides a comprehensive analysis of FAW-induced volatiles and the corresponding volatile biosynthetic genes potentially involved in indirect defense in rice. Evolution of the genetic basis governing volatile terpenoid biosynthesis for indirect defense is discussed.

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Amino, H; Tomitsuka, E; Kita, K

1999-08-04

We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

Plasma and serum concentrations of cytarabine administered via continuous intravenous infusion to dogs with meningoencephalomyelitis of unknown etiology.

PubMed

Early, P J; Crook, K I; Williams, L M; Davis, E G; Muñana, K R; Papich, M G; Messenger, K M

2017-08-01

The objective of this study was to evaluate the plasma and serum concentrations of cytarabine (CA) administered via constant rate infusion (CRI) in dogs with meningoencephalomyelitis of unknown etiology (MUE). Nineteen client-owned dogs received a CRI of CA at a dose of 25 mg/m 2 /h for 8 h as treatment for MUE. Dogs were divided into four groups, those receiving CA alone and those receiving CA in conjunction with other drugs. Blood samples were collected at 0, 1, 8, and 12 h after initiating the CRI. Plasma (n = 13) and serum (n = 11) cytarabine concentrations were measured by high-pressure liquid chromatography. The mean peak concentration (C MAX ) and area under the curve (AUC) after CRI administration were 1.70 ± 0.66 μg/mL and 11.39 ± 3.37 h·μg/mL, respectively, for dogs receiving cytarabine alone, 2.36 ± 0.35 μg/mL and 16.91 + 3.60 h·μg/mL for dogs administered cytarabine and concurrently on other drugs. Mean concentrations for all dogs were above 1.0 μg/mL at both the 1- and 8-h time points. The steady-state achieved with cytarabine CRI produces a consistent and prolonged exposure in plasma and serum, which is likely to produce equilibrium between blood and the central nervous system in dogs with a clinical diagnosis of MUE. Other medications commonly used to treat MUE do not appear to alter CA concentrations in serum and plasma. © 2016 John Wiley & Sons Ltd.

Accuracy of clinical diagnosis of Parkinson disease: A systematic review and meta-analysis.

PubMed

Rizzo, Giovanni; Copetti, Massimiliano; Arcuti, Simona; Martino, Davide; Fontana, Andrea; Logroscino, Giancarlo

2016-02-09

To evaluate the diagnostic accuracy of clinical diagnosis of Parkinson disease (PD) reported in the last 25 years by a systematic review and meta-analysis. We searched for articles published between 1988 and August 2014. Studies were included if reporting diagnostic parameters regarding clinical diagnosis of PD or crude data. The selected studies were subclassified based on different study setting, type of test diagnosis, and gold standard. Bayesian meta-analyses of available data were performed. We selected 20 studies, including 11 using pathologic examination as gold standard. Considering only these 11 studies, the pooled diagnostic accuracy was 80.6% (95% credible interval [CrI] 75.2%-85.3%). Accuracy was 73.8% (95% CrI 67.8%-79.6%) for clinical diagnosis performed mainly by nonexperts. Accuracy of clinical diagnosis performed by movement disorders experts rose from 79.6% (95% CrI 46%-95.1%) of initial assessment to 83.9% (95% CrI 69.7%-92.6%) of refined diagnosis after follow-up. Using UK Parkinson's Disease Society Brain Bank Research Center criteria, the pooled diagnostic accuracy was 82.7% (95% CrI 62.6%-93%). The overall validity of clinical diagnosis of PD is not satisfying. The accuracy did not significantly improve in the last 25 years, particularly in the early stages of disease, where response to dopaminergic treatment is less defined and hallmarks of alternative diagnoses such as atypical parkinsonism may not have emerged. Misclassification rate should be considered to calculate the sample size both in observational studies and randomized controlled trials. Imaging and biomarkers are urgently needed to improve the accuracy of clinical diagnosis in vivo. © 2016 American Academy of Neurology.

Effect of monochromatic light on circadian rhythmic expression of clock genes in the hypothalamus of chick.

PubMed

Jiang, Nan; Wang, Zixu; Cao, Jing; Dong, Yulan; Chen, Yaoxing

2017-08-01

To clarify the effect of monochromatic light on circadian clock gene expression in chick hypothalamus, a total 240 newly hatched chickens were reared under blue light (BL), green light (GL), red light (RL) and white light (WL), respectively. On the post-hatched day 14, 24-h profiles of seven core clock genes (cClock, cBmal1, cBmal2, cCry1, cCry2, cPer2 and cPer3) were measured at six time points (CT 0, CT 4, CT 8, CT 12, CT 16, CT 20, circadian time). We found all these clock genes expressed with a significant rhythmicity in different light wavelength groups. Meanwhile, cClock and cBmal1 showed a high level under GL, and followed a corresponding high expression of cCry1. However, RL decreased the expression levels of these genes. Be consistent with the mRNA level, CLOCK and BMAL1 proteins also showed a high level under GL. The CLOCK-like immunoreactive neurons were observed not only in the SCN, but also in the non-SCN brain region such as the nucleus anterior medialis hypothalami, the periventricularis nucleus, the paraventricular nucleus and the median eminence. All these results are consistent with the auto-regulatory circadian feedback loop, and indicate that GL may play an important role on the circadian time generation and development in the chick hypothalamus. Our results also suggest that the circadian clock in the chick hypothalamus such as non-SCN brain region were involved in the regulation of photo information. Copyright © 2017 Elsevier B.V. All rights reserved.

Establishment of a dietary exposure assay for evaluating the toxicity of insecticidal compounds to Apolygus lucorum (Hemiptera: Miridae).

PubMed

Zhao, Man; Li, Yunhe; Yuan, Xiangdong; Liang, Gemei; Wang, Bingjie; Liu, Chen; Khaing, Myint Myint

2018-06-01

With the commercialization of transgenic cotton that expresses Bt (Bacillus thuringiensis) insecticidal proteins, mirid bugs have become key pests in cotton and maize fields in China. Genetically engineered (GE) crops for controlling mirids are unavailable owing to a lack of suitable insecticidal genes. In this study, we developed and validated a dietary exposure assay for screening insecticidal compounds and for assessing the potential effects of insecticidal proteins produced by GE plants on Apolygus lucorum, one of the main mirid pests of Bt cotton and Bt maize. Diets containing potassium arsenate (PA) or the cysteine protease inhibitor E-64 were used as positive controls for validating the efficacy of the dietary exposure assay. The results showed that with increasing concentrations of PA or E-64, A. lucorum larval development time was prolonged and adult weight and fecundity were decreased, suggesting that the dietary exposure assay was useful for detecting the toxicity of insecticidal compounds to A. lucorum. This assay was then used to assess the toxicity of Cry1Ab, Cry1Ac, Cry1F, Cry2Aa, and Cry2Ab proteins, which have been transformed into several crops, against A. lucorum. The results showed that A. lucorum did not show a negative effect by feeding on an artificial diet containing any of the purified Cry proteins. No significant changes in the activities of digestive, detoxifying, or antioxidant enzymes were detected in A. lucorum that fed on a diet containing Cry proteins, but A. lucorum fitness was reduced when the insect fed on a diet containing E-64 or PA. These results demonstrate that A. lucorum is not sensitive to the tested Cry proteins and that the dietary exposure assay is useful for evaluating the toxicity of insecticidal compounds to this species. Copyright © 2018 Elsevier Ltd. All rights reserved.

Association of Pharmacological Treatments for Obesity With Weight Loss and Adverse Events

PubMed Central

Khera, Rohan; Murad, Mohammad Hassan; Chandar, Apoorva K.; Dulai, Parambir S.; Wang, Zhen; Prokop, Larry J.; Loomba, Rohit; Camilleri, Michael; Singh, Siddharth

2017-01-01

IMPORTANCE Five medications have been approved for the management of obesity, but data on comparative effectiveness are limited. OBJECTIVE To compare weight loss and adverse events among drug treatments for obesity using a systematic review and network meta-analysis. DATA SOURCES MEDLINE, EMBASE, Web of Science, Scopus, and Cochrane Central from inception to March 23, 2016; clinical trial registries. STUDY SELECTION Randomized clinical trials conducted among overweight and obese adults treated with US Food and Drug Administration–approved long-term weight loss agents (orlistat, lorcaserin, naltrexone-bupropion, phentermine-topiramate, or liraglutide) for at least 1 year compared with another active agent or placebo. DATA EXTRACTION AND SYNTHESIS Two investigators identified studies and independently abstracted data using a predefined protocol. A Bayesian network meta-analysis was performed and relative ranking of agents was assessed using surface under the cumulative ranking (SUCRA) probabilities. Quality of evidence was assessed using GRADE criteria. MAIN OUTCOMES AND MEASURES Proportions of patients with at least 5%weight loss and at least 10% weight loss, magnitude of decrease in weight, and discontinuation of therapy because of adverse events at 1 year. RESULTS Twenty-eight randomized clinical trials with 29018 patients (median age, 46 years; 74%women; median baseline body weight, 100.5 kg; median baseline body mass index, 36.1) were included. A median 23%of placebo participants had at least 5%weight loss vs 75%of participants taking phentermine-topiramate (odds ratio [OR], 9.22; 95%credible interval [CrI], 6.63–12.85; SUCRA, 0.95), 63%of participants taking liraglutide (OR, 5.54; 95%CrI, 4.16–7.78; SUCRA, 0.83), 55%taking naltrexone-bupropion (OR, 3.96; 95%CrI, 3.03–5.11; SUCRA, 0.60), 49%taking lorcaserin (OR, 3.10; 95%CrI, 2.38–4.05; SUCRA, 0.39), and 44%taking orlistat (OR, 2.70; 95%CrI, 2.34–3.09; SUCRA, 0.22). All active agents were associated with significant excess weight loss compared with placebo at 1 year—phentermine-topiramate, 8.8 kg (95%CrI, −10.20 to −7.42 kg); liraglutide, 5.3 kg (95%CrI, −6.06 to −4.52 kg); naltrexone-bupropion, 5.0 kg (95%CrI, −5.94 to −3.96 kg); lorcaserin, 3.2 kg (95%CrI, −3.97 to −2.46 kg); and orlistat, 2.6 kg (95%CrI, −3.04 to −2.16 kg). Compared with placebo, liraglutide (OR, 2.95; 95%CrI, 2.11–4.23) and naltrexone-bupropion (OR, 2.64; 95%CrI, 2.10–3.35) were associated with the highest odds of adverse event–related treatment discontinuation. High attrition rates (30%–45%in all trials) were associated with lower confidence in estimates. CONCLUSIONS AND RELEVANCE Among overweight or obese adults, orlistat, lorcaserin, naltrexone-bupropion, phentermine-topiramate, and liraglutide, compared with placebo, were each associated with achieving at least 5%weight loss at 52 weeks. Phentermine-topiramate and liraglutide were associated with the highest odds of achieving at least 5%weight loss. PMID:27299618

Behavioral and genetic correlates of the neural response to infant crying among human fathers

PubMed Central

Mascaro, Jennifer S.; Hackett, Patrick D.; Gouzoules, Harold; Lori, Adriana

2014-01-01

Although evolution has shaped human infant crying and the corresponding response from caregivers, there is marked variation in paternal involvement and caretaking behavior, highlighting the importance of understanding the neurobiology supporting optimal paternal responses to cries. We explored the neural response to infant cries in fathers of children aged 1–2, and its relationship with hormone levels, variation in the androgen receptor (AR) gene, parental attitudes and parental behavior. Although number of AR CAG trinucleotide repeats was positively correlated with neural activity in brain regions important for empathy (anterior insula and inferior frontal gyrus), restrictive attitudes were inversely correlated with neural activity in these regions and with regions involved with emotion regulation (orbitofrontal cortex). Anterior insula activity had a non-linear relationship with paternal caregiving, such that fathers with intermediate activation were most involved. These results suggest that restrictive attitudes may be associated with decreased empathy and emotion regulation in response to a child in distress, and that moderate anterior insula activity reflects an optimal level of arousal that supports engaged fathering. PMID:24336349

Comparative analyses of light-induced anthocyanin accumulation and gene expression between the ray florets and leaves in chrysanthemum.

PubMed

Hong, Yan; Yang, Li-Wen; Li, Meng-Ling; Dai, Si-Lan

2016-06-01

Light is one of the key environmental factors that affect anthocyanin biosynthesis. However, the underlying molecular mechanism remains unclear, and many problems regarding phenotypic change and corresponding gene regulation have not been solved. In the present study, comparative analyses of light-induced anthocyanin accumulation and gene expression between the ray florets and leaves were performed in Chrysanthemum × morifolium 'Purple Reagan'. After contrasting the variations in the flower color phenotype and relative pigment content, as well as expression patterns of structural and regulator genes responsible for anthocyanin biosynthesis and photoreceptor between different plant organs under light and dark conditions, we concluded that (1) both the capitulum and foliage are key organs responding to light for chrysanthemum coloration; (2) compared with flavones, shading makes a greater decrease on the anthocyanins accumulation; (3) most of the structural and regulatory genes in the light-induced anthocyanin pathway specifically express in the ray florets; and (4) CmCHS, CmF3H, CmF3'H, CmANS, CmDFR, Cm3GT, CmMYB5-1, CmMYB6, CmMYB7-1, CmbHLH24, CmCOP1 and CmHY5 are key genes for light-induced anthocyanin biosynthesis in chrysanthemum ray florets, while on the transcriptional level, the expressions of CmPHYA, CmPHYB, CmCRY1a, CmCRY1b and CmCRY2 are insignificantly changed. Moreover, the inferred comprehensive effect of multiple signals on the accumulation of anthocyanins and transmission channel of light signal that exist between the leaves and ray florets were further discussed. These results further our understanding of the relationship between the gene expression and light-induced anthocyanin biosynthesis, and lay foundations for the promotion of the molecular breeding of novel flower colors in chrysanthemums. Copyright © 2016. Published by Elsevier Masson SAS.

Twenty-seven nonoverlapping zinc finger cDNAs from human T cells map to nine different chromosomes with apparent clustering.

PubMed Central

Huebner, K; Druck, T; Croce, C M; Thiesen, H J

1991-01-01

cDNA clones encoding zinc finger structures were isolated by screening Molt4 and Jurkat cDNA libraries with zinc finger consensus sequences. Candidate clones were partially sequenced to verify the presence of zinc finger-encoding regions; nonoverlapping cDNA clones were chosen on the basis of sequences and genomic hybridization pattern. Zinc finger structure-encoding clones, which were designated by the term "Kox" and a number from 1 to 32 and which were apparently unique (i.e., distinct from each other and distinct from those isolated by other laboratories), were chosen for mapping in the human genome. DNAs from rodent-human somatic cell hybrids retaining defined complements of human chromosomes were analyzed for the presence of each of the Kox genes. Correlation between the presence of specific human chromosome regions and specific Kox genes established the chromosomal locations. Multiple Kox loci were mapped to 7q (Kox 18 and 25 and a locus detected by both Kox 8 cDNA and Kox 27 cDNA), 8q24 5' to the myc locus (Kox 9 and 32), 10cen----q24 (Kox 2, 15, 19, 21, 30, and 31), 12q13-qter (Kox 1 and 20), 17p13 (Kox 11 and 26), and 19q (Kox 5, 6, 10, 22, 24, and 28). Single Kox loci were mapped to 7p22 (Kox 3), 18q12 (Kox 17), 19p (Kox 13), 22q11 between IG lambda and BCR-1 (locus detected by both Kox 8 cDNA and Kox 27 cDNA), and Xp (Kox 14). Several of the Kox loci map to regions in which other zinc finger structure-encoding loci have already been localized, indicating possible zinc finger gene clusters. In addition, Kox genes at 8q24, 17p13, and 22q11--and perhaps other Kox genes--are located near recurrent chromosomal translocation breakpoints. Others, such as those on 7p and 7q, may be near regions specifically active in T cells. Images Figure 4 Figure 5 Figure 2 Figure 3 PMID:2014798

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Investigating the genetics of Bti resistance using mRNA tag sequencing: application on laboratory strains and natural populations of the dengue vector Aedes aegypti

PubMed Central

Paris, Margot; Marcombe, Sebastien; Coissac, Eric; Corbel, Vincent; David, Jean-Philippe; Després, Laurence

2013-01-01

Mosquito control is often the main method used to reduce mosquito-transmitted diseases. In order to investigate the genetic basis of resistance to the bio-insecticide Bacillus thuringiensis subsp. israelensis (Bti), we used information on polymorphism obtained from cDNA tag sequences from pooled larvae of laboratory Bti-resistant and susceptible Aedes aegypti mosquito strains to identify and analyse 1520 single nucleotide polymorphisms (SNPs). Of the 372 SNPs tested, 99.2% were validated using DNA Illumina GoldenGate® array, with a strong correlation between the allelic frequencies inferred from the pooled and individual data (r = 0.85). A total of 11 genomic regions and five candidate genes were detected using a genome scan approach. One of these candidate genes showed significant departures from neutrality in the resistant strain at sequence level. Six natural populations from Martinique Island were sequenced for the 372 tested SNPs with a high transferability (87%), and association mapping analyses detected 14 loci associated with Bti resistance, including one located in a putative receptor for Cry11 toxins. Three of these loci were also significantly differentiated between the laboratory strains, suggesting that most of the genes associated with resistance might differ between the two environments. It also suggests that common selected regions might harbour key genes for Bti resistance. PMID:24187584

Mutational analysis of the PTPN11 gene in Egyptian patients with Noonan syndrome.

PubMed

Essawi, Mona L; Ismail, Manal F; Afifi, Hanan H; Kobesiy, Maha M; El Kotoury, Ahmed; Barakat, Maged M

2013-11-01

Noonan syndrome (NS) is inherited as an autosomal dominant disorder with dysmorphic facies, short stature, and cardiac defects, which can be caused by missense mutations in the protein tyrosine phosphatase nonreceptor type 11 (PTPN11) gene, which encodes src homology region 2 domain containing tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factors and cytokines. The current study aimed to study the molecular characterization of the PTPN11 gene among Egyptian patients with Noonan syndrome. Eleven exons of the PTPN11 gene were amplified and screened by single stranded conformational polymorphism (SSCP). DNA samples showing band shift in SSCP were subjected to sequencing. Mutational analysis of the PTPN11 gene revealed T→C transition at position 854 in exon 8, predicting Phe285Ser substitution within PTP domain of SHP-2 protein, in one NS patient and -21C→T polymorphism in intron 7 in four other cases. Knowing that NS is phenotypically heterogeneous, molecular characterization of the PTPN11 gene should serve to establish NS diagnosis in patients with atypical features, although lack of a mutation does not exclude the possibility of NS. Copyright © 2012. Published by Elsevier B.V.

The maize lilliputian1 (lil1) gene, encoding a brassinosteroid cytochrome P450 C-6 oxidase, is involved in plant growth and drought response.

PubMed

Castorina, Giulia; Persico, Martina; Zilio, Massimo; Sangiorgio, Stefano; Carabelli, Laura; Consonni, Gabriella

2018-05-16

Brassinosteroids (BRs) are plant hormones involved in many developmental processes as well as in plant-environment interactions. Their role was investigated in this study through the analysis of lilliputian1-1 (lil1-1), a dwarf mutant impaired in BR biosynthesis in maize (Zea mays). We isolated lil1-1 through transposon tagging in maize. The action of lil1 was investigated through morphological and genetic analysis. Moreover, by comparing lil1-1 mutant and wild-type individuals grown under drought stress, the effect of BR reduction on the response to drought stress was examined. lil1-1 is a novel allele of the brassinosteroid-deficient dwarf1 (brd1) gene, encoding a brassinosteroid C-6 oxidase. We show in this study that lil1 is epistatic to nana plant1 (na1), a BR gene involved in earlier steps of the pathway. The lill-1 mutation causes alteration in the root gravitropic response, leaf epidermal cell density, epicuticular wax deposition and seedling adaptation to water scarcity conditions. Lack of active BR molecules in maize causes a pleiotropic effect on plant development and improves seedling tolerance of drought. BR-deficient maize mutants can thus be instrumental in unravelling novel mechanisms on which plant adaptations to abiotic stress are based.

Antibiotics influence the toxicity of the delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa armigera.

PubMed

Paramasiva, Inakarla; Sharma, Hari C; Krishnayya, Pulipaka Venkata

2014-07-24

The cotton bollworm, Helicoverpa armigera is one of the most important crop pests worldwide. It has developed high levels of resistance to synthetic insecticides, and hence, Bacillus thuringiensis (Bt) formulations are used as a safer pesticide and the Bt genes have been deployed in transgenic crops for controlling this pest. There is an apprehension that H. armigera might develop resistance to transgenic crops in future. Therefore, we studied the role of gut microbes by eliminating them with antibiotics in H. armigera larvae on the toxicity of Bt toxins against this pest. Commercial formulation of Bt (Biolep®) and the pure Cry1Ab and Cry1Ac toxin proteins were evaluated at ED50, LC50, and LC90 dosages against the H. armigera larvae with and without antibiotics (which removed the gut microbes). Lowest H. armigera larval mortality due to Bt formulation and the Bt toxins Cry1Ab and Cry1Ac was recorded in insects reared on diets with 250 and 500 μg ml-1 diet of each of the four antibiotics (gentamicin, penicillin, rifampicin, and streptomycin), while the highest larval mortality was recorded in insects reared on diets without the antibiotics. Mortality of H. armigera larvae fed on diets with Bt formulation and the δ-endotoxins Cry1Ab and Cry1Ac was inversely proportional to the concentration of antibiotics in the artificial diet. Nearly 30% reduction in larval mortality was observed in H. armigera larvae from F1 to F3 generation when the larvae were reared on diets without antibiotics (with gut microbes) and fed on 0.15% Bt or 12 μg Cry1Ab or Cry1Ac ml-1 diet, indicating development of resistance to Bt in the presence of gut microflora. However, there were no differences in larval mortality due to Bt, Cry1Ab or Cry1Ac across generations in insects when they were reared on diets with 250 μg of each antibiotic ml-1 diet (without gut microflora). The results suggested that antibiotics which eliminated gut microflora influenced the toxicity of Bt towards H. armigera, and any variation in diversity and abundance of gut microflora will have a major bearing on development of resistance to Bt toxins applied as foliar sprays or deployed in transgenic crops for pest management.

The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase.

PubMed

Devendran, Saravanan; Mythen, Sean M; Ridlon, Jason M

2018-06-01

Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydro-xyandrostenedione. A cortisol-inducible operon ( desABCD ) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were K m of 4.96 ± 0.57 µM and k cat of 0.87 ± 0.076 min -1 Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids. Copyright © 2018 Devendran et al.

Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718.

PubMed

Rang, Jie; He, Hao; Wang, Ting; Ding, Xuezhi; Zuo, Mingxing; Quan, Meifang; Sun, Yunjun; Yu, Ziquan; Hu, Shengbiao; Xia, Liqiu

2015-01-01

Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

Comparative safety of antiepileptic drugs for neurological development in children exposed during pregnancy and breast feeding: a systematic review and network meta-analysis.

PubMed

Veroniki, Areti Angeliki; Rios, Patricia; Cogo, Elise; Straus, Sharon E; Finkelstein, Yaron; Kealey, Ryan; Reynen, Emily; Soobiah, Charlene; Thavorn, Kednapa; Hutton, Brian; Hemmelgarn, Brenda R; Yazdi, Fatemeh; D'Souza, Jennifer; MacDonald, Heather; Tricco, Andrea C

2017-07-20

Compare the safety of antiepileptic drugs (AEDs) on neurodevelopment of infants/children exposed in utero or during breast feeding. Systematic review and Bayesian random-effects network meta-analysis (NMA). MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials were searched until 27 April 2017. Screening, data abstraction and quality appraisal were completed in duplicate by independent reviewers. 29 cohort studies including 5100 infants/children. Monotherapy and polytherapy AEDs including first-generation (carbamazepine, clobazam, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproate) and newer-generation (gabapentin, lamotrigine, levetiracetam, oxcarbazepine, topiramate, vigabatrin) AEDs. Epileptic women who did not receive AEDs during pregnancy or breast feeding served as the control group. Cognitive developmental delay and autism/dyspraxia were primary outcomes. Attention-deficit hyperactivity disorder, language delay, neonatal seizures, psychomotor developmental delay and social impairment were secondary outcomes. The NMA on cognitive developmental delay (11 cohort studies, 933 children, 18 treatments) suggested that among all AEDs only valproate was statistically significantly associated with more children experiencing cognitive developmental delay compared with control (OR=7.40, 95% credible interval (CrI) 3.00 to 18.46). The NMA on autism (5 cohort studies, 2551 children, 12 treatments) suggested that oxcarbazepine (OR 13.51, CrI 1.28 to 221.40), valproate (OR 17.29, 95% CrI 2.40 to 217.60), lamotrigine (OR 8.88, CrI 1.28 to 112.00) and lamotrigine+valproate (OR 132.70, CrI 7.41 to 3851.00) were associated with significantly greater odds of developing autism compared with control. The NMA on psychomotor developmental delay (11 cohort studies, 1145 children, 18 treatments) found that valproate (OR 4.16, CrI 2.04 to 8.75) and carbamazepine+phenobarbital+valproate (OR 19.12, CrI 1.49 to 337.50) were associated with significantly greater odds of psychomotor delay compared with control. Valproate alone or combined with another AED is associated with the greatest odds of adverse neurodevelopmental outcomes compared with control. Oxcarbazepine and lamotrigine were associated with increased occurrence of autism. Counselling is advised for women considering pregnancy to tailor the safest regimen. PROSPERO database (CRD42014008925). © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Comparative safety of antiepileptic drugs for neurological development in children exposed during pregnancy and breast feeding: a systematic review and network meta-analysis

PubMed Central

Veroniki, Areti Angeliki; Rios, Patricia; Cogo, Elise; Straus, Sharon E; Finkelstein, Yaron; Kealey, Ryan; Reynen, Emily; Soobiah, Charlene; Thavorn, Kednapa; Hutton, Brian; Hemmelgarn, Brenda R; Yazdi, Fatemeh; D'Souza, Jennifer; MacDonald, Heather; Tricco, Andrea C

2017-01-01

Objectives Compare the safety of antiepileptic drugs (AEDs) on neurodevelopment of infants/children exposed in utero or during breast feeding. Design and setting Systematic review and Bayesian random-effects network meta-analysis (NMA). MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials were searched until 27 April 2017. Screening, data abstraction and quality appraisal were completed in duplicate by independent reviewers. Participants 29 cohort studies including 5100 infants/children. Interventions Monotherapy and polytherapy AEDs including first-generation (carbamazepine, clobazam, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproate) and newer-generation (gabapentin, lamotrigine, levetiracetam, oxcarbazepine, topiramate, vigabatrin) AEDs. Epileptic women who did not receive AEDs during pregnancy or breast feeding served as the control group. Primary and secondary outcome measures Cognitive developmental delay and autism/dyspraxia were primary outcomes. Attention-deficit hyperactivity disorder, language delay, neonatal seizures, psychomotor developmental delay and social impairment were secondary outcomes. Results The NMA on cognitive developmental delay (11 cohort studies, 933 children, 18 treatments) suggested that among all AEDs only valproate was statistically significantly associated with more children experiencing cognitive developmental delay compared with control (OR=7.40, 95% credible interval (CrI) 3.00 to 18.46). The NMA on autism (5 cohort studies, 2551 children, 12 treatments) suggested that oxcarbazepine (OR 13.51, CrI 1.28 to 221.40), valproate (OR 17.29, 95% CrI 2.40 to 217.60), lamotrigine (OR 8.88, CrI 1.28 to 112.00) and lamotrigine+valproate (OR 132.70, CrI 7.41 to 3851.00) were associated with significantly greater odds of developing autism compared with control. The NMA on psychomotor developmental delay (11 cohort studies, 1145 children, 18 treatments) found that valproate (OR 4.16, CrI 2.04 to 8.75) and carbamazepine+phenobarbital+valproate (OR 19.12, CrI 1.49 to 337.50) were associated with significantly greater odds of psychomotor delay compared with control. Conclusions Valproate alone or combined with another AED is associated with the greatest odds of adverse neurodevelopmental outcomes compared with control. Oxcarbazepine and lamotrigine were associated with increased occurrence of autism. Counselling is advised for women considering pregnancy to tailor the safest regimen. Trial registration number PROSPERO database (CRD42014008925). PMID:28729328

Evidence for Horizontal Gene Transfer in Evolution of Elongation Factor Tu in Enterococci

PubMed Central

Ke, Danbing; Boissinot, Maurice; Huletsky, Ann; Picard, François J.; Frenette, Johanne; Ouellette, Marc; Roy, Paul H.; Bergeron, Michel G.

2000-01-01

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes. PMID:11092850

Large subunit of the ribonucleotide reductase gene is a virulent factor and plays a critical role in Marek's disease virus pathogenesis

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR) gene consisting of two subunits UL39 (RR1) and UL40 (RR2). Both RR1 and RR2 form an active holoenzyme and are necessary for enzyme activity. This gene was indentified by monoclonal antibody T81 in a gt11 MDV expression library and f...

Isolation of pheromone precursor genes of Magnaporthe grisea.

PubMed

Shen, W C; Bobrowicz, P; Ebbole, D J

1999-01-01

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia. Copyright 1999 Academic Press.

A Phase I Study of CPI-613 in Combination with High-Dose Cytarabine and Mitoxantrone for Relapsed or Refractory Acute Myeloid Leukemia.

PubMed

Pardee, Timothy S; Anderson, Rebecca G; Pladna, Kristin M; Isom, Scott; Ghiraldeli, Lais P; Miller, Lance D; Chou, Jeff W; Jin, Guangxu; Zhang, Wei; Ellis, Leslie R; Berenzon, Dmitriy; Howard, Dianna S; Hurd, David D; Manuel, Megan; Dralle, Sarah; Lyerly, Susan; Powell, Bayard L

2018-05-01

Purpose: CPI-613, a lipoate analogue that inhibits pyruvate dehydrogenase (PDH) and α-ketogluterate dehydrogenase (KGDH), has activity in patients with myeloid malignancies. This study explored the role of mitochondrial metabolism in chemotherapy response and determined the MTD, efficacy, and safety of CPI-613 combined with high-dose cytarabine and mitoxantrone in patients with relapsed or refractory acute myeloid leukemia. Experimental Design: The role of mitochondrial response to chemotherapy was assessed in cell lines and animal models. A phase I study of CPI-613 plus cytarabine and mitoxantrone was conducted in patients with relapsed or refractory AML. Results: Exposure to chemotherapy induced mitochondrial oxygen consumption that depended on PDH. CPI-613 sensitized AML cells to chemotherapy indicating that mitochondrial metabolism is a source of resistance. Loss of p53 did not alter response to CPI-613. The phase I study enrolled 67 patients and 62 were evaluable for response. The overall response rate was 50% (26CR+5CRi/62). Median survival was 6.7 months. In patients over 60 years old, the CR/CRi rate was 47% (15/32) with a median survival of 6.9 months. The response rate for patients with poor-risk cytogenetics also was encouraging with 46% (11/24 patients) achieving a CR or CRi. RNA sequencing analysis of a subset of baseline bone marrow samples revealed a gene expression signature consistent with the presence of B cells in the pretreatment marrow of responders. Conclusions: The addition of CPI-613 to chemotherapy is a promising approach in older patients and those with poor-risk cytogenetics. Clin Cancer Res; 24(9); 2060-73. ©2018 AACR . ©2018 American Association for Cancer Research.

Cry1F resistance in fall armyworm Spodoptera frugiperda: single gene versus pyramided Bt maize

USDA-ARS?s Scientific Manuscript database

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. af...

[Impacts of genetically modified soybean leaf residues on Folsomia candida.

PubMed

Zhou, Lin; Wang, Bai Feng; Liu, Xin Ying; Jiang, Ying; Wang, Da Ming; Feng, Shu Dan; Song, Xin Yuan

2016-09-01

When the genetically modified soybean is planted in the field, the expression product of exogenous gene could be exposed in the soil ecosystem and bring potential risk to the soil fauna, with the form of leaves and other debris. A few of genetically modified soybeans developed by China independently were used in our study as materials. They were Phytophthora-resistant soybean harboring hrpZm gene (B4J8049), leaf-feeding insect-resistant soybean harboring Cry1C gene (A2A8001) and Leguminivora glycinivorella-resistant soybean harboring Cry1Iem gene (C802). By feeding Folsomia candida with the three genetically modified soybeans for continuous 60 days, the surviving rate, reproductive rate and changes on the body length of F. candida were studied. The results showed that all the three genetically modified soybeans of B4J8049, A2A8001 and C802 had no significant adverse effects on the growth of F. candida, as an environmental indicator organism. It was initially inferred that they were environmentally safe under short-term exposure, which provided basic data of ecological safety for their wide cultivation.

A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chun, Sung Kook; Department of Biological Sciences, Seoul National University, Seoul, 151-747; Department of Brain & Cognitive Sciences, Seoul National University, Seoul, 151-747

Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes,more » were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer. - Highlights: • Cryptochrome inhibitor (KS15) has anti-tumor activity to human breast cancer cells. • KS15 induces differential changes in cell cycle regulators and pro-apoptotic genes. • KS15 inhibits MCF-7 cell growth and enhances susceptibility to anti-tumor drugs.« less

Population dynamics of Sesamia inferens on transgenic rice expressing Cry1Ac and CpTI in southern China.

PubMed

Han, Lanzhi; Liu, Peilei; Wu, Kongming; Peng, Yufa; Wang, Feng

2008-10-01

Genetically modified insect-resistant rice lines containing the cry1Ac gene from Bacillus thuringiensis (Bt) or the CpTI (cowpea trypsin inhibitor) gene developed for the management of lepidopterous pests are highly resistant to the major target pests, Chilo suppressalis (Walker), Cnaphalocrocis medinalis (Guenée), and Scirpophaga incertulas (Walker), in the main rice-growing areas of China. However, the effects of these transgenic lines on Sesamia inferens (Walker), an important lepidopterous rice pest, are currently unknown. Because different insect species have varying susceptibility to Bt insecticidal proteins that may affect population dynamics, research into the effects of these transgenic rice lines on the population dynamics of S. inferens was conducted in Fuzhou, southern China, in 2005 and 2006. The results of laboratory, field cage, and field plot experiments show that S. inferens has comparatively high susceptibility to the transgenic line during the early growing season, with significant differences observed in larval density and infestation levels between transgenic and control lines. Because of a decrease in Cry1Ac levels in the plant as it ages, the transgenic line provided only a low potential for population suppression late in the growing season. There is a correlation between the changing expression of Cry1Ac and the impact of transgenic rice on the population dynamics of S. inferens during the season. These results indicate that S. inferens may become a major pest in fields of prospective commercially released transgenic rice, and more attention should be paid to developing an effective alternative management strategy.

Effects of Lidocaine, Dexmedetomidine or Their Combination on the Minimum Alveolar Concentration of Sevoflurane in Dogs

PubMed Central

MORAN-MUÑOZ, Rafael; IBANCOVICHI, J. A.; Gutierrez-BLANCO, Eduardo; ACEVEDO-ARCIQUE, Carlos M.; Victoria MORA, J. Mauro; TENDILLO, Francisco J.; SANTOS-GONZALEZ, Martin; YAMASHITA, Kazuto

2014-01-01

ABSTRACT The aim of this study was to determine the effects of lidocaine (LIDO) and dexmedetomidine (DEX) or their combination (LIDO–DEX), administered by constant-rate infusion (CRI), on the minimum alveolar concentration (MAC) of sevoflurane in dogs. Seven healthy mongrel dogs were used with a 2-week washout interval between treatments in this study. Anesthesia was induced with propofol and maintained with sevoflurane in oxygen, and MAC of sevoflurane was determined after 90 min equilibration period in the dogs (SEV-MACBASAL). Then, sevoflurane MAC was determined again in the dogs after 45 min equilibration period of one of the following treatments: an intravenous loading dose of lidocaine 2 mg/kg followed by 6 mg/kg/hr CRI (SEV-MACLIDO); an intravenous loading dose of dexmedetomidine 2 µg/kg followed by 2 µg/kg/hr CRI (SEV-MACDEX); or their combination (SEV-MACLIDO-DEX). These SEV-MACs were determined in duplicate. Data were analyzed using ANOVA and post hoc Tuckey test when appropriate. The SEV-MACBASAL was 1.82 ± 0.06%, SEV-MACLIDO was 1.38 ± 0.08%, SEV-MACDEX was 1.22 ± 0.10%, and SEV-MACLIDO-DEX was 0.78 ± 0.06%. The CRI administration of lidocaine, dexmedetomidine and their combination produced a significant reduction in the MAC of sevoflurane by 26.1 ± 9.0% (P<0.0001), 43.7 ± 11.8% (P<0.0002) and 54.4 ± 9.8% (P<0.0001), respectively. The MAC reduction was significantly greater after the CRI combination of lidocaine and dexmedetomidine when compared with lidocaine CRI (P<0.0001) or dexmedetomidine CRI treatments (P<0.025). PMID:24572631

The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction.

PubMed

Shu, Yinghua; Ma, Honghui; Du, Yan; Li, Zhixian; Feng, Yuanjiao; Wang, Jianwu

2011-11-01

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction. Copyright © 2011 Elsevier Ltd. All rights reserved.

The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B.

PubMed

Grabarczyk, Piotr; Winkler, Passorn; Delin, Martin; Sappa, Praveen K; Bekeschus, Sander; Hildebrandt, Petra; Przybylski, Grzegorz K; Völker, Uwe; Hammer, Elke; Schmidt, Christian A

2018-03-01

The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization. Copyright © 2018 American Society for Microbiology.

Genetic determinants restricting the reassortment of heterologous NSP2 genes into the simian rotavirus SA11 genome.

PubMed

Mingo, Rebecca; Zhang, Shu; Long, Courtney P; LaConte, Leslie E W; McDonald, Sarah M

2017-08-24

Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784-820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784-820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.

Circadian expression of clock and putative clock-controlled genes in skeletal muscle of the zebrafish.

PubMed

Amaral, Ian P G; Johnston, Ian A

2012-01-01

To identify circadian patterns of gene expression in skeletal muscle, adult male zebrafish were acclimated for 2 wk to a 12:12-h light-dark photoperiod and then exposed to continuous darkness for 86 h with ad libitum feeding. The increase in gut food content associated with the subjective light period was much diminished by the third cycle, enabling feeding and circadian rhythms to be distinguished. Expression of zebrafish paralogs of mammalian transcriptional activators of the circadian mechanism (bmal1, clock1, and rora) followed a rhythmic pattern with a ∼24-h periodicity. Peak expression of rora paralogs occurred at the beginning of the subjective light period [Zeitgeber time (ZT)07 and ZT02 for roraa and rorab], whereas the highest expression of bmal1 and clock paralogs occurred 12 h later (ZT13-15 and ZT16 for bmal and clock paralogs). Expression of the transcriptional repressors cry1a, per1a/1b, per2, per3, nr1d2a/2b, and nr1d1 also followed a circadian pattern with peak expression at ZT0-02. Expression of the two paralogs of cry2 occurred in phase with clock1a/1b. Duplicated genes had a high correlation of expression except for paralogs of clock1, nr1d2, and per1, with cry1b showing no circadian pattern. The highest expression difference was 9.2-fold for the activator bmal1b and 51.7-fold for the repressor per1a. Out of 32 candidate clock-controlled genes, only myf6, igfbp3, igfbp5b, and hsf2 showed circadian expression patterns. Igfbp3, igfbp5b, and myf6 were expressed in phase with clock1a/1b and had an average of twofold change in expression from peak to trough, whereas hsf2 transcripts were expressed in phase with cry1a and had a 7.2-fold-change in expression. The changes in expression of clock and clock-controlled genes observed during continuous darkness were also observed at similar ZTs in fish exposed to a normal photoperiod in a separate control experiment. The role of circadian clocks in regulating muscle maintenance and growth are discussed.

The fa2 gene and molecular markers mapping in the gp segment of the Pisum linkage group V.

PubMed

Gawłowska, M; Święcicki, W

2016-08-01

Review studies on the world Pisum genetic resources have shown that stem fasciation is controlled by three loci, i.e., fa1 (LGIV; Wt 10006 - type line of the Polish Gene Bank), fa2 (LGV, the line Wt 12185), and fas (LGIII, the line Shtambovii). Outstanding advantages of this character (e.g., pods gathered in upper part of a stem) resulted in breeding some cultivars. Preliminary investigations suggested linkages of the newly described fa2 gene within the gp-U segment. Based on the further linkage test crosses, it was stated that the fa2 is localized between the gp and Pis_Gen_9_3_1 markers (in the LGV). Additionally, four molecular markers (AD175, AB146, AC58, and AD280) and the morphological marker lk were also localized in this segment. Moreover, rms5, lum3, and cri were found to map on the other side of gp with tight linkage observed between lum3 and cri.

Polymorphisms of genes encoding P2X7R, IL-1B, OPG and RANK in orthodontic-induced apical root resorption.

PubMed

Pereira, S; Lavado, N; Nogueira, L; Lopez, M; Abreu, J; Silva, H

2014-10-01

Orthodontic-induced external apical root resorption (EARR) is a complex phenotype determined by poorly defined mechanical and patient intrinsic factors. The aim of this work was to construct a multifactorial integrative model, including clinical and genetic susceptibility factors, to analyze the risk of developing this common orthodontic complication. This retrospective study included 195 orthodontic patients. Using a multiple-linear regression model, where the dependent variable was the maximum% of root resorption (%EARRmax) for each patient, we assessed the contribution of nine clinical variables and four polymorphisms of genes involved in bone and tooth root remodeling (rs1718119 from P2RX7, rs1143634 from IL1B, rs3102735 from TNFRSF11B, encoding OPG, and rs1805034 from TNFRSF11A, encoding RANK). Clinical and genetic variables explained 30% of%EARRmax variability. The variables with the most significant unique contribution to the model were: gender (P < 0.05), treatment duration (P < 0.001), premolar extractions (P < 0.01), Hyrax appliance (P < 0.001) and GG genotype of rs1718119 from P2RX7 gene (P < 0.01). Age, overjet, tongue thrust, skeletal class II and the other polymorphisms made minor contributions. This study highlights the P2RX7 gene as a possible factor of susceptibility to EARR. A more extensive genetic profile may improve this model. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Excessive infant crying doubles the risk of mood and behavioral problems at age 5: evidence for mediation by maternal characteristics.

PubMed

Smarius, Laetitia Joanna Clara Antonia; Strieder, Thea G A; Loomans, Eva M; Doreleijers, Theo A H; Vrijkotte, Tanja G M; Gemke, Reinoud J; van Eijsden, Manon

2017-03-01

The onset of behavioral problems starts in early life. This study examined whether excessive infant crying (maternal ratings) is a determinant of emotional and behavioral problems at age 5-6 years. In the Amsterdam Born Children and their Development (ABCD) study, a large prospective, observational, population-based multiethnic birth cohort, excessive infant crying (crying for three or more hours per 24 h day over the past week) during the 13th week after birth (range 11-25 weeks, SD 2 weeks), maternal burden of infant care and maternal aggressive behavior (either angry speaking, or physical aggression) was assessed using a questionnaire. Children's behavioral and emotional problems at the age of 5-6 were assessed by Goodman's Strengths and Difficulties Questionnaire (SDQ), by the subscale of generalized anxiety of the preschool anxiety scale (PAS), and by the Short Mood and Feelings Questionnaire (SMFQ). Inclusion criterion was singleton birth. Exclusion criteria were preterm born babies or congenital disorders. Among 3389 children, excessive infant crying (n = 102) was associated with a twofold increased risk of the overall problem behavior, conduct problems, hyperactivity, and mood problems at the age of 5-6 [ORs between 1.75 (95 % CI 1.09-2.81) and 2.12 (95 % CI 1.30-3.46)]. This association was mediated by maternal burden of infant care (change in odds' ratio 1-17 %) and maternal aggressive behavior (change in odds' ratio 4-10 %). There was no effect modification by the child's gender or maternal parity. Excessive infant crying was not associated with general anxiety problems. Excessive infant crying doubles the risk of behavioral, hyperactivity, and mood problems at the age of 5-6, as reported by their mother. Maternal burden of infant care partially mediates the association between excessive crying and behavioral and mood problems. Special care for mothers with a high burden of care for their excessive crying infant, notwithstanding their own good health, can be a feasible strategy for possible prevention of mood and behavioral problems in their children later in life.

A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges.

PubMed

Boualem, Adnane; Troadec, Christelle; Camps, Céline; Lemhemdi, Afef; Morin, Halima; Sari, Marie-Agnes; Fraenkel-Zagouri, Rina; Kovalski, Irina; Dogimont, Catherine; Perl-Treves, Rafael; Bendahmane, Abdelhafid

2015-11-06

Understanding the evolution of sex determination in plants requires identifying the mechanisms underlying the transition from monoecious plants, where male and female flowers coexist, to unisexual individuals found in dioecious species. We show that in melon and cucumber, the androecy gene controls female flower development and encodes a limiting enzyme of ethylene biosynthesis, ACS11. ACS11 is expressed in phloem cells connected to flowers programmed to become female, and ACS11 loss-of-function mutants lead to male plants (androecy). CmACS11 represses the expression of the male promoting gene CmWIP1 to control the development and the coexistence of male and female flowers in monoecious species. Because monoecy can lead to dioecy, we show how a combination of alleles of CmACS11 and CmWIP1 can create artificial dioecy. Copyright © 2015, American Association for the Advancement of Science.

Functional characterization of a prokaryotic Kir channel.

PubMed

Enkvetchakul, Decha; Bhattacharyya, Jaya; Jeliazkova, Iana; Groesbeck, Darcy K; Cukras, Catherine A; Nichols, Colin G

2004-11-05

The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.

Post-Transcriptional Regulation of the Trypanosome Heat Shock Response by a Zinc Finger Protein

PubMed Central

Droll, Dorothea; Minia, Igor; Fadda, Abeer; Singh, Aditi; Stewart, Mhairi; Queiroz, Rafael; Clayton, Christine

2013-01-01

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3′-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures. PMID:23592996

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes

PubMed Central

Tang, Huiwu; Zheng, Xingmei; Li, Chuliang; Xie, Xianrong; Chen, Yuanling; Chen, Letian; Zhao, Xiucai; Zheng, Huiqi; Zhou, Jiajian; Ye, Shan; Guo, Jingxin; Liu, Yao-Guang

2017-01-01

New gene origination is a major source of genomic innovations that confer phenotypic changes and biological diversity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility (CMS), which can promote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and function remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c (previously named WA352) and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary trajectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with substoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COX11, resulting in premature programmed cell death in the anther tapetum and male sterility. Furthermore, we show that the sequences that encode the COX11-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the formation and evolution of new CMS genes via a “multi-recombination/protogene formation/functionalization” mechanism involving gradual variations in the structure, sequence, copy number, and function. PMID:27725674

[Correlation between thermostability of the xylanase EvXyn11(TS) and its N-terminal disulfide bridge].

PubMed

Min, Rou; Li, Jianfang; Gao, Shujuan; Zhang, Huimin; Wu, Jing; Wu, Minchen

2013-04-04

To reveal the correlation between thermostability of xylanase EvXyn11(TS) and its N-terminal disulfide bridge, an EvXyn11(TS)-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis. Multiple homology alignment of protein primary structures between the EvXyn11(TS) and several GH family 11 xylanases displayed that, in their N-termini, only EvXyn11(TS) contained a disulfide bridge (Cys5-Cys32), whose effect on the xylanase thermostability was predicted by molecular dynamics simulation. We constructed a gene Syxyn11(M), encoding the mutated xylanase (EvXyn11(M)) without N-terminal disulfide bridge. Then, Syxyn11 and Syxyn11(M) were expressed in Pichia pastoris GS115, and temperature and pH properties of the expressed enzymes were analyzed. The analytical results displayed that the temperature optimum of EvXyn11(M) was 70 degrees C, which was 15 degrees C lower than that of EvXyn11(TS). The half-life (t1/2(90)) of EvXyn11(TS) at 90 degrees C was 32 min, while the t1/2(70) of EvXyn11(M) at 70 degrees C was only 8.0 min. The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11(TS) was first predicted by molecular dynamics simulation, and confirmed by site-directed mutagenesis. This work provided a novel strategy to improve thermostabilities of the mesophilic family 11 xylanases with high specific activities.

The correlation between cellulose allomorphs (I and II) and conversion after removal of hemicellulose and lignin of lignocellulose.

PubMed

Song, Yanliang; Zhang, Jingzhi; Zhang, Xu; Tan, Tianwei

2015-10-01

H2SO4, NaOH and H3PO4 were applied to decompose lignocellulose samples (giant reeds, pennisetum and cotton stalks) to investigate the correlation between cellulose allomorphs (cellulose I and II) and conversion of cellulose. The effect of removal of hemicellulose and lignin on the surface morphology, crystallinity index (CrI), cellulose allomorphs (cellulose I and II), and enzymatic hydrolysis under different pretreatments was also studied. CrI caused by H3PO4 pretreatment reached 11.19%, 24.93% and 8.15% for the three samples, respectively. Corn stalk showed highest conversion of cellulose among three samples, irrespective of the pretreatment used. This accounted for the widely use of corn stalk as the renewable crop substrate to synthesize biofuels like ethanol. CrI of cellulose I (CrI-I) negatively affects cellulose conversion but CrI of cellulose II (CrI-II) positively affects cellulose conversion. It contributes to make the strategy to transform cellulose I to cellulose II and enhancing enzymatic hydrolysis of lignocellulose. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antigen S1, encoded by the MIC1 gene, is characterized as an epitope of human CD59, enabling measurement of mutagen-induced intragenic deletions in the AL cell system

NASA Technical Reports Server (NTRS)

Wilson, A. B.; Seilly, D.; Willers, C.; Vannais, D. B.; McGraw, M.; Waldren, C. A.; Hei, T. K.; Davies, A.; Chatterjee, A. (Principal Investigator)

1999-01-01

S1 cell membrane antigen is encoded by the MIC1 gene on human chromosome 11. This antigen has been widely used as a marker for studies in gene mapping or in analysis of mutagen-induced gene deletions/mutations, which utilized the human-hamster hybrid cell-line, AL-J1, carrying human chromosome 11. Evidence is presented here which identifies S1 as an epitope of CD59, a cell membrane complement inhibiting protein. E7.1 monoclonal antibody, specific for the S1 determinant, was found to react strongly with membrane CD59 in Western blotting, and to bind to purified, urinary form of CD59 in ELISAs. Cell membrane expression of S1 on various cell lines always correlated with that of CD59 when examined by immunofluorescent staining. In addition, E7.1 antibody inhibited the complement regulatory function of CD59. Identification of S1 protein as CD59 has increased the scope of the AL cell system by enabling analysis of intragenic mutations, and multiplex PCR analysis of mutated cells is described, showing variable loss of CD59 exons.

Exploring the Midgut Transcriptome and Brush Border Membrane Vesicle Proteome of the Rice Stem Borer, Chilo suppressalis (Walker)

PubMed Central

Peng, Chuanhua; Wang, Xiaoping; Li, Fei; Lin, Yongjun

2012-01-01

The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac. PMID:22666467

Effect of Roux-en-Y gastric bypass on the distribution and hormone expression of small-intestinal enteroendocrine cells in obese patients with type 2 diabetes.

PubMed

Rhee, Nicolai A; Wahlgren, Camilla D; Pedersen, Jens; Mortensen, Brynjulf; Langholz, Ebbe; Wandall, Erik P; Friis, Steffen U; Vilmann, Peter; Paulsen, Sarah J; Kristiansen, Viggo B; Jelsing, Jacob; Dalbøge, Louise S; Poulsen, Steen S; Holst, Jens J; Vilsbøll, Tina; Knop, Filip K

2015-10-01

We studied the impact of Roux-en-Y gastric bypass (RYGB) on the density and hormonal gene expression of small-intestinal enteroendocrine cells in obese patients with type 2 diabetes. Twelve patients with diabetes and 11 age- and BMI-matched controls underwent RYGB followed by enteroscopy ~10 months later. Mucosal biopsies taken during surgery and enteroscopy were immunohistochemically stained for glucagon-like peptide-1 (GLP-1), peptide YY (PYY), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and prohormone convertase 2 (PC2) and the expression of GCG (encoding preproglucagon), PYY, CCK, GIP, GHRL (encoding ghrelin), SCT (encoding secretin), NTS (encoding neurotensin) and NR1H4 (encoding farnesoid X receptor) was evaluated. The density of cells immunoreactive for GLP-1, CCK and GIP increased in patients after RYGB and the density of those immunoreactive for GLP-1, PYY, CCK and PC2 increased in controls. In both groups, GHRL, SCT and GIP mRNA was reduced after RYGB while PYY, CCK, NTS and NR1H4 gene expression was unaltered. GCG mRNA was upregulated in both groups. Numerous alterations in the distribution of enteroendocrine cells and their expression of hormonal genes are seen after RYGB and include increased density of GLP-1-, PYY-, CCK-, GIP- and PC2-positive cells, reduced gene expression of GHRL, SCT and GIP and increased expression of GCG.

Behavioral and genetic correlates of the neural response to infant crying among human fathers.

PubMed

Mascaro, Jennifer S; Hackett, Patrick D; Gouzoules, Harold; Lori, Adriana; Rilling, James K

2014-11-01

Although evolution has shaped human infant crying and the corresponding response from caregivers, there is marked variation in paternal involvement and caretaking behavior, highlighting the importance of understanding the neurobiology supporting optimal paternal responses to cries. We explored the neural response to infant cries in fathers of children aged 1-2, and its relationship with hormone levels, variation in the androgen receptor (AR) gene, parental attitudes and parental behavior. Although number of AR CAG trinucleotide repeats was positively correlated with neural activity in brain regions important for empathy (anterior insula and inferior frontal gyrus), restrictive attitudes were inversely correlated with neural activity in these regions and with regions involved with emotion regulation (orbitofrontal cortex). Anterior insula activity had a non-linear relationship with paternal caregiving, such that fathers with intermediate activation were most involved. These results suggest that restrictive attitudes may be associated with decreased empathy and emotion regulation in response to a child in distress, and that moderate anterior insula activity reflects an optimal level of arousal that supports engaged fathering. © The Author (2013). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Cry1F resistance among lepidopteran pests: a model for improved resistance management?

PubMed

Vélez, Ana M; Vellichirammal, Neetha Nanoth; Jurat-Fuentes, Juan Luis; Siegfried, Blair D

2016-06-01

The Cry1Fa protein from the bacterium Bacillus thuringiensis (Bt) is known for its potential to control lepidopteran pests, especially through transgenic expression in maize and cotton. The maize event TC1507 expressing the cry1Fa toxin gene became commercially available in the United States in 2003 for the management of key lepidopteran pests including the European corn borer, Ostrinia nubilalis, and the fall armyworm, Spodoptera frugiperda. A high-dose/refuge strategy has been widely adopted to delay evolution of resistance to event TC1507 and other transgenic Bt crops. Efficacy of this strategy depends on the crops expressing a high dose of the Bt toxin to targeted pests and adjacent refuges of non-Bt host plants serving as a source of abundant susceptible insects. While this strategy has proved effective in delaying O. nubilalis resistance, field-evolved resistance to event TC1507 has been reported in S. frugiperda populations in Puerto Rico, Brazil, and the southeastern United States. This paper examines available information on resistance to Cry1Fa in O. nubilalis and S. frugiperda and discusses how this information identifies opportunities to refine resistance management recommendations for Bt maize. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

PubMed Central

Oh, Eun Joong; Skerker, Jeffrey M.; Kim, Soo Rin; Wei, Na; Turner, Timothy L.; Maurer, Matthew J.; Arkin, Adam P.

2016-01-01

ABSTRACT Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1. We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. IMPORTANCE In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering. PMID:27084006

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

PubMed

Oh, Eun Joong; Skerker, Jeffrey M; Kim, Soo Rin; Wei, Na; Turner, Timothy L; Maurer, Matthew J; Arkin, Adam P; Jin, Yong-Su

2016-06-15

Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Activity of a Brazilian strain of Bacillus thuringiensis israelensis against the cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae).

PubMed

Monnerat, R; Martins, E; Praça, L; Dumas, V; Berry, C

2012-02-01

A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC50 values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.

Human cytomegalovirus inhibits antigen presentation by a sequential multistep process.

PubMed Central

Ahn, K; Angulo, A; Ghazal, P; Peterson, P A; Yang, Y; Früh, K

1996-01-01

The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8855296

Permanent Neonatal Diabetes Caused by Dominant, Recessive, or Compound Heterozygous SUR1 Mutations with Opposite Functional Effects

PubMed Central

Ellard, Sian ; Flanagan, Sarah E. ; Girard, Christophe A. ; Patch, Ann-Marie ; Harries, Lorna W. ; Parrish, Andrew ; Edghill, Emma L. ; Mackay, Deborah J. G. ; Proks, Peter ; Shimomura, Kenju ; Haberland, Holger ; Carson, Dennis J. ; Shield, Julian P. H. ; Hattersley, Andrew T. ; Ashcroft, Frances M. 

2007-01-01

Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell KATP channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the KATP channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present. PMID:17668386

Non-functional plastid ndh gene fragments are present in the nuclear genome of Norway spruce (Picea abies L. Karsch): insights from in silico analysis of nuclear and organellar genomes.

PubMed

Ranade, Sonali Sachin; García-Gil, María Rosario; Rosselló, Josep A

2016-04-01

Many genes have been lost from the prokaryote plastidial genome during the early events of endosymbiosis in eukaryotes. Some of them were definitively lost, but others were relocated and functionally integrated to the host nuclear genomes through serial events of gene transfer during plant evolution. In gymnosperms, plastid genome sequencing has revealed the loss of ndh genes from several species of Gnetales and Pinaceae, including Norway spruce (Picea abies). This study aims to trace the ndh genes in the nuclear and organellar Norway spruce genomes. The plastid genomes of higher plants contain 11 ndh genes which are homologues of mitochondrial genes encoding subunits of the proton-pumping NADH-dehydrogenase (nicotinamide adenine dinucleotide dehydrogenase) or complex I (electron transport chain). Ndh genes encode 11 NDH polypeptides forming the Ndh complex (analogous to complex I) which seems to be primarily involved in chloro-respiration processes. We considered ndh genes from the plastidial genome of four gymnosperms (Cryptomeria japonica, Cycas revoluta, Ginkgo biloba, Podocarpus totara) and a single angiosperm species (Arabidopsis thaliana) to trace putative homologs in the nuclear and organellar Norway spruce genomes using tBLASTn to assess the evolutionary fate of ndh genes in Norway spruce and to address their genomic location(s), structure, integrity and functionality. The results obtained from tBLASTn were subsequently analyzed by performing homology search for finding ndh specific conserved domains using conserved domain search. We report the presence of non-functional plastid ndh gene fragments, excepting ndhE and ndhG genes, in the nuclear genome of Norway spruce. Regulatory transcriptional elements like promoters, TATA boxes and enhancers were detected in the upstream regions of some ndh fragments. We also found transposable elements in the flanking regions of few ndh fragments suggesting nuclear rearrangements in those regions. These evidences support the hypothesis that, at least in Picea, ndh translocations from the plastid to the nuclear genome have occurred, and that there might have been a functional machinery at some time during evolution to accommodate them within a nuclear-encoded environment, or attempts to form it.

Distinct Calcium Signaling Pathways Regulate Calmodulin Gene Expression in Tobacco1

PubMed Central

van der Luit, Arnold H.; Olivari, Claudio; Haley, Ann; Knight, Marc R.; Trewavas, Anthony J.

1999-01-01

Cold shock and wind stimuli initiate Ca2+ transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca2+ pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca2+ transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca2+ signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca2+ dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca2+ signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm. PMID:10557218

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

PubMed

de Oliveira, Raquel S; Oliveira-Neto, Osmundo B; Moura, Hudson F N; de Macedo, Leonardo L P; Arraes, Fabrício B M; Lucena, Wagner A; Lourenço-Tessutti, Isabela T; de Deus Barbosa, Aulus A; da Silva, Maria C M; Grossi-de-Sa, Maria F

2016-01-01

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

Influence of transgenic rice expressing a fused Cry1Ab/1Ac protein on frogs in paddy fields.

PubMed

Wang, Jia-Mei; Chen, Xiu-Ping; Liang, Yu-Yong; Zhu, Hao-Jun; Ding, Jia-Tong; Peng, Yu-Fa

2014-11-01

As genetic engineering in plants is increasingly used to control agricultural pests, it is important to determine whether such transgenic plants adversely affect non-target organisms within and around cultivated fields. The cry1Ab/1Ac fusion gene from Bacillus thuringiensis (Bt) has insecticidal activity and has been introduced into rice line Minghui 63 (MH63). We evaluated the effect of transgenic cry1Ab/1Ac rice (Huahui 1, HH1) on paddy frogs by comparing HH1 and MH63 rice paddies with and without pesticide treatment. The density of tadpoles in rice fields was surveyed at regular intervals, and Cry1Ab/1Ac protein levels were determined in tissues of tadpoles and froglets collected from the paddy fields. In addition, Rana nigromaculata froglets were raised in purse nets placed within these experimental plots. The survival, body weight, feeding habits, and histological characteristics of the digestive tract of these froglets were analyzed. We found that the tadpole density was significantly decreased immediately after pesticide application, and the weight of R. nigromaculata froglets of pesticide groups was significantly reduced compared with no pesticide treatment, but we found no differences between Bt and non-Bt rice groups. Moreover, no Cry1Ab/1Ac protein was detected in tissue samples collected from 192 tadpoles and froglets representing all four experimental groups. In addition, R. nigromaculata froglets raised in purse seines fed primarily on stem borer and non-target insects, and showed no obvious abnormality in the microstructure of their digestive tracts. Based on these results, we conclude that cultivation of transgenic cry1Ab/1Ac rice does not adversely affect paddy frogs.

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis)

PubMed Central

de Oliveira, Raquel S.; Oliveira-Neto, Osmundo B.; Moura, Hudson F. N.; de Macedo, Leonardo L. P.; Arraes, Fabrício B. M.; Lucena, Wagner A.; Lourenço-Tessutti, Isabela T.; de Deus Barbosa, Aulus A.; da Silva, Maria C. M.; Grossi-de-Sa, Maria F.

2016-01-01

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests. PMID:26925081

Genome analysis of Daldinia eschscholtzii strains UM 1400 and UM 1020, wood-decaying fungi isolated from human hosts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, Chai Ling; Yew, Su Mei; Ngeow, Yun Fong

Background: Daldinia eschscholtzii is a wood-inhabiting fungus that causes wood decay under certain conditions. It has a broad host range and produces a large repertoire of potentially bioactive compounds. However, there is no extensive genome analysis on this fungal species. Results: Two fungal isolates (UM 1400 and UM 1020) from human specimens were identified as Daldinia eschscholtzii by morphological features and ITS-based phylogenetic analysis. Both genomes were similar in size with 10,822 predicted genes in UM 1400 (35.8 Mb) and 11,120 predicted genes in UM 1020 (35.5 Mb). A total of 751 gene families were shared among both UM isolates,more » including gene families associated with fungus-host interactions. In the CAZyme comparative analysis, both genomes were found to contain arrays of CAZyme related to plant cell wall degradation. Genes encoding secreted peptidases were found in the genomes, which encode for the peptidases involved in the degradation of structural proteins in plant cell wall. In addition, arrays of secondary metabolite backbone genes were identified in both genomes, indicating of their potential to produce bioactive secondary metabolites. Both genomes also contained an abundance of gene encoding signaling components, with three proposed MAPK cascades involved in cell wall integrity, osmoregulation, and mating/filamentation. Besides genomic evidence for degrading capability, both isolates also harbored an array of genes encoding stress response proteins that are potentially significant for adaptation to living in the hostile environments. In conclusion: Our genomic studies provide further information for the biological understanding of the D. eschscholtzii and suggest that these wood-decaying fungi are also equipped for adaptation to adverse environments in the human host.« less

Genome analysis of Daldinia eschscholtzii strains UM 1400 and UM 1020, wood-decaying fungi isolated from human hosts

DOE PAGES

Chan, Chai Ling; Yew, Su Mei; Ngeow, Yun Fong; ...

2015-11-18

Background: Daldinia eschscholtzii is a wood-inhabiting fungus that causes wood decay under certain conditions. It has a broad host range and produces a large repertoire of potentially bioactive compounds. However, there is no extensive genome analysis on this fungal species. Results: Two fungal isolates (UM 1400 and UM 1020) from human specimens were identified as Daldinia eschscholtzii by morphological features and ITS-based phylogenetic analysis. Both genomes were similar in size with 10,822 predicted genes in UM 1400 (35.8 Mb) and 11,120 predicted genes in UM 1020 (35.5 Mb). A total of 751 gene families were shared among both UM isolates,more » including gene families associated with fungus-host interactions. In the CAZyme comparative analysis, both genomes were found to contain arrays of CAZyme related to plant cell wall degradation. Genes encoding secreted peptidases were found in the genomes, which encode for the peptidases involved in the degradation of structural proteins in plant cell wall. In addition, arrays of secondary metabolite backbone genes were identified in both genomes, indicating of their potential to produce bioactive secondary metabolites. Both genomes also contained an abundance of gene encoding signaling components, with three proposed MAPK cascades involved in cell wall integrity, osmoregulation, and mating/filamentation. Besides genomic evidence for degrading capability, both isolates also harbored an array of genes encoding stress response proteins that are potentially significant for adaptation to living in the hostile environments. In conclusion: Our genomic studies provide further information for the biological understanding of the D. eschscholtzii and suggest that these wood-decaying fungi are also equipped for adaptation to adverse environments in the human host.« less

A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes.

PubMed

Sahoo, Dipak K; Abeysekara, Nilwala S; Cianzio, Silvia R; Robertson, Alison E; Bhattacharyya, Madan K

2017-01-01

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

The location and translocation of ndh genes of chloroplast origin in the Orchidaceae family

PubMed Central

Lin, Choun-Sea; Chen, Jeremy J. W.; Huang, Yao-Ting; Chan, Ming-Tsair; Daniell, Henry; Chang, Wan-Jung; Hsu, Chen-Tran; Liao, De-Chih; Wu, Fu-Huei; Lin, Sheng-Yi; Liao, Chen-Fu; Deyholos, Michael K.; Wong, Gane Ka-Shu; Albert, Victor A.; Chou, Ming-Lun; Chen, Chun-Yi; Shih, Ming-Che

2015-01-01

The NAD(P)H dehydrogenase complex is encoded by 11 ndh genes in plant chloroplast (cp) genomes. However, ndh genes are truncated or deleted in some autotrophic Epidendroideae orchid cp genomes. To determine the evolutionary timing of the gene deletions and the genomic locations of the various ndh genes in orchids, the cp genomes of Vanilla planifolia, Paphiopedilum armeniacum, Paphiopedilum niveum, Cypripedium formosanum, Habenaria longidenticulata, Goodyera fumata and Masdevallia picturata were sequenced; these genomes represent Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae subfamilies. Four orchid cp genome sequences were found to contain a complete set of ndh genes. In other genomes, ndh deletions did not correlate to known taxonomic or evolutionary relationships and deletions occurred independently after the orchid family split into different subfamilies. In orchids lacking cp encoded ndh genes, non cp localized ndh sequences were identified. In Erycina pusilla, at least 10 truncated ndh gene fragments were found transferred to the mitochondrial (mt) genome. The phenomenon of orchid ndh transfer to the mt genome existed in ndh-deleted orchids and also in ndh containing species. PMID:25761566

The protocadherin 11X/Y (PCDH11X/Y) gene pair as determinant of cerebral asymmetry in modern Homo sapiens.

PubMed

Priddle, Thomas H; Crow, Timothy J

2013-06-01

Annett's right-shift theory proposes that human cerebral dominance (the functional and anatomical asymmetry or torque along the antero-posterior axis) and handedness are determined by a single "right-shift" gene. Familial transmission of handedness and specific deviations of cerebral dominance in sex chromosome aneuploidies implicate a locus within an X-Y homologous region of the sex chromosomes. The Xq21.3/Yp11.2 human-specific region of homology includes the protocadherin 11X/Y (PCDH11X/Y) gene pair, which encode cell adhesion molecules subject to accelerated evolution following the separation of the human and chimpanzee lineages six million years ago. PCDH11X and PCDH11Y, differentially regulated by retinoic acid, are highly expressed in the ventricular zone, subplate, and cortical plate of the developing cerebral cortex. Both proteins interact with β-catenin, a protein that plays a role in determining axis formation and regulating cortical size. In this way, the PCDH11X/Y gene pair determines cerebral asymmetry by initiating the right shift in Homo sapiens. © 2013 New York Academy of Sciences.

DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions.

PubMed

Nzeduru, Chinyere V; Ronca, Sandra; Wilkinson, Mike J

2012-01-01

Transgenes encoding for insecticidal crystal (Cry) proteins from the soil-dwelling bacterium Bacillus Thuringiensis have been widely introduced into Genetically Modified (GM) crops to confer protection against insect pests. Concern that these transgenes may also harm beneficial or otherwise valued insects (so-called Non Target Organisms, NTOs) represents a major element of the Environmental Risk Assessments (ERAs) used by all countries prior to commercial release. Compiling a comprehensive list of potentially susceptible NTOs is therefore a necessary part of an ERA for any Cry toxin-containing GM crop. In partly-characterised and biodiverse countries, NTO identification is slowed by the need for taxonomic expertise and time to enable morphological identifications. This limitation represents a potentially serious barrier to timely adoption of GM technology in some developing countries. We consider Bt Cry1A cowpea (Vigna unguiculata) in Nigeria as an exemplar to demonstrate how COI barcoding can provide a simple and cost-effective means of addressing this problem. Over a period of eight weeks, we collected 163 insects from cowpea flowers across the agroecological and geographic range of the crop in Nigeria. These individuals included 32 Operational Taxonomic Units (OTUs) spanning four Orders and that could mostly be assigned to genus or species level. They included 12 Lepidopterans and two Coleopterans (both potentially sensitive to different groups of Cry proteins). Thus, barcode-assisted diagnoses were highly harmonised across groups (typically to genus or species level) and so were insensitive to expertise or knowledge gaps. Decisively, the entire study was completed within four months at a cost of less than 10,000 US$. The broader implications of the findings for food security and the capacity for safe adoption of GM technology are briefly explored.

Biotechnologically generating 'super chickpea' for food and nutritional security.

PubMed

Acharjee, Sumita; Sarmah, Bidyut Kumar

2013-06-01

Chickpea productivity is affected by various constraints that are biotic (Helicoverpa, Aphids, Callosobruchus, Bromus and Orobanche) and abiotic (drought and salinity). In addition, the grains of this legume are deficient in sulfur amino acids, methionine and cysteine. The possibilities for genetic improvement by marker-assisted breeding and selection approaches are limited in chickpeas due to their sexually incompatible gene pool. Transgenic chickpeas expressing either the cry1Ac/b or the cry2Aa gene and the bean α-amylase inhibitor gene are resistant to Helicoverpa and bruchids, respectively, but these chickpeas have yet to be commercialized. Unfortunately, attempts to generate transgenic chickpeas with increased tolerance to drought and salinity or with increased methionine content have been less successful. The commercialization of transgenic chickpeas containing a single transgene may not give adequate yield advantage, as chickpeas are affected by many production constraints in the field and in storage. Gene pyramiding by incorporating two or more genes may be useful because improving one trait at a time will be time-consuming, labor-intensive and costly. Use of modern multi-gene vectors that contain recognition sites for zinc finger nucleases (ZFNs) and homing endonucleases may simplify the incorporation of multiple genes into chickpeas. This approach necessitates a collaborative effort between individuals, public and private organizations to generate 'super chickpeas' that harbor multiple transgenic traits. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Alterations of the PPP2R1B gene located at 11q23 in human colorectal cancers

PubMed Central

Takagi, Y; Futamura, M; Yamaguchi, K; Aoki, S; Takahashi, T; Saji, S

2000-01-01

BACKGROUND/AIMS—In 1998 the PPP2R1B gene encoding the A subunit of the serine/threonine protein phosphatase was identified as a putative tumour suppressor gene in lung and colon cancer in the chromosome region 11q22-24. The aim of the present study was to determine the type of alterations in primary rectal cancers as well as colon cancers and the correlation between these alterations and clinicopathological data.
METHODS—Mutation analyses of the PPP2R1B gene sequence encoding the binding sites of the catalytic C subunit (Huntington elongation A subunit TOR (HEAT) repeats 11-15) and partial binding sites of the regulatory B subunit were carried out on cDNA samples from 30 primary colorectal cancer specimens and corresponding normal tissues using a combination of the polymerase chain reaction and subsequent direct DNA sequencing.
RESULTS—Five missense mutations producing amino acid substitutions were detected in the four colon cancer cases (13.3%; four of 30 colorectal cancers): 15glycine (GGT) to alanine (GCT) and 499leucine (TTA) to isoleucine (ATA) in the same case, and 498valine (GTG) to glutamic acid (GAG), 500valine (GTA) to glycine (GGA), and 365serine (TCT) to proline (CCT). Of these five mutations, three (60%) were located in HEAT repeat 13 and four (80%) showed T to other nucleotide substitutions. In addition, a normal polymorphism, 478leucine, was found. No correlation was found between these mutations and clinicopathological data.
CONCLUSION—Our results suggest that the PPP2R1B gene is one of the true targets at 11q23, and its inactivation is involved in the development of all types of colorectal cancers.


Keywords: PPP2R1B gene; colorectal cancer; tumour suppressor gene; protein phosphatase PMID:10896920

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

PubMed

Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

2004-04-01

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. Copyright 2004 Wiley-Liss, Inc.

Mapping of aldose reductase gene sequences to human chromosomes 1, 3, 7, 9, 11, and 13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bateman, J.B.; Kojis, T.; Heinzmann, C.

1993-09-01

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, the authors mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q43, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other activemore » genes, non-aldose reductase homologous sequences, or pseudogenes. 24 refs., 3 figs., 2 tabs.« less

Genomic analysis of the type VI secretion systems in Pseudomonas spp.: novel clusters and putative effectors uncovered.

PubMed

Barret, Matthieu; Egan, Frank; Fargier, Emilie; Morrissey, John P; O'Gara, Fergal

2011-06-01

Bacteria encode multiple protein secretion systems that are crucial for interaction with the environment and with hosts. In recent years, attention has focused on type VI secretion systems (T6SSs), which are specialized transporters widely encoded in Proteobacteria. The myriad of processes associated with these secretion systems could be explained by subclasses of T6SS, each involved in specialized functions. To assess diversity and predict function associated with different T6SSs, comparative genomic analysis of 34 Pseudomonas genomes was performed. This identified 70 T6SSs, with at least one locus in every strain, except for Pseudomonas stutzeri A1501. By comparing 11 core genes of the T6SS, it was possible to identify five main Pseudomonas phylogenetic clusters, with strains typically carrying T6SSs from more than one clade. In addition, most strains encode additional vgrG and hcp genes, which encode extracellular structural components of the secretion apparatus. Using a combination of phylogenetic and meta-analysis of transcriptome datasets it was possible to associate specific subsets of VgrG and Hcp proteins with each Pseudomonas T6SS clade. Moreover, a closer examination of the genomic context of vgrG genes in multiple strains highlights a number of additional genes associated with these regions. It is proposed that these genes may play a role in secretion or alternatively could be new T6S effectors.

Comprehensive in silico allergenicity assessment of novel protein engineered chimeric Cry proteins for safe deployment in crops.

PubMed

Rathinam, Maniraj; Singh, Shweta; Pattanayak, Debasis; Sreevathsa, Rohini

2017-08-02

Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants.

Variation in performance on cry1Ab-transformed and nontransgenic rice varieties among populations of Scirpophaga incertulas (Lepidoptera: Pyralidae) from Luzon Island, Philippines.

PubMed

Bentur, J S; Cohen, M B; Gould, F

2000-12-01

We quantified variation in performance under greenhouse conditions among seven populations of Scirpophaga incertulas (Walker) from Luzon Island, Philippines, on three rice varieties: 'IR58' transformed with the cry1Ab gene from Bacillus thuringiensis Berliner, and nontransgenic IR58 and IR62. On IR62, S. incertutas performance did not differ among provinces for any of the 10 parameters measured, but there was a significant effect of town within province for one parameter, 20-d-old larval weight. Larval survival after 48 h on cy1Ab-transformed IR58 did not differ significantly among provinces, but did differ significantly among towns within a province. There was no geographic variation in larval survival after 48 h on control plants of IR58. Surviving insects from the cry1Ab-transformed IR58 were transferred to IR62 to complete development. There was no geographic variation in the percentage of insects completing development to adult emergence and the time required by the transferred female insects to complete development. However, there was variation among provinces in male developmental time. The absence of geographic variation on nontransgenic IR58 and the very limited variation on IR62 indicated that there was little variation in general vigor among the S. incertulas populations and thus that the variation in performance oil cry1Ab-transformed IR58 was probably attributable to differences in susceptibility to Cry1Ab.

The Dynein Gene Family in Chlamydomonas Reinhardtii

PubMed Central

Porter, M. E.; Knott, J. A.; Myster, S. H.; Farlow, S. J.

1996-01-01

To correlate dynein heavy chain (Dhc) genes with flagellar mutations and gain insight into the function of specific dynein isoforms, we placed eight members of the Dhc gene family on the genetic map of Chlamydomonas. Using a PCR-based strategy, we cloned 11 Dhc genes from Chlamydomonas. Comparisons with other Dhc genes indicate that two clones correspond to genes encoding the alpha and beta heavy chains of the outer dynein arm. Alignment of the predicted amino acid sequences spanning the nucleotide binding site indicates that the remaining nine clones can be subdivided into three groups that are likely to include representatives of the inner-arm Dhc isoforms. Gene-specific probes reveal that each clone represents a single-copy gene that is expressed as a transcript of the appropriate size (>13 kb) sufficient to encode a high molecular weight Dhc polypeptide. The expression of all nine genes is upregulated in response to deflagellation, suggesting a role in axoneme assembly or motility. Restriction fragment length polymorphisms between divergent C. reinhardtii strains have been used to place each Dhc gene on the genetic map of Chlamydomonas. These studies lay the groundwork for correlating defects in different Dhc genes with specific flagellar mutations. PMID:8889521

Gene amplification of the transcription factor DP1 and CTNND1 in human lung cancer.

PubMed

Castillo, Sandra D; Angulo, Barbara; Suarez-Gauthier, Ana; Melchor, Lorenzo; Medina, Pedro P; Sanchez-Verde, Lydia; Torres-Lanzas, Juan; Pita, Guillermo; Benitez, Javier; Sanchez-Cespedes, Montse

2010-09-01

The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168.

Cloning and characterization of an inulinase gene from the marine yeast Candida membranifaciens subsp. flavinogenie W14-3 and its expression in Saccharomyces sp. W0 for ethanol production.

PubMed

Zhang, Lin-Lin; Tan, Mei-Juan; Liu, Guang-Lei; Chi, Zhe; Wang, Guang-Yuan; Chi, Zhen-Ming

2015-04-01

The INU1 gene encoding an exo-inulinase from the marine-derived yeast Candida membranifaciens subsp. flavinogenie W14-3 was cloned and characterized. It had an open reading frame of 1,536 bp long encoding an inulinase. The coding region of it was not interrupted by any intron. The cloned gene encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids and a calculated molecular mass of 57.8 kDa. The protein sequence deduced from the inulinase gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP FS and Q. The protein also had six conserved putative N-glycosylation sites. The deduced inulinase from the yeast strain W14-3 was found to be closely related to that from Candida kutaonensis sp. nov. KRF1, Kluyveromyces marxianus, and Cryptococcus aureus G7a. The inulinase gene with its signal peptide encoding sequence was subcloned into the pMIRSC11 expression vector and expressed in Saccharomyces sp. W0. The recombinant yeast strain W14-3-INU-112 obtained could produce 16.8 U/ml of inulinase activity and 12.5 % (v/v) ethanol from 250 g/l of inulin within 168 h. The monosaccharides were detected after the hydrolysis of inulin with the crude inulinase (the yeast culture). All the results indicated that the cloned gene and the recombinant yeast strain W14-3-INU-112 had potential applications in biotechnology.

The mapping of the human 52-kD Ro/SSA autoantigen gene to human chromosome II, and its polymorphisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frank, M.B.; Itoh, Kazuko; Fujisaku, Atsushi

1993-01-01

Autoantibodies to the ribonucleoprotein Ro/SSA occur in nearly half of the patients with systemic lupus erythematosus and are associated with lymphopenia, photosensitive dermatitis, and pulmonary and renal disease, which suggests that they have an immunopathologic role. The majority of Ro/SSA precipitin-positive patients produce serum antibodies that bind to the 60-kD and 52-kD Ro/SSA proteins. The authors previously isolated and determined the nucleotide sequence of a cDNA clone that encodes the 52-kD form of the human Ro/SSA protein. In the present study, they have determined the chromosomal location of the gene by in situ hybridization to the end of the shortmore » arm of chromosome 11. Hybridization of portions of the cDNA probe to restriction enzyme-digested DNA indicated the gene is composed of at least three exons. The exon encoding the putative zinc fingers of this protein was found to be distinct from that which encodes the leucine zipper. An RFLP of this gene was identified and is associated with the presence of lupus, primarily in black Americans. 60 refs., 3 figs., 3 tabs.« less

Virulence and extended-spectrum β-lactamase encoding genes in Escherichia coli recovered from chicken meat intended for hospitalized human consumption.

PubMed

Younis, Gamal A; Elkenany, Rasha M; Fouda, Mohamed A; Mostafa, Noura F

2017-10-01

This study describes the prevalence of Escherichia coli in frozen chicken meat intended for human consumption with emphasis on their virulence determinants through detection of the virulence genes and recognition of the extended-spectrum β-lactamase (ESBL) encoding genes ( bla OXA and bla TEM genes). A total of 120 frozen chicken meat samples were investigated for isolation of E. coli . All isolates were subjected to biochemical and serological tests. Eight serotypes isolated from samples were analyzed for the presence of various virulence genes ( stx1, stx2 , and eae A genes) using multiplex polymerase chain reaction (PCR) technique. Moreover, the strains were evaluated for the ESBL encoding genes ( bla TEM and bla OXA ). Overall, 11.66% (14/120) chicken meat samples carried E. coli according to cultural and biochemical properties. The most predominant serotypes were O78 and O128: H2 (21.5%, each), followed by O121: H7 and O44: H18. Molecular method detected that 2 strains (25%) harbored stx1 , 3 strains (37.5%) stx2 , and 3 strains (37.5%) both stx1 and stx2 , while 1 (12.5%) strain carried eae A gene. Particularly, only O26 serotype had all tested virulence genes ( stx1, stx2, and eae A ). The results revealed that all examined 8 serotypes were Shiga toxin-producing E. coli (STEC). The ESBL encoding genes ( bla TEM and bla OXA ) of STEC were detected in 4 (50%) isolates by multiplex PCR. The overall incidence of bla TEM and bla OXA genes was 3 (37.5%) and 2 (25%) isolates. The present study indicates the prevalence of virulent and ESBL-producing E. coli in frozen chicken meat intended for hospitalized human consumption due to poor hygienic measures and irregular use of antibiotics. Therefore, the basic instructions regarding good hygienic measures should be adapted to limit public health hazard.

Gene encoding the collagen type I and thrombospondin receptor CD36 is located on chromosome 7q11. 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernandez-Ruiz, E.; Armesilla, A.L.; Sanchez-Madrid, F.

The human CD36 is a member of a gene family of structurally related glycoproteins and functions as a receptor for collagen type I and thrombospondin. CD36 also binds to red blood cells infected with the human malaria parasite Plasmodium falciparum. In the present study, the CD36 gene was assigned to chromosome 7 by using the polymerase chain reaction with DNA from human-hamster somatic cell hybrids. Furthermore, the use of a CD36 genomic probe has allowed the localization of the CD36 locus to the 7q11.2 band by fluorescence in situ hybridization coupled with GTG-banding. 14 refs., 2 figs.

First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize.

PubMed

Fantozzi, Anna; Ermolli, Monica; Marini, Massimiliano; Scotti, Domenico; Balla, Branko; Querci, Maddalena; Langrell, Stephen R H; Van den Eede, Guy

2007-02-21

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes.

PubMed

Azevedo, Analice C; Bento, Cláudia B P; Ruiz, Jeronimo C; Queiroz, Marisa V; Mantovani, Hilário C

2015-10-01

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Circadian pacemaking in cells and circuits of the suprachiasmatic nucleus.

PubMed

Hastings, M H; Brancaccio, M; Maywood, E S

2014-01-01

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker of the brain. It co-ordinates the daily rhythms of sleep and wakefulness, as well as physiology and behaviour, that set the tempo to our lives. Disturbance of this daily pattern, most acutely with jet-lag but more insidiously with rotational shift-work, can have severely deleterious effects for mental function and long-term health. The present review considers recent developments in our understanding of the properties of the SCN that make it a robust circadian time-keeper. It first focuses on the intracellular transcriptional/ translational feedback loops (TTFL) that constitute the cellular clockwork of the SCN neurone. Daily timing by these loops pivots around the negative regulation of the Period (Per) and Cryptochrome (Cry) genes by their protein products. The period of the circadian cycle is set by the relative stability of Per and Cry proteins, and this can be controlled by both genetic and pharmacological interventions. It then considers the function of these feedback loops in the context of cytosolic signalling by cAMP and intracellular calcium ([Ca(2+) ]i ), which are both outputs from, and inputs to, the TTFL, as well as the critical role of vasoactive intestinal peptide (VIP) signalling in synchronising cellular clocks across the SCN. Synchronisation by VIP in the SCN is paracrine, operating over an unconventionally long time frame (i.e. 24 h) and wide spatial domain, mediated via the cytosolic pathways upstream of the TTFL. Finally, we show how intersectional pharmacogenetics can be used to control G-protein-coupled signalling in individual SCN neurones, and how manipulation of Gq/[Ca(2+) ]i -signalling in VIP neurones can re-programme the circuit-level encoding of circadian time. Circadian pacemaking in the SCN therefore provides an unrivalled context in which to understand how a complex, adaptive behaviour can be organised by the dynamic activity of a relatively few gene products, operating in a clearly defined neuronal circuit, with both cell-autonomous and emergent, circuit-level properties. © 2014 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of The British Society for Neuroendocrinology.

Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library.

PubMed

Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

2018-04-01

Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 10 7  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL -1 , respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.

Characterization of CpSte11, a MAPKKK gene of Cryphonectria parasitica, and initial evidence of its involvement in the pheromone response pathway.

PubMed

Park, Jin-Ah; Kim, Jung-Mi; Park, Seung-Moon; Kim, Dae-Hyuk

2012-04-01

The gene CpSte11 of Cryphonectria parasitica, which encodes a yeast Ste11 homologue, was cloned and characterized. Gene replacement analysis revealed a high frequency of CpSte11 null mutants. When compared with the wild-type parent strain, CpSte11 null mutants showed no difference in terms of growth rate or pigmentation. However, CpSte11 null mutants showed a marked decrease in both the number and size of stromal pustules on chestnut twigs. The virulence test showed that, in comparison with those of the wild-type and virus-infected hypovirulent strains, CpSte11 null mutants produced necrotic areas of intermediate size. Disruption of the CpSte11 gene also resulted in defects in female fertility. Down-regulation of transcripts for the mating pheromone precursor gene, Mf2/2, and mating response transcription factors, such as cpst12 and pro1, was observed in CpSte11 null mutants. The down-regulation of Mf2/2, cpst12 and pro1 was also observed in the mutant phenotype of Cpmk2, a mating response Fus3-like mitogen-activated protein kinase (MAPK) gene, but not in the mutant of Cpmk1, a high-osmolarity glycerol Hog1-like MAPK gene. These results indicate that the cloned CpSte11 gene is functionally involved in the mating response pathway and acts through downstream targets, including Cpmk2, cpst12, pro1 and Mf2/2. However, the characteristics of the CpSte11 null mutant were fully phenocopied only in the cpst12 null mutant, but not in other studied null mutants of components of the putative mating response pathway. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

Arabidopsis ESK1 encodes a novel regulator of freezing tolerance.

PubMed

Xin, Zhanguo; Mandaokar, Ajin; Chen, Junping; Last, Robert L; Browse, John

2007-03-01

The eskimo1 (esk1) mutation of Arabidopsis resulted in a 5.5 degrees C improvement in freezing tolerance in the absence of cold acclimation. Here we show that the increase in freezing tolerance is not associated with any increase in the ability to survive drought or salt stresses, which are similar to freezing in their induction of cellular dehydration. Genome-wide comparisons of gene expression between esk1-1 and wild type indicate that mutations at esk1 result in altered expression of transcription factors and signaling components and of a set of stress-responsive genes. Interestingly, the list of 312 genes regulated by ESK1 shows greater overlap with sets of genes regulated by salt, osmotic and abscisic acid treatments than with genes regulated by cold acclimation or by the transcription factors CBF3 and ICE1, which have been shown to control genetic pathways for freezing tolerance. Map-based cloning identified the esk1 locus as At3g55990. The wild-type ESK1 gene encodes a 57-kDa protein and is a member of a large gene family of DUF231 domain proteins whose members encode a total of 45 proteins of unknown function. Our results indicate that ESK1 is a novel negative regulator of cold acclimation. Mutations in the ESK1 gene provide strong freezing tolerance through genetic regulation that is apparently very different from previously described genetic mechanisms of cold acclimation.

Human HST1 (HSTF1) gene maps to chromosome band 11q13 and coamplifies with the INT2 gene in human cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Michihiro C.; Wada, Makio; Satoh, Hitoshi

1988-07-01

The human HST1 gene, previously designated the hst gene, and now assigned the name HSTF1 for heparin-binding secretory transforming factor in human gene nomenclature, was originally identified as a transforming gene in DNAs from human stomach cancers by transfection assay with mouse NIH 3T3 cells. The amino acid sequence of the product deduced from DNA sequences of the HST1 cDNA and genomic clones had approximately 40% homology to human basic and acidic fibroblast growth factors and mouse Int-2-encoded protein. The authors have mapped the human HST1 gene to chromosome 11 at band q13.3 by Southern blot hybridization analysis of amore » panel of human and mouse somatic cell hybrids and in situ hybridization with an HST1 cDNA probe. The HST1 gene was found to be amplified in DNAs obtained from a stomach cancer and a vulvar carcinoma cell line, A431. In all of these samples of DNA, the INT2 gene, previously mapped to human chromosome 11q13, was also amplified to the same degree as the HST1 gene.« less

phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens.

PubMed

Elsheimer-Matulova, Marta; Varmuzova, Karolina; Kyrova, Kamila; Havlickova, Hana; Sisak, Frantisek; Rahman, Masudur; Rychlik, Ivan

2015-09-17

Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the interactions between chickens and S. enterica is therefore important for vaccine design and subsequent decrease in the incidence of human salmonellosis. In this study we therefore characterized the interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested the response of HD11 chicken macrophage-like cell line to S. Enteritidis infection monitoring the transcription of 36 genes related to immune response. All the mutants and the wild type strain induced inflammatory signaling in the HD11 cell line though the response to SPI1 mutant infection was different from the rest of the mutants. When newly hatched chickens were inoculated, the phoP as well as the SPI1 mutant did not induce an expression of any of the tested genes in the cecum. Despite this, such chickens were protected against challenge with wild-type S. Enteritidis. On the other hand, inoculation of chickens with the aroA or SPI2 mutant induced expression of 27 and 18 genes, respectively, including genes encoding immunoglobulins. Challenge of chickens inoculated with these two mutants resulted in repeated induction of 11 and 13 tested genes, respectively, including the genes encoding immunoglobulins. In conclusion, SPI1 and phoP mutants induced protective immunity without inducing an inflammatory response and antibody production. Inoculation of chickens with the SPI2 and aroA mutants also led to protective immunity but was associated with inflammation and antibody production. The differences in interaction between the mutants and chicken host can be used for a more detailed understanding of the chicken immune system.

A fully decompressed synthetic bacteriophage øX174 genome assembled and archived in yeast.

PubMed

Jaschke, Paul R; Lieberman, Erica K; Rodriguez, Jon; Sierra, Adrian; Endy, Drew

2012-12-20

The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic 'drydock' within which to maintain and manipulate clonal lineages of other obligate lytic phage. Copyright © 2012 Elsevier Inc. All rights reserved.

Dubin-Johnson syndrome and intrahepatic cholestasis of pregnancy in a Sri Lankan family: a case report.

PubMed

Kularatnam, Grace Angeline Malarnangai; Warawitage, Dilanthi; Vidanapathirana, Dinesha Maduri; Jayasena, Subashini; Jasinge, Eresha; de Silva, Nalika; Liyanarachchi, Kirinda Liyana Arachchige Manoj Sanjeeva; Wickramasinghe, Pujitha; Devgun, Manjit Singh; Barbu, Veronique; Lascols, Olivier

2017-09-18

Dubin-Johnson syndrome and intrahepatic cholestasis of pregnancy are rare chronic liver disorders. Dubin-Johnson syndrome may manifest as conjugated hyperbilirubinemia, darkly pigmented liver, presence of abnormal pigment in the parenchyma of hepatocytes and abnormal distribution of the coproporphyrin isomers I and III in the urine. Intrahepatic cholestatic jaundice of pregnancy presents as pruritus, abnormal liver biochemistry and increased serum bile acids. A Sri Lankan girl presented with recurrent episodes of jaundice. She had conjugated hyperbilirubinaemia with diffuse, coarse brown pigments in the hepatocytes. Urine coproporphyrin examination suggested Dubin-Johnson syndrome. Genetic studies confirmed missense homozygous variant p.Trp709Arg in the ATP-binding cassette sub-family C member 2 gene ABCC2 that encodes the Multidrug resistance-associated protein 2 that causes Dubin-Johnson syndrome. The gene study of the mother revealed the same missense variant in ABCC2/MRP2 but with a heterozygous status, and in addition a homozygous missense variant p.Val444Ala in the ATP-binding cassette, sub-family B member 11 gene ABCB11 that encodes the bile salt export pump. Dubin-Johnson syndrome should be considered when the common causes for conjugated hyperbilirubinaemia have been excluded, and patient has an increased percentage of direct bilirubin relative to total bilirubin concentration. Its early diagnosis prevents repeated hospital admissions and investigations. Knowledge of a well known homozygous variant in ABCB11 gene could help in the management of pregnancy.

CHLORELLA VIRUSES

PubMed Central

Yamada, Takashi; Onimatsu, Hideki; Van Etten, James L.

2007-01-01

Chlorella viruses or chloroviruses are large, icosahedral, plaque‐forming, double‐stranded‐DNA—containing viruses that replicate in certain strains of the unicellular green alga Chlorella. DNA sequence analysis of the 330‐kbp genome of Paramecium bursaria chlorella virus 1 (PBCV‐1), the prototype of this virus family (Phycodnaviridae), predict ∼366 protein‐encoding genes and 11 tRNA genes. The predicted gene products of ∼50% of these genes resemble proteins of known function, including many that are completely unexpected for a virus. In addition, the chlorella viruses have several features and encode many gene products that distinguish them from most viruses. These products include: (1) multiple DNA methyltransferases and DNA site‐specific endonucleases, (2) the enzymes required to glycosylate their proteins and synthesize polysaccharides such as hyaluronan and chitin, (3) a virus‐encoded K+ channel (called Kcv) located in the internal membrane of the virions, (4) a SET domain containing protein (referred to as vSET) that dimethylates Lys27 in histone 3, and (5) PBCV‐1 has three types of introns; a self‐splicing intron, a spliceosomal processed intron, and a small tRNA intron. Accumulating evidence indicates that the chlorella viruses have a very long evolutionary history. This review mainly deals with research on the virion structure, genome rearrangements, gene expression, cell wall degradation, polysaccharide synthesis, and evolution of PBCV‐1 as well as other related viruses. PMID:16877063

Circadian clock-deficient mice as a tool for exploring disease etiology.

PubMed

Doi, Masao

2012-01-01

One of the most significant conceptual changes brought about by the analysis of circadian clock-deficient mice is that abnormalities in the circadian clock are linked not only to sleep arousal disorder but also to a wide variety of common diseases, including hypertension, diabetes, obesity, and cancer. It has recently been shown that the disruption of the two cryptochrome genes Cry1 and Cry2-core elements of the circadian clock-induces salt-dependent hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland. This adrenal disorder occurs as a result of increased expression of Hsd3b6, a newly identified steroidogenic enzyme that regulates aldosterone production within the adrenal zona glomerular cells. Importantly, this enzyme is functionally conserved in humans, and the pathophysiologic condition of human idiopathic hyperaldosteronism resembles that of Cry1/2-deficient mice. This review highlights the potential utility of circadian clock-deficient mice as a tool for exploring hitherto unknown disease etiology linked to the circadian clock.

Neonatal Diabetes: An Expanding List of Genes Allows for Improved Diagnosis and Treatment

PubMed Central

Naylor, Rochelle N.; Philipson, Louis H.; Bell, Graeme I.

2011-01-01

There has been major progress in recent years uncovering the genetic causes of diabetes presenting in the first year of life. Twenty genes have been identified to date. The most common causes accounting for the majority of cases are mutations in the genes encoding the two subunits of the ATP-sensitive potassium channel (KATP), KCNJ11 and ABCC8, and the insulin gene (INS), as well as abnormalities in chromosome 6q24. Patients with activating mutations in KCNJ11 and ABCC8 can be treated with oral sulfonylureas in lieu of insulin injections. This compelling example of personalized genetic medicine leading to improved glucose regulation and quality of life may—with continued research—be repeated for other forms of neonatal diabetes in the future. PMID:21993633

SNP-based Microdeletion and Aneuploidy RegisTry (SMART)

ClinicalTrials.gov

2017-09-20

22q11 Deletion Syndrome; DiGeorge Syndrome; Trisomy 21; Trisomy 18; Trisomy 13; Monosomy X; Sex Chromosome Abnormalities; Cri-du-Chat Syndrome; Angelman Syndrome; Prader-Willi Syndrome; 1p36 Deletion Syndrome

The effects of Jiao-Tai-Wan on sleep, inflammation and insulin resistance in obesity-resistant rats with chronic partial sleep deprivation.

PubMed

Zou, Xin; Huang, Wenya; Lu, Fuer; Fang, Ke; Wang, Dingkun; Zhao, Shuyong; Jia, Jiming; Xu, Lijun; Wang, Kaifu; Wang, Nan; Dong, Hui

2017-03-23

Jiao-Tai-Wan (JTW), composed of Rhizome Coptidis and Cortex Cinnamomi, is a classical traditional Chinese prescription for treating insomnia. Several in vivo studies have concluded that JTW could exert its therapeutical effect in insomnia rats. However, the specific mechanism is still unclear. The present study aimed to explore the effect of JTW on sleep in obesity-resistant (OR) rats with chronic partial sleep deprivation (PSD) and to clarify its possible mechanism. JTW was prepared and the main components contained in the granules were identified by 3D-High Performance Liquid Chromatography (3D-HPLC) assay. The Male Sprague-Dawley (SD) rats underwent 4 h PSD by environmental noise and the treatment with low and high doses of JTW orally for 4 weeks, respectively. Then sleep structure was analyzed by electroencephalographic (EEG). Inflammation markers including high-sensitivity C reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were examined in the rat plasma. Meanwhile, metabolic parameters as body weight increase rate, fasting plasma glucose (FPG), fasting insulin (FINS) levels and insulin resistance index (HOMA-IR) were measured. The expressions of clock gene cryptochromes (Cry1 and Cry2) and inflammation gene nuclear factor-κB (NF-κB) in peripheral blood monocyte cells (PBMC) were also determined. The result showed that the administration of JTW significantly increased total sleep time and total slow wave sleep (SWS) time in OR rats with PSD. Furthermore, the treatment with JTW reversed the increase in the markers of systemic inflammation and insulin resistance caused by sleep loss. These changes were also associated with the up-regulation of Cry1 mRNA and Cry 2 mRNA and the down-regulation of NF-κB mRNA expression in PBMC. This study suggests that JTW has the beneficial effects of improving sleep, inflammation and insulin sensitivity. The mechanism appears to be related to the modulation of circadian clock and inflammation genes expressions in PBMC.

Spread of imipenem-resistant Acinetobacter baumannii co-expressing OXA-23 and GES-11 carbapenemases in Lebanon.

PubMed

Hammoudi, D; Moubareck, C Ayoub; Hakime, N; Houmani, M; Barakat, A; Najjar, Z; Suleiman, M; Fayad, N; Sarraf, R; Sarkis, D Karam

2015-07-01

The acquisition of carbapenemases by Acinetobacter baumannii is reported increasingly worldwide, but data from Lebanon are limited. The aims of this study were to evaluate the prevalence of imipenem-resistant A. baumannii in Lebanon, identify resistance determinants, and detect clonal relatedness. Imipenem-resistant A. baumannii were collected from nine Lebanese hospitals during 2012. Antimicrobial susceptibility, the cloxacillin effect, and ethylenediaminetetraacetic acid (EDTA) synergy were determined. Genes encoding carbapenemases and insertion sequence ISAba1 were screened via PCR sequencing. ISAba1 position relative to genes encoding Acinetobacter-derived cephalosporinases (ADCs) and OXA-23 was studied by PCR mapping. Clonal linkage was examined by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Out of 724 A. baumannii isolated in 2012, 638 (88%) were imipenem-resistant. Of these, 142 were analyzed. Clavulanic acid-imipenem synergy suggested carbapenem-hydrolyzing extended-spectrum β-lactamase. A positive cloxacillin test indicated ADCs, while EDTA detection strips were negative. Genotyping indicated that 90% of isolates co-harbored blaOXA-23 and blaGES-11. The remaining strains had blaOXA-23, blaOXA-24, blaGES-11, or blaOXA-24 with blaGES-11. ISAba1 was located upstream of blaADC and blaOXA-23 in 97% and 100% of isolates, respectively. ERIC-PCR fingerprinting revealed 18 pulsotypes spread via horizontal gene transfer and clonal dissemination. This survey established baseline evidence of OXA-23 and GES-11-producing A. baumannii in Lebanon, indicating the need for further surveillance. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key Nitrate Regulators[OPEN

PubMed Central

Zhao, Lufei; Zhang, Chengfei; Li, Zehui; Lei, Zhao; Liu, Fei; Guan, Peizhu; Crawford, Nigel M.

2016-01-01

We show that NITRATE REGULATORY GENE2 (NRG2), which we identified using forward genetics, mediates nitrate signaling in Arabidopsis thaliana. A mutation in NRG2 disrupted the induction of nitrate-responsive genes after nitrate treatment by an ammonium-independent mechanism. The nitrate content in roots was lower in the mutants than in the wild type, which may have resulted from reduced expression of NRT1.1 (also called NPF6.3, encoding a nitrate transporter/receptor) and upregulation of NRT1.8 (also called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest that NRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2 directly interacts with the nitrate regulator NLP7 in the nucleus, but nuclear retention of NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysis revealed that genes involved in four nitrogen-related clusters including nitrate transport and response to nitrate were differentially expressed in the nrg2 mutants. A nitrogen compound transport cluster containing some members of the NRT/PTR family was regulated by both NRG2 and NRT1.1, while no nitrogen-related clusters showed regulation by both NRG2 and NLP7. Thus, NRG2 plays a key role in nitrate regulation in part through modulating NRT1.1 expression and may function with NLP7 via their physical interaction. PMID:26744214

The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.E.; Beck, T.W.; Brennscheidt, U.

1994-03-01

The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. The authors have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776more » nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors. 65 refs., 7 figs., 1 tab.« less

LRIG2 Mutations Cause Urofacial Syndrome

PubMed Central

Stuart, Helen M.; Roberts, Neil A.; Burgu, Berk; Daly, Sarah B.; Urquhart, Jill E.; Bhaskar, Sanjeev; Dickerson, Jonathan E.; Mermerkaya, Murat; Silay, Mesrur Selcuk; Lewis, Malcolm A.; Olondriz, M. Beatriz Orive; Gener, Blanca; Beetz, Christian; Varga, Rita E.; Gülpınar, Ömer; Süer, Evren; Soygür, Tarkan; Özçakar, Zeynep B.; Yalçınkaya, Fatoş; Kavaz, Aslı; Bulum, Burcu; Gücük, Adnan; Yue, Wyatt W.; Erdogan, Firat; Berry, Andrew; Hanley, Neil A.; McKenzie, Edward A.; Hilton, Emma N.; Woolf, Adrian S.; Newman, William G.

2013-01-01

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract. PMID:23313374

An Arabidopsis mutation in translation elongation factor 2 causes superinduction of CBF/DREB1 transcription factor genes but blocks the induction of their downstream targets under low temperatures.

PubMed

Guo, Yan; Xiong, Liming; Ishitani, Manabu; Zhu, Jian-Kang

2002-05-28

Low temperature regulates gene expression in bacteria, yeast, and animals as well as in plants. However, the signal transduction cascades mediating the low temperature responses are not well understood in any organism. To identify components in low temperature signaling genetically, we isolated Arabidopsis thaliana mutants in which cold-responsive genes are no longer induced by low temperatures. One of these mutations, los1-1, specifically blocks low temperature-induced transcription of cold-responsive genes. Surprisingly, cold-induced expression of the early response transcriptional activators, C-repeat/dehydration responsive element binding factors (CBF/DREB1s), is enhanced by the los1-1 mutation. The los1-1 mutation also reduces the capacity of plants to develop freezing tolerance but does not impair the vernalization response. Genetic analysis indicated that los1-1 is a recessive mutation in a single nuclear gene. The LOS1 gene encodes a translation elongation factor 2-like protein. Protein labeling studies show that new protein synthesis is blocked in los1-1 mutant plants specifically in the cold. These results reveal a critical role of new protein synthesis in the proper transduction of low temperature signals. Our results also suggest that cold-induced transcription of CBF/DREB1s is feedback inhibited by their gene products or by products of their downstream target genes.

Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes.

PubMed

Ricardo, Elisabete; Costa-de-Oliveira, Sofia; Dias, Ana Silva; Guerra, José; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

2009-06-01

Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida. The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 microg mL(-1)); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1, CDR2, MDR1, encoding for efflux pumps, and ERG11, encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly (P>0.05), probably acting as a Cdrp blocker.

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

PubMed Central

Tabashnik, Bruce E.; Fabrick, Jeffrey A.; Unnithan, Gopalan C.; Yelich, Alex J.; Masson, Luke; Zhang, Jie; Bravo, Alejandra; Soberón, Mario

2013-01-01

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both. PMID:24244692

Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the detection of 20 drug resistance encoding genes.

PubMed

Murugan, Nandagopal; Malathi, Jambulingam; Therese, K Lily; Madhavan, Hajib NarahariRao

2018-02-01

Pseudomonas aeruginosa (P. aeruginosa) is a menacing opportunistic, nosocomial pathogen; become a growing concern as conventional antimicrobial therapy is now futile against it. Multi-drug resistant P. aeruginosa (MDRPA) has distinctive resistance mechanisms such as production of β-lactamases, repression of porin genes and over-expression of efflux pumps. The focus of this study is to standardize and application of multiplex PCR (mPCR) to detect the presence of betalactamase genes encoding bla Tem , bla OXA , bla CTX-M-15 , bla Vim , bla Ges , bla Veb , bla DIM , AmpC and Efflux pump genes encoding Mex A,B-oprM, Mex C,D-oprJ, Mex X,Y-oprN, oprD, nfxB, MexR. A total of 200 clinical isolates of P. aeruginosa were tested for the presence of the above mentioned genes genotypically through mPCR and characterized by phenotypic methods for ESBL and MBL production. Out of 200 isolates, 163 (81.5%) nfxB regulator gene, 102 (51%) MexA, 96 (48%) MexC, 93 (46.5%) MexB, 86 (43%) MexD, 81 (40.5%) OprM, 74 (37%) OprJ, 72 (36%) OprD and MexR, 53 (26.5%) Mex X and OprN, 49 (24.5%) MexY gene. Betalactamase genes 145 (72.5%) bla Tem , 67 (33.5%) bla OXA, 35 (17.5%) blaVim, 25(12.50%), 23 (11.50%) blaVeb, 21 (11.5%) blaGes, 14 (7%) Ctx-m and 10 (5%) AmpC and 5 (2.5%) blaDim-1 gene were tested positive by mPCR. Phenotypically 38 (19%) and 29 (14.5%) out of 200 tested positive for ESBL and MBL production. Application of this mPCR on clinical specimens is fast, accurate, specific and low-cost reliable tool for the screening, where culture negative Eubacterial PCR positive cases for an early molecular detection of drug resistance mechanism assisting the clinician to treat the disease with appropriate antibiotic selection. Copyright © 2017. Published by Elsevier Taiwan.

SGR2, a Phospholipase-Like Protein, and ZIG/SGR4, a SNARE, Are Involved in the Shoot Gravitropism of Arabidopsis

PubMed Central

Kato, Takehide; Morita, Miyo Terao; Fukaki, Hidehiro; Yamauchi, Yoshiro; Uehara, Michiko; Niihama, Mitsuru; Tasaka, Masao

2002-01-01

In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid–preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism. PMID:11826297

Gene Expression by the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough Grown on an Iron Electrode under Cathodic Protection Conditions▿ †

PubMed Central

Caffrey, Sean M.; Park, Hyung Soo; Been, Jenny; Gordon, Paul; Sensen, Christoph W.; Voordouw, Gerrit

2008-01-01

The genome sequence of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a c-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type D. vulgaris grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of −1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the hyn-1 genes for [NiFe] hydrogenase 1 and the hyd genes, encoding [Fe] hydrogenase. The hmc genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the hys genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with hyn-1, hyd, and hmc mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in D. vulgaris-mediated iron corrosion. PMID:18310429

Bayesian Source Attribution of Salmonellosis in South Australia.

PubMed

Glass, K; Fearnley, E; Hocking, H; Raupach, J; Veitch, M; Ford, L; Kirk, M D

2016-03-01

Salmonellosis is a significant cause of foodborne gastroenteritis in Australia, and rates of illness have increased over recent years. We adopt a Bayesian source attribution model to estimate the contribution of different animal reservoirs to illness due to Salmonella spp. in South Australia between 2000 and 2010, together with 95% credible intervals (CrI). We excluded known travel associated cases and those of rare subtypes (fewer than 20 human cases or fewer than 10 isolates from included sources over the 11-year period), and the remaining 76% of cases were classified as sporadic or outbreak associated. Source-related parameters were included to allow for different handling and consumption practices. We attributed 35% (95% CrI: 20-49) of sporadic cases to chicken meat and 37% (95% CrI: 23-53) of sporadic cases to eggs. Of outbreak-related cases, 33% (95% CrI: 20-62) were attributed to chicken meat and 59% (95% CrI: 29-75) to eggs. A comparison of alternative model assumptions indicated that biases due to possible clustering of samples from sources had relatively minor effects on these estimates. Analysis of source-related parameters showed higher risk of illness from contaminated eggs than from contaminated chicken meat, suggesting that consumption and handling practices potentially play a bigger role in illness due to eggs, considering low Salmonella prevalence on eggs. Our results strengthen the evidence that eggs and chicken meat are important vehicles for salmonellosis in South Australia. © 2015 Society for Risk Analysis.

Structure of Full-length Drosophila Cryptochrome

PubMed Central

Zoltowski, Brian D.; Vaidya, Anand T.; Top, Deniz; Widom, Joanne; Young, Michael W.; Crane, Brian R.

2011-01-01

The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions. PMID:22080955

Metadata Analysis of Phanerochaete chrysosporium Gene Expression Data Identified Common CAZymes Encoding Gene Expression Profiles Involved in Cellulose and Hemicellulose Degradation.

PubMed

Kameshwar, Ayyappa Kumar Sista; Qin, Wensheng

2017-01-01

In literature, extensive studies have been conducted on popular wood degrading white rot fungus, Phanerochaete chrysosporium about its lignin degrading mechanisms compared to the cellulose and hemicellulose degrading abilities. This study delineates cellulose and hemicellulose degrading mechanisms through large scale metadata analysis of P. chrysosporium gene expression data (retrieved from NCBI GEO) to understand the common expression patterns of differentially expressed genes when cultured on different growth substrates. Genes encoding glycoside hydrolase classes commonly expressed during breakdown of cellulose such as GH-5,6,7,9,44,45,48 and hemicellulose are GH-2,8,10,11,26,30,43,47 were found to be highly expressed among varied growth conditions including simple customized and complex natural plant biomass growth mediums. Genes encoding carbohydrate esterase class enzymes CE (1,4,8,9,15,16) polysaccharide lyase class enzymes PL-8 and PL-14, and glycosyl transferases classes GT (1,2,4,8,15,20,35,39,48) were differentially expressed in natural plant biomass growth mediums. Based on these results, P. chrysosporium, on natural plant biomass substrates was found to express lignin and hemicellulose degrading enzymes more than cellulolytic enzymes except GH-61 (LPMO) class enzymes, in early stages. It was observed that the fate of P. chrysosporium transcriptome is significantly affected by the wood substrate provided. We believe, the gene expression findings in this study plays crucial role in developing genetically efficient microbe with effective cellulose and hemicellulose degradation abilities.

Identification and characterization of NF-YB family genes in tung tree.

PubMed

Yang, Susu; Wang, Yangdong; Yin, Hengfu; Guo, Haobo; Gao, Ming; Zhu, Huiping; Chen, Yicun

2015-12-01

The NF-YB transcription factor gene family encodes a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that strongly binds to the CCAAT box promoter element. Studies on model plants have shown that NF-YB proteins participate in important developmental and physiological processes, but little is known about NF-YB proteins in trees. Here, we identified seven NF-YB transcription factor-encoding genes in Vernicia fordii, an important oilseed tree in China. A phylogenetic analysis separated the genes into two groups; non-LEC1 type (VfNF-YB1, 5, 7, 9, 11, 13) and LEC1-type (VfNF-YB 14). A gene structure analysis showed that VfNF-YB 5 has three introns and the other genes have no introns. The seven VfNF-YB sequences contain highly conserved domains, a disordered region at the N terminus, and two long helix structures at the C terminus. Phylogenetic analyses showed that VfNF-YB family genes are highly homologous to GmNF-YB genes, and many of them are closely related to functionally characterized NF-YBs. In expression analyses of various tissues (root, stem, leaf, and kernel) and the root during pathogen infection, VfNF-YB1, 5, and 11 were dominantly expressed in kernels, and VfNF-YB7 and 9 were expressed only in the root. Different VfNF-YB family genes showed different responses to pathogen infection, suggesting that they play different roles in the pathogen response. Together, these findings represent the first extensive evaluation of the NF-YB family in tung tree and provide a foundation for dissecting the functions of VfNF-YB genes in seed development, stress adaption, fatty acid synthesis, and pathogen response.

Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays.

PubMed

Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi

2017-01-01

Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T 1 and T 2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T 2 progenies and PCR-identified Apr gene segregation in T 2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods.

Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments.

PubMed

Matthijnssens, Jelle; Ciarlet, Max; Rahman, Mustafizur; Attoui, Houssam; Bányai, Krisztián; Estes, Mary K; Gentsch, Jon R; Iturriza-Gómara, Miren; Kirkwood, Carl D; Martella, Vito; Mertens, Peter P C; Nakagomi, Osamu; Patton, John T; Ruggeri, Franco M; Saif, Linda J; Santos, Norma; Steyer, Andrej; Taniguchi, Koki; Desselberger, Ulrich; Van Ranst, Marc

2008-01-01

Recently, a classification system was proposed for rotaviruses in which all the 11 genomic RNA segments are used (Matthijnssens et al. in J Virol 82:3204-3219, 2008). Based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. A nomenclature for the comparison of complete rotavirus genomes was considered in which the notations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx are used for the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding genes, respectively. This classification system is an extension of the previously applied genotype-based system which made use of the rotavirus gene segments encoding VP4, VP7, VP6, and NSP4. In order to assign rotavirus strains to one of the established genotypes or a new genotype, a standard procedure is proposed in this report. As more human and animal rotavirus genomes will be completely sequenced, new genotypes for each of the 11 gene segments may be identified. A Rotavirus Classification Working Group (RCWG) including specialists in molecular virology, infectious diseases, epidemiology, and public health was formed, which can assist in the appropriate delineation of new genotypes, thus avoiding duplications and helping minimize errors. Scientists discovering a potentially new rotavirus genotype for any of the 11 gene segments are invited to send the novel sequence to the RCWG, where the sequence will be analyzed, and a new nomenclature will be advised as appropriate. The RCWG will update the list of classified strains regularly and make this accessible on a website. Close collaboration with the Study Group Reoviridae of the International Committee on the Taxonomy of Viruses will be maintained.

Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

PubMed Central

Wei, Hengling; Li, Wei; Sun, Xiwei; Zhu, Shuijin; Zhu, Jun

2013-01-01

Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes. PMID:23936305

Transcriptional analysis of Penaeus stylirostris densovirus genes

USDA-ARS?s Scientific Manuscript database

Penaeus stylirostris densovirus (PstDNV) genome contains three open reading frames (ORFs), left, middle, and right, which encode a non-structural (NS) protein, an unknown protein, and a capsid protein (CP), respectively. Transcription mapping revealed that P2, P11 and P61 promoters transcribe the le...

Using Bayesian Models to Assess the Effects of Under-reporting of Cannabis Use on the Association with Birth Defects, National Birth Defects Prevention Study, 1997–2005

PubMed Central

van Gelder, Marleen M. H. J.; Rogier, A.; Donders, T.; Devine, Owen; Roeleveld, Nel; Reefhuis, Jennita

2015-01-01

Background Studies on associations between periconceptional cannabis exposure and birth defects have mainly relied on self-reported exposure. Therefore, the results may be biased due to underreporting of the exposure. The aim of this study was to quantify the potential effects of this form of exposure misclassification. Methods Using multivariable logistic regression, we re-analyzed associations between periconceptional cannabis use and 20 specific birth defects using data from the National Birth Defects Prevention Study from 1997–2005 for 13 859 case infants and 6556 control infants. For seven birth defects, we implemented four Bayesian models based on various assumptions concerning the sensitivity of self-reported cannabis use to estimate odds ratios (ORs), adjusted for confounding and underreporting of the exposure. We used information on sensitivity of self-reported cannabis use from the literature for prior assumptions. Results The results unadjusted for underreporting of the exposure showed an association between cannabis use and anencephaly (posterior OR 1.9 [95% credible interval (CRI) 1.1, 3.2]) which persisted after adjustment for potential exposure misclassification. Initially, no statistically significant associations were observed between cannabis use and the other birth defect categories studied. Although adjustment for underreporting did not notably change these effect estimates, cannabis use was associated with esophageal atresia (posterior OR 1.7 [95% CRI 1.0, 2.9]), diaphragmatic hernia (posterior OR 1.8 [95% CRI 1.1, 3.0]) and gastroschisis (posterior OR 1.7 [95% CRI 1.2, 2.3]) after correction for exposure misclassification. Conclusions Underreporting of the exposure may have obscured some cannabis-birth defect associations in previous studies. However, the resulting bias is likely to be limited. PMID:25155701

Using bayesian models to assess the effects of under-reporting of cannabis use on the association with birth defects, national birth defects prevention study, 1997-2005.

PubMed

van Gelder, Marleen M H J; Donders, A Rogier T; Devine, Owen; Roeleveld, Nel; Reefhuis, Jennita

2014-09-01

Studies on associations between periconceptional cannabis exposure and birth defects have mainly relied on self-reported exposure. Therefore, the results may be biased due to under-reporting of the exposure. The aim of this study was to quantify the potential effects of this form of exposure misclassification. Using multivariable logistic regression, we re-analysed associations between periconceptional cannabis use and 20 specific birth defects using data from the National Birth Defects Prevention Study from 1997-2005 for 13 859 case infants and 6556 control infants. For seven birth defects, we implemented four Bayesian models based on various assumptions concerning the sensitivity of self-reported cannabis use to estimate odds ratios (ORs), adjusted for confounding and under-reporting of the exposure. We used information on sensitivity of self-reported cannabis use from the literature for prior assumptions. The results unadjusted for under-reporting of the exposure showed an association between cannabis use and anencephaly (posterior OR 1.9 [95% credible interval (CRI) 1.1, 3.2]) which persisted after adjustment for potential exposure misclassification. Initially, no statistically significant associations were observed between cannabis use and the other birth defect categories studied. Although adjustment for under-reporting did not notably change these effect estimates, cannabis use was associated with esophageal atresia (posterior OR 1.7 [95% CRI 1.0, 2.9]), diaphragmatic hernia (posterior OR 1.8 [95% CRI 1.1, 3.0]), and gastroschisis (posterior OR 1.7 [95% CRI 1.2, 2.3]) after correction for exposure misclassification. Under-reporting of the exposure may have obscured some cannabis-birth defect associations in previous studies. However, the resulting bias is likely to be limited. © 2014 John Wiley & Sons Ltd.

Intact Arabidopsis RPB1 functions in stem cell niches maintenance and cell cycling control.

PubMed

Zhang, Qian-Qian; Li, Ying; Fu, Zhao-Ying; Liu, Xun-Biao; Yuan, Kai; Fang, Ying; Liu, Yan; Li, Gang; Zhang, Xian-Sheng; Chong, Kang; Ge, Lei

2018-05-12

Plant meristem activity depends on accurate execution of transcriptional networks required for establishing optimum functioning of stem cell niches. An Arabidopsis mutant card1-1 (constitutive auxin response with DR5:GFP) that encodes a truncated RPB1 (RNA Polymerase II's largest subunit) with shortened C-terminal domain (CTD) was identified. Phosphorylation of the CTD repeats of RPB1 is coupled to transcription in eukaryotes. Here we uncover that the truncated CTD of RPB1 disturbed cell cycling and enlarged the size of shoot and root meristem. The defects in patterning of root stem cell niche in card1-1 indicates that intact CTD of RPB1 is necessary for fine-tuning the specific expression of genes responsible for cell-fate determination. The gene-edited plants with different CTD length of RPB1, created by CRISPR-CAS9 technology, confirmed that both the full length and the DK-rich tail of RPB1's CTD play roles in the accurate transcription of CYCB1;1 encoding a cell-cycle marker protein in root meristem and hence participate in maintaining root meristem size. Our experiment proves that the intact RPB1 CTD is necessary for stem cell niche maintenance, which is mediated by transcriptional regulation of cell cycling genes. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana.

PubMed

Yu, Jingyin; Tehrim, Sadia; Zhang, Fengqi; Tong, Chaobo; Huang, Junyan; Cheng, Xiaohui; Dong, Caihua; Zhou, Yanqiu; Qin, Rui; Hua, Wei; Liu, Shengyi

2014-01-03

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana. Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species. This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yang; National Institute of Biological Sciences, Beijing 102206; Xiong, Wei

Although a deficiency in CRY1 or CRY2 correlates with a shorter or longer circadian period, the regulation of CRY proteins in the circadian period has not been well studied. In this study, we found that both CRY1 and CRY2 were able to rescue oscillation in CRY null cells and that they displayed different periods. Furthermore, we demonstrated that protein nuclear import rates, not protein stability, regulate the period-length at the cellular level. Co-transfection of CRY1 and CRY2 in various ratios in the same cells gives rise to the predicted period length in a dose-dependent manner. Given the distinct characteristics ofmore » the C-terminal tails of the CRY1 and CRY2 proteins, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period. - Highlights: • Rhythmic CRY2, like CRY1, in the correct CRY1 phase is sufficient to rescue clock oscillation in CRY null cells. • The short-period mammalian CRY2 protein is more stable than the CRY1 protein. • The N-terminal polypeptide of CRY2 contributes to its stability and Per2 repression, but it does not affect the period. • The C-terminal tails of CRYs regulate their protein stability and nuclear import, but the import rate governs the period. • The ratio, rather than the absolute amounts of CRY1 and CRY2 proteins, determines the period in mammalian cells.« less

Recurrent Deletions and Reciprocal Duplications of 10q11.21q11.23 Including CHAT and SLC18A3 are Likely Mediated by Complex Low-Copy Repeats

PubMed Central

Stankiewicz, Paweł; Kulkarni, Shashikant; Dharmadhikari, Avinash V.; Sampath, Srirangan; Bhatt, Samarth S.; Shaikh, Tamim H.; Xia, Zhilian; Pursley, Amber N.; Cooper, M. Lance; Shinawi, Marwan; Paciorkowski, Alex R.; Grange, Dorothy K.; Noetzel, Michael J.; Saunders, Scott; Simons, Paul; Summar, Marshall; Lee, Brendan; Scaglia, Fernando; Fellmann, Florence; Martinet, Danielle; Beckmann, Jacques S.; Asamoah, Alexander; Platky, Kathryn; Sparks, Susan; Martin, Ann S.; Madan-Khetarpal, Suneeta; Hoover, Jacqueline; Medne, Livija; Bonnemann, Carsten G.; Moeschler, John B.; Vallee, Stephanie E.; Parikh, Sumit; Irwin, Polly; Dalzell, Victoria P.; Smith, Wendy E.; Banks, Valerie C.; Flannery, David B.; Lovell, Carolyn M.; Bellus, Gary A.; Golden-Grant, Kathryn; Gorski, Jerome L.; Kussmann, Jennifer L.; McGregor, Tracy L.; Hamid, Rizwan; Pfotenhauer, Jean; Ballif, Blake C.; Shaw, Chad A.; Kang, Sung-Hae L.; Bacino, Carlos A.; Patel, Ankita; Rosenfeld, Jill A.; Cheung, Sau Wai; Shaffer, Lisa G.

2013-01-01

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers. PMID:21948486

Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene.

PubMed

Lopez, M; Eberlé, F; Mattei, M G; Gabert, J; Birg, F; Bardin, F; Maroc, C; Dubreuil, P

1995-04-03

The human poliovirus (PV) receptor (PVR) is a member of the immunoglobulin (Ig) superfamily with unknown cellular function. We have isolated a human PVR-related (PRR) cDNA. The deduced amino acid (aa) sequence of PRR showed, in the extracellular region, 51.7 and 54.3% similarity with human PVR and with the murine PVR homolog, respectively. The cDNA coding sequence is 1.6-kb long and encodes a deduced 57-kDa protein; this protein has a structural organization analogous to that of PVR, that is, one V- and two C-set Ig domains, with a conserved number of aa. Northern blot analysis indicated that a major 5.9-kb transcript is present in all normal human tissues tested. In situ hybridization showed that the PRR gene is located at bands q23-q24 of human chromosome 11.

Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

PubMed

Tzagoloff, A; Shtanko, A

1995-06-01

Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

Nucleic acids encoding human trithorax protein

DOEpatents

Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

2001-01-01

In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

Cloning and functional characterization of the Xenopus orthologue of the Treacher Collins syndrome (TCOF1) gene product.

PubMed

Gonzales, Bianca; Yang, Hushan; Henning, Dale; Valdez, Benigno C

2005-10-10

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development caused by mutations in the TCOF1 gene, which encodes the nucleolar phosphoprotein treacle. We previously reported a function for mammalian treacle in ribosomal DNA gene transcription by its interaction with upstream binding factor. As an initial step in the development of a TCS model for frog the cDNA that encodes the Xenopus laevis treacle was cloned. Although the derived amino acid sequence shows a poor homology with its mammalian orthologues, Xenopus treacle has 11 highly homologous direct repeats near the center of the protein molecule similar to those present in its human, dog and mouse orthologues. Comparison of their amino acid compositions indicates conservation of predominant specific amino acid residues. Antisense-mediated down-regulation of treacle expression in X. laevis oocytes resulted in inhibition of rDNA gene transcription. The results suggest evolutionary conservation of the function of treacle in ribosomal RNA biogenesis in higher eukaryotes.

Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23.

PubMed

Peoples, R J; Cisco, M J; Kaplan, P; Francke, U

1998-01-01

We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

FMRFamide-related peptides in potato cyst nematodes.

PubMed

Kimber, M J; Fleming, C C; Bjourson, A J; Halton, D W; Maule, A G

2001-09-03

This study presents data demonstrating the presence of FMRFamide-related peptides (FaRPs) in potato cyst nematodes (PCN). Five transcripts of FaRP encoding genes, designated gpflp-1 to gpflp-5, were characterised using RACE. In terms of ORFs, gpflp-1 was 444 base pairs (bp) long and coded for four copies of the FaRP, PF3 (KSAYMRFamide) whilst gpflp-2 was 309 bp long and encoded one copy of the peptide, KNKFEFIRFamide. gpflp-3 (420 bp) Encoded two copies of KHEYLRFamide (AF2) and the genes gpflp-4 and gpflp-5 encoded a total of 11 FaRPs, most of which are novel to PCN. FMRFamide-related peptide (FaRP)-like immunoreactivity was observed in both PCN species, Globodera pallida and Globodera rostochiensis, using an antiserum raised against the invertebrate peptide, FMRFamide. Immunopositive neurones were found throughout the central nervous system in the ventral and dorsal nerve cords and the circumpharyngeal and perianal nerve rings. Reactive neurones were also present peripherally, innervating the highly muscular pharynx with a nerve net and ring-like structures. Positive immunostaining was also observed in neurones running toward the stylet protractor muscles and/or the anterior sensory apparatus. This study implicates a role for FaRPs in feeding, host penetration and sensory function of PCN. This is the first study to characterise FaRP encoding genes from a plant-parasitic nematode using a targeted PCR based RACE approach and further underlines the importance and diversity of this neuropeptide group in the phylum Nematoda.

Transgenic potato plants expressing cry3A gene confer resistance to Colorado potato beetle.

PubMed

Mi, Xiaoxiao; Ji, Xiangzhuo; Yang, Jiangwei; Liang, Lina; Si, Huaijun; Wu, Jiahe; Zhang, Ning; Wang, Di

2015-07-01

The Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a fatal pest, which is a quarantine pest in China. The CPB has now invaded the Xinjiang Uygur Autonomous Region and is constantly spreading eastward in China. In this study, we developed transgenic potato plants expressing cry3A gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the cry3A gene expressed in leaves, stems and roots of the transgenic plants under the control of CaMV 35S promoter, while they expressed only in leaves and stems under the control of potato leaf and stem-specific promoter ST-LS1. The mortality of the larvae was higher (28% and 36%) on the transgenic plant line 35S1 on the 3rd and 4th days, and on ST3 (48%) on the 5th day after inoculation with instar larvae. Insect biomass accumulation on the foliage of the transgenic plant lines 35S1, 35S2 and ST3 was significantly lower (0.42%, 0.43% and 0.42%). Foliage consumption was lowest on transgenic lines 35S8 and ST2 among all plant foliage (7.47 mg/larvae/day and 12.46 mg/larvae/day). The different transgenic plant foliages had varied inhibition to larval growth. The survivors on the transgenic lines obviously were smaller than their original size and extremely weak. The transgenic potato plants with CPB resistance could be used to develop germplasms or varieties for controlling CPB damage and halting its spread in China. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

GTF2IRD2 is located in the Williams–Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix–loop–helix repeats

PubMed Central

Makeyev, Aleksandr V.; Erdenechimeg, Lkhamsuren; Mungunsukh, Ognoon; Roth, Jutta J.; Enkhmandakh, Badam; Ruddle, Frank H.; Bayarsaihan, Dashzeveg

2004-01-01

Williams–Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix–loop–helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix–loop–helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams–Beuren syndrome critical region. PMID:15243160

GTF2IRD2 is located in the Williams-Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix-loop-helix repeats.

PubMed

Makeyev, Aleksandr V; Erdenechimeg, Lkhamsuren; Mungunsukh, Ognoon; Roth, Jutta J; Enkhmandakh, Badam; Ruddle, Frank H; Bayarsaihan, Dashzeveg

2004-07-27

Williams-Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix-loop-helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix-loop-helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams-Beuren syndrome critical region.

OPAQUE11 Is a Central Hub of the Regulatory Network for Maize Endosperm Development and Nutrient Metabolism[OPEN

PubMed Central

Feng, Fan; Qi, Weiwei; Lv, Yuanda; Yan, Shumei; Xu, Liming; Yang, Wenyao; Yuan, Yue; Chen, Yihan

2018-01-01

Maize (Zea mays) endosperm is a primary tissue for nutrient storage and is highly differentiated during development. However, the regulatory networks of endosperm development and nutrient metabolism remain largely unknown. Maize opaque11 (o11) is a classic seed mutant with a small and opaque endosperm showing decreased starch and protein accumulation. We cloned O11 and found that it encodes an endosperm-specific bHLH transcription factor (TF). Loss of function of O11 significantly affected transcription of carbohydrate/amino acid metabolism and stress response genes. Genome-wide binding site analysis revealed 9885 O11 binding sites distributed over 6033 genes. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 259 O11-modulated target genes. O11 was found to directly regulate key TFs in endosperm development (NKD2 and ZmDOF3) and nutrient metabolism (O2 and PBF). Moreover, O11 directly regulates cyPPDKs and multiple carbohydrate metabolic enzymes. O11 is an activator of ZmYoda, suggesting its regulatory function through the MAPK pathway in endosperm development. Many stress-response genes are also direct targets of O11. In addition, 11 O11-interacting proteins were identified, including ZmIce1, which coregulates stress response targets and ZmYoda with O11. Therefore, this study reveals an endosperm regulatory network centered around O11, which coordinates endosperm development, metabolism and stress responses. PMID:29436476

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Bt Proteins Have No Detrimental Effects on Larvae of the Green Lacewing, Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae).

PubMed

Ali, I; Zhang, S; Muhammad, M S; Iqbal, M; Cui, J-J J-J

2018-06-01

Biosafety of a genetically modified crop is required to be assessed prior to its commercialization. For this, a suitable artificial diet was developed and used to establish a dietary exposure test for assessing the toxicity of midgut-active Bt insecticidal proteins on Chrysopa pallens (Rambur). Subsequently, this dietary exposure test was used to evaluate the toxicity of the proteins Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa on C. pallens larvae. Temporal stability, bioactivity, and the intake of the insecticidal proteins were confirmed by enzyme-linked immunosorbent assay and a sensitive-insect bioassay. The life history characteristics, such as survival, pupation, adult emergence, 7-day larval weight, larval developmental time, and emerged male and female fresh weights remained unaffected, when C. pallens were fed the pure artificial diet (negative control) and the artificial diets containing 200 μg/g of each purified protein: Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, or Vip3Aa. On the contrary, all of the life history characteristics of C. pallens larvae were adversely affected when fed artificial diet containing boric acid (positive control). The results demonstrate that diets containing the tested concentrations of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa have null effects on C. pallens larvae. The outcome indicates that genetically modified crops expressing the tested Bt proteins are safe for the lacewing, C. pallens.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Transcriptional analysis of the multicopy hao gene coding for hydroxylamine oxidoreductase in Nitrosomonas sp. strain ENI-11.

PubMed

Hirota, Ryuichi; Kuroda, Akio; Ikeda, Tsukasa; Takiguchi, Noboru; Ohtake, Hisao; Kato, Junichi

2006-08-01

The nitrifying bacterium Nitrosomonas sp. strain ENI-11 has three copies of the gene encoding hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)) on its genome. Broad-host-range reporter plasmids containing transcriptional fusion genes between hao copies and lacZ were constructed to analyze the expression of each hydroxylamine oxidoreductase gene (hao) copy individually and quantitatively. beta-Galactosidase assays of ENI-11 harboring reporter plasmids revealed that all hao copies were transcribed in the wild-type strain. Promoter analysis of hao copies revealed that transcription of hao(3) was highest among the hao copies. Expression levels of hao(1) and hao(2) were 40% and 62% of that of hao(3) respectively. Transcription of hao(1) was negatively regulated, whereas a portion of hao(3) transcription was read through transcription from the rpsT promoter. When energy-depleted cells were incubated in the growth medium, only hao(3) expression increased. This result suggests that it is hao(3) that is responsible for recovery from energy-depleted conditions in Nitrosomonas sp. strain ENI-11.

A single type of cadherin is involved in Bacillus thuringiensis toxicity in Plutella xylostella.

PubMed

Park, Y; Herrero, S; Kim, Y

2015-12-01

Cadherins have been described as one the main functional receptors for the toxins of the entomopathogenic bacterium, Bacillus thuringiensis (Bt). With the availability of the whole genome of Plutella xylostella, different types of cadherins have been annotated. In this study we focused on determining those members of the cadherin-related proteins that potentially play a role in the mode of action of Bt toxins. For this, we mined the genome of P. xylostella to identify these putative cadherins. The genome screening revealed 52 genes that were annotated as cadherin or cadherin-like genes. Further analysis revealed that six of these putative cadherins had three motifs common to all Bt-related cadherins: a signal peptide, cadherin repeats and a transmembrane domain. From the six selected cadherins, only P. xylostella cadherin 1 (PxCad1) was expressed in the larval midgut and only the silencing of this gene by RNA interference (double-stranded RNA feeding) reduce toxicity and binding to the midgut of the Cry1Ac type toxin from Bt. These results indicate that from the whole set of cadherin-related genes identified in P. xylostella, only PxCad1 is associated with the Cry1Ac mode of action. © 2015 The Royal Entomological Society.

Prokaryote-derived protein inhibitors of peptidases: a sketchy occurrence and mostly unknown function

PubMed Central

Kantyka, Tomasz; Rawlings, Neil D.; Potempa, Jan

2010-01-01

In metazoan organisms protein inhibitors of peptidases are important factors essential for regulation of proteolytic activity. In vertebrates genes encoding peptidase inhibitors constitute up to 1% of genes reflecting a need for tight and specific control of proteolysis especially in extracellular body fluids. In stark contrast unicellular organisms, both prokaryotic and eukaryotic consistently contain only few, if any, genes coding for putative peptidase inhibitors. This may seem perplexing in the light of the fact that these organisms produce large numbers of proteases of different catalytic classes with the genes constituting up to 6% of the total gene count with the average being about 3%. Apparently, however, a unicellular life-style is fully compatible with other mechanisms of regulation of proteolysis and does not require protein inhibitors to control their intracellular and extracellular proteolytic activity. So in prokaryotes occurrence of genes encoding different types of peptidase inhibitors is infrequent and often scattered among phylogenetically distinct orders or even phyla of microbiota. Genes encoding proteins homologous to alpha-2-macroglobulin (family I39), serine carboxypeptidase Y inhibitor (family I51), alpha-1-peptidase inhibitor (family I4) and ecotin (family I11) are the most frequently represented in Bacteria. Although several of these gene products were shown to possess inhibitory activity, with an exception of ecotin and staphostatins, the biological function of microbial inhibitors is unclear. In this review we present distribution of protein inhibitors from different families among prokaryotes, describe their mode of action and hypothesize on their role in microbial physiology and interactions with hosts and environment. PMID:20558234

Directed evolution of polymerase function by compartmentalized self-replication.

PubMed

Ghadessy, F J; Ong, J L; Holliger, P

2001-04-10

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

Mycobacterium ahvazicum sp. nov., the nineteenth species of the Mycobacterium simiae complex.

PubMed

Bouam, Amar; Heidarieh, Parvin; Shahraki, Abodolrazagh Hashemi; Pourahmad, Fazel; Mirsaeidi, Mehdi; Hashemzadeh, Mohamad; Baptiste, Emeline; Armstrong, Nicholas; Levasseur, Anthony; Robert, Catherine; Drancourt, Michel

2018-03-07

Four slowly growing mycobacteria isolates were isolated from the respiratory tract and soft tissue biopsies collected in four unrelated patients in Iran. Conventional phenotypic tests indicated that these four isolates were identical to Mycobacterium lentiflavum while 16S rRNA gene sequencing yielded a unique sequence separated from that of M. lentiflavum. One representative strain AFP-003 T was characterized as comprising a 6,121,237-bp chromosome (66.24% guanosine-cytosine content) encoding for 5,758 protein-coding genes, 50 tRNA and one complete rRNA operon. A total of 2,876 proteins were found to be associated with the mobilome, including 195 phage proteins. A total of 1,235 proteins were found to be associated with virulence and 96 with toxin/antitoxin systems. The genome of AFP-003 T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases and 11 genes encoding for bacteriocins. Two regions encoding putative prophages and three OriC regions separated by the dnaA gene were predicted. Strain AFP-003 T genome exhibits 86% average nucleotide identity with Mycobacterium genavense genome. Genetic and genomic data indicate that strain AFP-003 T is representative of a novel Mycobacterium species that we named Mycobacterium ahvazicum, the nineteenth species of the expanding Mycobacterium simiae complex.

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

PubMed

Bel, Yolanda; Sheets, Joel J; Tan, Sek Yee; Narva, Kenneth E; Escriche, Baltasar

2017-06-01

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens ) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125 -iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. Copyright © 2017 Bel et al.

Relation of Candidate Genes that Encode for Endothelial Function to Migraine and Stroke: The Stroke Prevention in Young Women Study

PubMed Central

MacClellan, Leah R.; Howard, Timothy D.; Cole, John W.; Stine, O. Colin; Giles, Wayne H.; O’Connell, Jeffery R.; Wozniak, Marcella A.; Stern, Barney J.; Mitchell, Braxton D.; Kittner, Steven J.

2009-01-01

Background and Purpose Migraine with aura is a risk factor for ischemic stroke but the mechanism by which these disorders are associated remains unclear. Both disorders exhibit familial clustering, which may imply a genetic influence on migraine and stroke risk. Genes encoding for endothelial function are promising candidate genes for migraine and stroke susceptibility because of the importance of endothelial function in regulating vascular tone and cerebral blood flow. Methods Using data from the Stroke Prevention in Young Women (SPYW) study, a population-based case-control study including 297 women aged 15–49 years with ischemic stroke and 422 women without stroke, we evaluated whether polymorphisms in genes regulating endothelial function, including endothelin-1 (EDN), endothelin receptor type B (EDNRB), and nitric oxide synthase-3 (NOS3), confer susceptibility to migraine and stroke. Results EDN SNPs rs1800542 and rs10478723 were associated with increased stroke susceptibility in Caucasians, (OR = 2.1 (95% CI, 1.1 to 4.2) and OR = 2.2 (95% CI, 1.1 to 4.4); p = 0.02 and 0.02, respectively) as were EDNRB SNPs rs4885493 and rs10507875, (OR = 1.7 (95% CI, 1.1 to 2.7) and OR = 2.4 (95% CI, 1.4 to 4.3); p = 0.01 and 0.002, respectively). Only one of the tested SNPs (NOS3 - rs3918166) was associated with both migraine and stroke. Conclusions In our study population, variants in EDN and EDNRB were associated with stroke susceptibility in Caucasian but not in African-American women. We found no evidence that these genes mediate the association between migraine and stroke. PMID:19661472

21 CFR 172.785 - Listeria-specific bacteriophage preparation.

Code of Federal Regulations, 2013 CFR

2013-04-01

... preparation. The additive may be safely used as an antimicrobial agent specific for Listeria monocytogenes (L... 725.421(d). No sequences derived from genes encoding bacterial 16S ribosomal RNA are present in the..., as determined by the “Method to Determine Microbial Contamination,” dated July 11, 2003, and printed...

21 CFR 172.785 - Listeria-specific bacteriophage preparation.

Code of Federal Regulations, 2011 CFR

2011-04-01

... preparation. The additive may be safely used as an antimicrobial agent specific for Listeria monocytogenes (L... 725.421(d). No sequences derived from genes encoding bacterial 16S ribosomal RNA are present in the..., as determined by the “Method to Determine Microbial Contamination,” dated July 11, 2003, and printed...

21 CFR 172.785 - Listeria-specific bacteriophage preparation.

Code of Federal Regulations, 2014 CFR

2014-04-01

... used as an antimicrobial agent specific for Listeria monocytogenes (L. monocytogenes) in accordance... 725.421(d). No sequences derived from genes encoding bacterial 16S ribosomal RNA are present in the..., as determined by the “Method to Determine Microbial Contamination,” dated July 11, 2003, and printed...

21 CFR 172.785 - Listeria-specific bacteriophage preparation.

Code of Federal Regulations, 2012 CFR

2012-04-01

... preparation. The additive may be safely used as an antimicrobial agent specific for Listeria monocytogenes (L... 725.421(d). No sequences derived from genes encoding bacterial 16S ribosomal RNA are present in the..., as determined by the “Method to Determine Microbial Contamination,” dated July 11, 2003, and printed...

Transcription Mapping of the Kaposi’s Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Genome in a Body Cavity-Based Lymphoma Cell Line (BC-1)

PubMed Central

Sarid, Ronit; Flore, Ornella; Bohenzky, Roy A.; Chang, Yuan; Moore, Patrick S.

1998-01-01

Kaposi’s sarcoma-associated herpesvirus (KSHV) gene transcription in the BC-1 cell line (KSHV and Epstein-Barr virus coinfected) was examined by using Northern analysis with DNA probes extending across the viral genome except for a 3-kb unclonable rightmost region. Three broad classes of viral gene transcription have been identified. Class I genes, such as those encoding the v-cyclin, latency-associated nuclear antigen, and v-FLIP, are constitutively transcribed under standard growth conditions, are unaffected by tetradecanoylphorbol acetate (TPA) induction, and presumably represent latent viral transcripts. Class II genes are primarily clustered in nonconserved regions of the genome and include small polyadenylated RNAs (T0.7 and T1.1) as well as most of the virus-encoded cytokines and signal transduction genes. Class II genes are transcribed without TPA treatment but are induced to higher transcription levels by TPA treatment. Class III genes are primarily structural and replication genes that are transcribed only following TPA treatment and are presumably responsible for lytic virion production. These results indicate that BC-1 cells have detectable transcription of a number of KSHV genes, particularly nonconserved genes involved in cellular signal transduction and regulation, during noninduced (latent) virus culture. PMID:9444993

Homez, a homeobox leucine zipper gene specific to the vertebrate lineage.

PubMed

Bayarsaihan, Dashzeveg; Enkhmandakh, Badam; Makeyev, Aleksandr; Greally, John M; Leckman, James F; Ruddle, Frank H

2003-09-02

This work describes a vertebrate homeobox gene, designated Homez (homeodomain leucine zipper-encoding gene), that encodes a protein with an unusual structural organization. There are several regions within Homez, including three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The gene is ubiquitously expressed in human and murine tissues, although the expression pattern is more restricted during mouse development. Genomic analysis revealed that human and mouse genes are located at 14q11.2 and 14C, respectively, and are composed of two exons. The zebrafish and pufferfish homologs share high similarity to mammalian sequences, particularly within the homeodomain sequences. Based on homology of homeodomains and on the similarity in overall protein structure, we delineate Homez and members of ZHX family of zinc finger homeodomain factors as a subset within the superfamily of homeobox-containing proteins. The type and composition of homeodomains in the Homez subfamily are vertebrate-specific. Phylogenetic analysis indicates that Homez lineage was separated from related genes >400 million years ago before separation of ray- and lobe-finned fishes. We apply a duplication-degeneration-complementation model to explain how this family of genes has evolved.

NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in Mus musculus

PubMed Central

Sutter, Carrie Hayes; Olesen, Kristin M; Kensler, Thomas W

2018-01-01

Diurnal oscillation of intracellular redox potential is known to couple metabolism with the circadian clock, yet the responsible mechanisms are not well understood. We show here that chemical activation of NRF2 modifies circadian gene expression and rhythmicity, with phenotypes similar to genetic NRF2 activation. Loss of Nrf2 function in mouse fibroblasts, hepatocytes and liver also altered circadian rhythms, suggesting that NRF2 stoichiometry and/or timing of expression are important to timekeeping in some cells. Consistent with this concept, activation of NRF2 at a circadian time corresponding to the peak generation of endogenous oxidative signals resulted in NRF2-dependent reinforcement of circadian amplitude. In hepatocytes, activated NRF2 bound specific enhancer regions of the core clock repressor gene Cry2, increased Cry2 expression and repressed CLOCK/BMAL1-regulated E-box transcription. Together these data indicate that NRF2 and clock comprise an interlocking loop that integrates cellular redox signals into tissue-specific circadian timekeeping. PMID:29481323