Sample records for spot mutant isolates
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Arendrup, Maiken Cavling; Park, Steven; Brown, Steven; Pfaller, Michael; Perlin, David S.
2011-01-01
Disk diffusion testing has recently been standardized by the CLSI, and susceptibility breakpoints have been established for several antifungal compounds. For caspofungin, 5-μg disks are approved, and for micafungin, 10-μg disks are under evaluation. We evaluated the performances of caspofungin and micafungin disk testing using a panel of Candida isolates with and without known FKS echinocandin resistance mechanisms. Disk diffusion and microdilution assays were performed strictly according to CLSI documents M44-A2 and M27-A3. Eighty-nine clinical Candida isolates were included: Candida albicans (20 isolates/10 mutants), C. glabrata (19 isolates/10 mutants), C. dubliniensis (2 isolates/1 mutant), C. krusei (16 isolates/3 mutants), C. parapsilosis (14 isolates/0 mutants), and C. tropicalis (18 isolates/4 mutants). Quality control strains were C. parapsilosis ATCC 22019 and C. krusei ATCC 6258. The correlations between zone diameters and MIC results were good for both compounds, with identical susceptibility classifications for 93.3% of the isolates by applying the current CLSI breakpoints. However, the numbers of fks hot spot mutant isolates misclassified as being susceptible (S) (very major errors [VMEs]) were high (61% for caspofungin [S, ≥11 mm] and 93% for micafungin [S, ≥14 mm]). Changing the disk diffusion breakpoint to S at ≥22 mm significantly improved the discrimination. For caspofungin, 1 VME was detected (a C. tropicalis isolate with an F76S substitution) (3.5%), and for micafungin, 10 VMEs were detected, the majority of which were for C. glabrata (8/10). The broadest separation between zone diameter ranges for wild-type (WT) and mutant isolates was seen for caspofungin (6 to 12 mm versus −4 to 7 mm). In conclusion, caspofungin disk diffusion testing with a modified breakpoint led to excellent separation between WT and mutant isolates for all Candida species. PMID:21357293
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Garcia-Effron, Guillermo; Park, Steven; Perlin, David S
2011-05-01
Echinocandins are highly bound to serum proteins, altering their antifungal properties. The addition of 50% human serum to the MIC assay improves the identification of echinocandin-resistant Candida spp. harboring fks hot spot mutations. However, this modification cannot readily be applied to the method of the CLSI M27-A3 document due to safety and standardization difficulties. The aim of this study was to evaluate commercial bovine serum albumin (BSA) as a safe and standardized alternative to human serum. A collection of 28 echinocandin-susceptible strains, 10 Candida parapsilosis sensu lato strains (with naturally reduced echinocandin susceptibility), and 40 FKS hot spot mutants was used in this work. When RPMI 1640 was used for susceptibility testing, wild-type strains and fks mutants showed MIC range overlaps (-2, -1, and -3 2-fold-dilution steps separated these populations for anidulafungin, caspofungin, and micafungin, respectively). On the other hand, the addition of BSA to RPMI 1640 differentially increased echinocandin MIC values for these groups of strains, allowing better separation between populations, with no MIC range overlaps for any of the echinocandin drugs tested. Moreover, the use of RPMI-BSA reduced the number of fks hot spot mutant isolates for which MIC values were less than or equal to the upper limit for the wild type (very major errors) from 9, 2, and 7 with RPMI alone to 3, 0, and 3 for anidulafungin, caspofungin, and micafungin, respectively. When RPMI-BSA was used to study the susceptibility of C. parapsilosis sensu lato species to echinocandins, the strains behaved as anidulafungin- and micafungin-resistant isolates (MIC, ≥8 μg/ml). These data support the need for a revision of the CLSI protocol for in vitro testing of echinocandin susceptibility in order to identify all or most of the fks hot spot mutants. Also, caspofungin could be used as a surrogate marker of reduced susceptibility to echinocandins.
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Garcia-Effron, Guillermo; Park, Steven; Perlin, David S.
2011-01-01
Echinocandins are highly bound to serum proteins, altering their antifungal properties. The addition of 50% human serum to the MIC assay improves the identification of echinocandin-resistant Candida spp. harboring fks hot spot mutations. However, this modification cannot readily be applied to the method of the CLSI M27-A3 document due to safety and standardization difficulties. The aim of this study was to evaluate commercial bovine serum albumin (BSA) as a safe and standardized alternative to human serum. A collection of 28 echinocandin-susceptible strains, 10 Candida parapsilosis sensu lato strains (with naturally reduced echinocandin susceptibility), and 40 FKS hot spot mutants was used in this work. When RPMI 1640 was used for susceptibility testing, wild-type strains and fks mutants showed MIC range overlaps (−2, −1, and −3 2-fold-dilution steps separated these populations for anidulafungin, caspofungin, and micafungin, respectively). On the other hand, the addition of BSA to RPMI 1640 differentially increased echinocandin MIC values for these groups of strains, allowing better separation between populations, with no MIC range overlaps for any of the echinocandin drugs tested. Moreover, the use of RPMI-BSA reduced the number of fks hot spot mutant isolates for which MIC values were less than or equal to the upper limit for the wild type (very major errors) from 9, 2, and 7 with RPMI alone to 3, 0, and 3 for anidulafungin, caspofungin, and micafungin, respectively. When RPMI-BSA was used to study the susceptibility of C. parapsilosis sensu lato species to echinocandins, the strains behaved as anidulafungin- and micafungin-resistant isolates (MIC, ≥8 μg/ml). These data support the need for a revision of the CLSI protocol for in vitro testing of echinocandin susceptibility in order to identify all or most of the fks hot spot mutants. Also, caspofungin could be used as a surrogate marker of reduced susceptibility to echinocandins. PMID:21383097
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Arendrup, Maiken Cavling; Garcia-Effron, Guillermo; Lass-Flörl, Cornelia; Lopez, Alicia Gomez; Rodriguez-Tudela, Juan-Luis; Cuenca-Estrella, Manuel; Perlin, David S.
2010-01-01
This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC50) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants. PMID:19884370
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Characterization and fine mapping of a light-dependent leaf lesion mimic mutant 1 in rice.
Wang, Jing; Ye, Bangquan; Yin, Junjie; Yuan, Can; Zhou, Xiaogang; Li, Weitao; He, Min; Wang, Jichun; Chen, Weilan; Qin, Peng; Ma, Bintian; Wang, Yuping; Li, Shigui; Chen, Xuewei
2015-12-01
Plants that spontaneously produce lesion mimics or spots, without any signs of obvious adversity, such as pesticide and mechanical damage, or pathogen infection, are so-called lesion mimic mutants (lmms). In rice, many lmms exhibit enhanced resistance to pathogens, which provides a unique opportunity to uncover the molecular mechanism underlying lmms. We isolated a rice light-dependent leaf lesion mimic mutant 1 (llm1). Lesion spots appeared in the leaves of the llm1 mutant at the tillering stage. Furthermore, the mutant llm1 had similar agronomic traits to wild type rice. Trypan blue and diamiobenzidine staining analyses revealed that the lesion spot formation on the llm1 mutant was due to programmed cell death and reactive oxygen species. The chloroplasts were severely damaged in the llm1 mutant, suggesting that chloroplast damage was associated with the formation of lesion spots in llm1. More importantly, llm1 exhibited enhanced resistance to bacterial blight pathogens within increased expression of pathogenesis related genes (PRs). Using a map-based cloning approach, we delimited the LLM1 locus to a 121-kb interval between two simple sequence repeat markers, RM17470 and RM17473, on chromosome 4. We sequenced the candidate genes on the interval and found that a base mutation had substituted adenine phosphate for thymine in the last exon of LOC_Os04g52130, which led to an amino acid change (Asp(388) to Val) in the llm1 mutant. Our investigation showed that the putative coproporphyrinogen III oxidase (CPOX) encoded by LOC_Os04g52130 was produced by LLM1 and that amino acid Asp(388) was essential for CPOX function. Our study provides the basis for further investigations into the mechanism underlying lesion mimic initiation associated with LLM1. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
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Vogt, Stefanie L.; Green, Christopher; Stevens, Katarzyna M.; Day, Brad; Erickson, David L.; Woods, Donald E.; Storey, Douglas G.
2011-01-01
The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3′,5′-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa. PMID:21788391
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Sokoro, AbdulRazaq A H; Lepage, Joyce; Antonishyn, Nick; McDonald, Ryan; Rockman-Greenberg, Cheryl; Irvine, James; Lehotay, Denis C
2010-12-01
Mutations in the SLC25A15 gene, encoding the human inner mitochondrial membrane ornithine transporter, are thought to be responsible for hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome, a rare autosomal recessive condition. HHH syndrome has been detected in several small, isolated communities in northern Saskatchewan (SK). To determine the incidence of HHH syndrome in these communities, a PCR method was set up to detect F188Δ, the common French-Canadian mutation. Neonatal blood spots collected from all newborns from the high risk area were genotyped for the F188Δ mutation for seven consecutive years. Using DNA analysis, we estimated that the heterozygote frequency for the mutant allele for HHH syndrome to be about 1 in 19 individuals, predicting one affected child with HHH syndrome for approximately every 1,500 individuals (1 in 1,550 live births; 1 child every 12 years) in this isolated population. The frequency for the mutant allele for HHH syndrome in this isolated community is probably the highest in the world for this rare disorder. We determined that ornithine levels, by tandem mass spectrometry, were not abnormal in newborns with F188Δ mutation, carriers and normals. Ornithine rises to abnormally high levels at some time after birth well past the time that the newborn screening blood spot is collected. The timing or the reasons for the delayed rise of ornithine in affected children with HHH syndrome have not been determined. Newborn screening for HHH Syndrome in this high risk population is only possible by detection of the mutant allele using DNA analysis.
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Distinct Rayleigh scattering from hot spot mutant p53 proteins reveals cancer cells.
Jun, Ho Joon; Nguyen, Anh H; Kim, Yeul Hong; Park, Kyong Hwa; Kim, Doyoun; Kim, Kyeong Kyu; Sim, Sang Jun
2014-07-23
The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Zhang, Zhifang; Zhu, Zengrong; Ma, Zhonghua; Li, Hongye
2009-05-31
Sixty-five isolates of Pencillium digitatum (Pers.:Fr) Sacc., a causative agent of green mold of postharvest citrus, were collected from various locations in Zhejiang province in 2000, 2005 and 2006, and assayed for their sensitivity to the quinone outside inhibitor (QoI) fungicide azoxystrobin. The results showed that azoxystrobin is highly effective against P. digitatum, in vitro, and that the effective concentrations resulting in reduction of conidial germination and mycelial growth by 50% (EC(50)) averaged 0.0426 microg/ml and 0.0250 microg/ml, respectively. Twenty-eight azoxystrobin-resistant mutants were obtained by UV mutagenesis and subsequent selection on medium amended with azoxystrobin (12 microg/ml) and salicylhydroxamic acid. All obtained mutants were highly resistant to azoxystrobin and their resistance was genetically stable. Analysis of the cytochrome b gene structure of P. digitatum (Pdcyt b) showed the absence of type I intron in the first hot spot region of mutation. These results indicate that P. digitatum is likely to evolve high levels of resistance to azoxystrobin after its application. Analysis of partial sequences of Pdcyt b from both the azoxystrobin-sensitive parental isolate and the 28 azoxystrobin-resistant mutants revealed that a point mutation, which leads to the substitution at code 143 of alanine for glycine (G143A), is responsible for the observed azoxystrobin resistance in the laboratory mutants. Based on this point mutation, two allele-specific PCR primers were designed and optimized for allele-specific PCR detection of azoxystrobin-resistant isolates of P. digitatum.
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Almási, Asztéria; Nemes, Katalin; Csömör, Zsófia; Tóbiás, István; Palkovics, László; Salánki, Katalin
2017-06-01
The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.
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Li, Chao; Yang, Jun; Zhou, Wei; Chen, Xiao-Lin; Huang, Jin-Guang; Cheng, Zhi-Hua; Zhao, Wen-Sheng; Zhang, Yan; Peng, You-Liang
2014-11-01
Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.
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Koana, Takao; Takahashi, Takashi; Tsujimura, Hidenobu
2012-03-01
The third instar larvae of Drosophila were irradiated with X rays, and the somatic mutation frequency in their wings was measured after their eclosion. In the flies with normal DNA repair and apoptosis functions, 0.2 Gy irradiation at 0.05 Gy/min reduced the frequency of the so-called small spot (mutant cell clone with reduced reproductive activity) compared with that in the sham-irradiated flies. When apoptosis was suppressed using the baculovirus p35 gene, the small spot frequency increased four times in the sham-irradiated control group, but the reduction by the 0.2-Gy irradiation was still evident. In a non-homologous end joining-deficient mutant, the small spot frequency was also reduced by 0.2 Gy radiation. In a mutant deficient in single-strand break repair, no reduction in the small spot frequency by 0.2 Gy radiation was observed, and the small spot frequency increased with the radiation dose. Large spot (mutant cell clone with normal reproductive activity) frequency was not affected by suppression of apoptosis and increased monotonically with radiation dose in wild-type larvae and in mutants for single- or double-strand break repair. It is hypothesized that some of the small spots resulted from single-strand damage and, in wild-type larvae, 0.2 Gy radiation activated the normal single-strand break repair gene, which reduced the background somatic mutation frequency.
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Fan, Lili; Fu, Kehe; Yu, Chuanjin; Li, Yingying; Li, Yaqian; Chen, Jie
2015-05-01
Mutant T66 was isolated from 450 mutants (constructed with Agrobacterium tumefaciens-mediated transformation method) of Trichoderma harzianum. Maize seeds coated with T66 were more susceptible to Curvularia lunata when compared with those coated with wild-type (WT) strain. The disease index of maize treated with T66 and WT were 62.5 and 42.1%, respectively. Further research showed T-DNA has inserted into the ORF of one gene, which resulted in the functional difference between WT and T66. The gene was cloned and named Thc6, which encodes a novel 327 amino acid protein. To investigate its function, we obtained knockout, complementation, and overexpression mutants of Thc6. Challenge inoculation studies suggested that the Thc6 overexpression mutant can reduce the disease index of maize inbred line Huangzao 4 against the leaf spot pathogen (C. lunata). Meanwhile, The Thc6 mutants were found to affect the resistance of maize inbred line Huangzao 4 against C. lunata by enhancing the activation of jasmonate-responsive genes expression. Liquid chromatography-mass spectrometry (LC-MS) data further confirmed that the concentration of jasmonate in the induced maize exhibits a parallel change tendency with the expression level of defense-related genes. Hence, the Thc6 gene could be participated in the induced resistance of maize inbred line Huangzao 4 against C. lunata infection through a jasmonic acid-dependent pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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di Rienzo, Valentina; Bubici, Giovanni; Montemurro, Cinzia; Cillo, Fabrizio
2018-01-01
In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.
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Gérando, H Máté de; Fayolle-Guichard, F; Rudant, L; Millah, S K; Monot, F; Ferreira, Nicolas Lopes; López-Contreras, A M
2016-06-01
Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.
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Zhu, Yuanyuan; Liang, Xinle; Zhang, Hong; Feng, Wei; Liu, Ye; Zhang, Fuming; Linhardt, Robert J
2017-05-02
Aspergillus oryzae koji plays a crucial role in fermented food products due to the hydrolytic activities of secreted enzymes. In the present study, we performed a comparative secretome analysis of the industrial strain of Aspergillus oryzae 3.042 and its spontaneous mutantZJGS-LZ-21. One hundred and fifty two (152) differential protein spots were excised (p<0.05), and 25 proteins were identified. Of the identified proteins, 91.3% belonged to hydrolytic enzymes acting on carbohydrates or proteins. Consistent with their enzyme activities, the expression of 14 proteins involved in the degradation of cellulose, hemicellulose, starch and proteins, increased in the ZJGS-LZ-21isolate. In particular, increased levels of acid protease (Pep) may favor the degradation of soy proteins in acidic environments and promote the cleavage of allergenic soybean proteins in fermentation, resulting in improvements of product safety and quality. The ZJGS-LZ-21 isolate showed higher protein secretion and increased hydrolytic activities than did strain 3.042, indicating its promising application in soybean paste fermentation. Copyright © 2017 Elsevier B.V. All rights reserved.
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Ponmani, Thangaraj; Munavar, M Hussain
2014-06-01
The pheS5 Ts mutant of Escherichia coli defined by a G293 → A293 transition, which is responsible for thermosensitive Phenylalanyl-tRNA synthetase has been well studied at both biochemical and molecular level but genetic analyses pertaining to suppressors of pheS5 were hard to come by. Here we have systematically analyzed a spectrum of Temperature-insensitive derivatives isolated from pheS5 Ts mutant and identified two intragenic suppressors affecting the same base pair coordinate G673 (pheS19 defines G673 → T673 ; Gly225 → Cys225 and pheS28 defines G673 → C673 ; Gly225 → Arg225). In fact in the third derivative, the intragenic suppressor originally named pheS43 (G673 → C673 transversion) is virtually same as pheS28. In the fourth case, the very pheS5 lesion itself has got changed from A293 → T293 (named pheS40). Cloning of pheS(+), pheS5, pheS5-pheS19, pheS5-pheS28 alleles into pBR322 and introduction of these clones into pheS5 mutant revealed that excess of double mutant protein is not at all good for the survival of cells at 42°C. These results clearly indicate a pivotal role for Gly225 in the structural/functional integrity of alpha subunit of E. coli PheRS enzyme and it is proposed that G673 might define a hot spot for intragenic suppressors of pheS5. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
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de Ronde, Dryas; Pasquier, Adrien; Ying, Su; Butterbach, Patrick; Lohuis, Dick; Kormelink, Richard
2014-02-01
Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)-based resistance. The observation that NSs from two natural resistance-breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance-inducing wild-type strains (NSs(RI) ), amino acid reversions in NSs from resistance-breaking strains (NSs(RB)), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw-mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N-terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs(RI) and NSs(RB). Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs(RI) showed that Avr functionality could not simply be transferred between species. Although deletion of the C-terminal domain rendered NSs completely dysfunctional, only a few single-amino-acid mutations in the C-terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference. © 2013 BSPP AND JOHN WILEY & SONS LTD.
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Vicente, J G; Everett, B; Roberts, S J
2006-07-01
ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.
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BODYGUARD is required for the biosynthesis of cutin in Arabidopsis.
Jakobson, Liina; Lindgren, Leif Ove; Verdier, Gaëtan; Laanemets, Kristiina; Brosché, Mikael; Beisson, Fred; Kollist, Hannes
2016-07-01
The cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis. An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated. The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an α/β-hydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated ω-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
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Johnson, Michael E; Katiyar, Santosh K; Edlind, Thomas D
2011-08-01
Echinocandins represent a new antifungal group with potent activity against Candida species. These lipopeptides inhibit the synthesis of β-1,3-glucan, the major cell wall polysaccharide. Acquired resistance or reduced echinocandin susceptibility (RES) is rare and associated with mutations in two "hot spot" regions of Fks1 or Fks2, the probable β-1,3-glucan synthases. In contrast, many fungi demonstrate intrinsic RES for reasons that remain unclear. We are using Saccharomyces cerevisiae to understand the basis for RES by modeling echinocandin-Fks interaction. Previously characterized mutations confer cross-RES; we screened for mutations conferring differential RES, implying direct interaction of that Fks residue with a variable echinocandin side chain. One mutant (in an fks1Δ background) exhibited ≥16-fold micafungin and anidulafungin versus caspofungin RES. Sequencing identified a novel Fks2 mutation, W714L/Y715N. Equivalent W695L/Y696N and related W695L/F/C mutations in Fks1 generated by site-directed mutagenesis and the isolation of a W695L-equivalent mutation in Candida glabrata confirmed the role of the new "hot spot 3" in RES. Further mutagenesis expanded hot spot 3 to Fks1 residues 690 to 700, yielding phenotypes ranging from cross-RES to differential hypersusceptibility. Fks1 sequences from intrinsically RES Scedosporium species revealed W695F-equivalent substitutions; Fks1 hybrids expressing Scedosporium prolificans hot spot 3 confirmed that this substitution imparts RES.
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Angelini, Sandra; My, Laetitia; Bouveret, Emmanuelle
2012-01-01
In bacteria, Acyl Carrier Protein (ACP) is the central cofactor for fatty acid biosynthesis. It carries the acyl chain in elongation and must therefore interact successively with all the enzymes of this pathway. Yet, ACP also interacts with proteins of diverse unrelated function. Among them, the interaction with SpoT has been proposed to be involved in regulating ppGpp levels in the cell in response to fatty acid synthesis inhibition. In order to better understand this mechanism, we screened for ACP mutants unable to interact with SpoT in vivo by bacterial two-hybrid, but still functional for fatty acid synthesis. The position of the selected mutations indicated that the helix II of ACP is responsible for the interaction with SpoT. This suggested a mechanism of recognition similar to one used for the enzymes of fatty acid synthesis. Consistently, the interactions tested by bacterial two-hybrid of ACP with fatty acid synthesis enzymes were also affected by the mutations that prevented the interaction with SpoT. Yet, interestingly, the corresponding mutant strains were viable, and the phenotypes of one mutant suggested a defect in growth regulation. PMID:22558350
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Horn, Nikki; Wegmann, Udo; Dertli, Enes; Mulholland, Francis; Collins, Samuel R A; Waldron, Keith W; Bongaerts, Roy J; Mayer, Melinda J; Narbad, Arjan
2013-01-01
As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.
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Carlisle, L; Steel, K; Forge, A
1990-11-01
Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.
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Baltes, Nina; Tonpitak, Walaiporn; Gerlach, Gerald-F.; Hennig-Pauka, Isabel; Hoffmann-Moujahid, Astrid; Ganter, Martin; Rothkötter, Hermann-J.
2001-01-01
Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniae single mutants (ΔexbB and ΔureC) and a newly constructed A. pleuropneumoniae double (ΔureC ΔexbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniae ΔexbB mutant nor the double ΔureC ΔexbB mutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniae in the porcine respiratory tract. The A. pleuropneumoniae ΔureC mutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniae ΔureC mutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number of A. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae ΔureC mutant than in the BALF of those infected with the parent strain. These results imply that A. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniae parent strain. PMID:11119539
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Hou, Xingsheng; McMillan, Mary; Coumans, Joëlle V F; Poljak, Anne; Raftery, Mark J; Pereg, Lily
2014-01-01
FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.
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Coumans, Joëlle V. F.; Poljak, Anne; Raftery, Mark J.; Pereg, Lily
2014-01-01
FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA − strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome. PMID:25502569
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Aguilera-Cogley, Vidal Antonio; Berbegal, Mónica; Català, Santiago; Brentu, Francis Collison; Armengol, Josep; Vicent, Antonio
2017-01-01
Greasy spot of citrus, caused by Zasmidium citri-griseum (= Mycosphaerella citri), is widely distributed in the Caribbean Basin, inducing leaf spots, premature defoliation, and yield loss. Greasy spot-like symptoms were frequently observed in humid citrus-growing regions in Panama as well as in semi-arid areas in Spain, but disease aetiology was unknown. Citrus-growing areas in Panama and Spain were surveyed and isolates of Mycosphaerellaceae were obtained from citrus greasy spot lesions. A selection of isolates from Panama (n = 22) and Spain (n = 16) was assembled based on their geographical origin, citrus species, and affected tissue. The isolates were characterized based on multi-locus DNA (ITS and EF-1α) sequence analyses, morphology, growth at different temperatures, and independent pathogenicity tests on the citrus species most affected in each country. Reference isolates and sequences were also included in the analysis. Isolates from Panama were identified as Z. citri-griseum complex, and others from Spain attributed to Amycosphaerella africana. Isolates of the Z. citri-griseum complex had a significantly higher optimal growth temperature (26.8°C) than those of A. africana (19.3°C), which corresponded well with their actual biogeographical range. The isolates of the Z. citri-griseum complex from Panama induced typical greasy spot symptoms in 'Valencia' sweet orange plants and the inoculated fungi were reisolated. No symptoms were observed in plants of the 'Ortanique' tangor inoculated with A. africana. These results demonstrate the presence of citrus greasy spot, caused by Z. citri-griseum complex, in Panama whereas A. africana was associated with greasy spot-like symptoms in Spain.
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Transcriptional specificity in various p53-mutant cells.
Okaichi, Kumio; Izumi, Nanaka; Takamura, Yuma; Fukui, Shoichi; Kudo, Takashi
2013-03-01
Mutation of the tumor suppressor gene p53 is the most common genetic alteration observed in human tumors. However, the relationship between the mutation point of p53 and the transcriptional specificity is not so obvious. We prepared Saos-2 cells with various mutations of p53 that are found in human tumors, and examined the resulting transcriptional alterations in the cells. Loss of function and gain of function were observed in all p53 mutants. Hot-spot mutations of p53 are frequently found in tumor cells. We compared hot-spot mutations and other mutations of p53 and found that a more than 2-fold transcription of CADPS2, PIWIL4 and TRIM9 was induced by hot spot mutations, but not by other mutations. As PIWIL4 suppresses the p16(INK4A) and ARF pathway, restraining cell growth and genomic instability, induction of PIWIL4 expression may be one reason why hot-spot mutations are frequently found in tumor cells.
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G-protein control of the ribosome-associated stress response protein SpoT.
Jiang, Mengxi; Sullivan, Susan M; Wout, Patrice K; Maddock, Janine R
2007-09-01
The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.
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Aguilera-Cogley, Vidal Antonio; Berbegal, Mónica; Català, Santiago; Brentu, Francis Collison; Armengol, Josep
2017-01-01
Greasy spot of citrus, caused by Zasmidium citri-griseum (= Mycosphaerella citri), is widely distributed in the Caribbean Basin, inducing leaf spots, premature defoliation, and yield loss. Greasy spot-like symptoms were frequently observed in humid citrus-growing regions in Panama as well as in semi-arid areas in Spain, but disease aetiology was unknown. Citrus-growing areas in Panama and Spain were surveyed and isolates of Mycosphaerellaceae were obtained from citrus greasy spot lesions. A selection of isolates from Panama (n = 22) and Spain (n = 16) was assembled based on their geographical origin, citrus species, and affected tissue. The isolates were characterized based on multi-locus DNA (ITS and EF-1α) sequence analyses, morphology, growth at different temperatures, and independent pathogenicity tests on the citrus species most affected in each country. Reference isolates and sequences were also included in the analysis. Isolates from Panama were identified as Z. citri-griseum complex, and others from Spain attributed to Amycosphaerella africana. Isolates of the Z. citri-griseum complex had a significantly higher optimal growth temperature (26.8°C) than those of A. africana (19.3°C), which corresponded well with their actual biogeographical range. The isolates of the Z. citri-griseum complex from Panama induced typical greasy spot symptoms in ‘Valencia’ sweet orange plants and the inoculated fungi were reisolated. No symptoms were observed in plants of the ‘Ortanique’ tangor inoculated with A. africana. These results demonstrate the presence of citrus greasy spot, caused by Z. citri-griseum complex, in Panama whereas A. africana was associated with greasy spot-like symptoms in Spain. PMID:29236789
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Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.
Jakobi, Stephan; Nguyen, Tran Xuan Phong; Debaene, François; Metz, Alexander; Sanglier-Cianférani, Sarah; Reuter, Klaus; Klebe, Gerhard
2014-10-01
Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. © 2014 Wiley Periodicals, Inc.
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Iwata, Masaki; Taira, Wataru; Hiyama, Atsuki; Otaki, Joji M
2015-06-01
The nymphalid groundplan has been proposed to explain diverse butterfly wing color patterns. In this model, each symmetry system is composed of a core element and a pair of paracore elements. The development of this elemental configuration has been explained by the induction model for positional information. However, the diversity of color patterns in other butterfly families in relation to the nymphalid groundplan has not been thoroughly examined. Here, we examined aberrant color pattern phenotypes of a lycaenid butterfly, Zizeeria maha, from mutagenesis and plasticity studies as well as from field surveys. In several mutants, the third and fourth spot arrays were coordinately positioned much closer to the discal spot in comparison to the normal phenotype. In temperature-shock types, the third and fourth array spots were elongated inwardly or outwardly from their normal positions. In field-caught spontaneous mutants, small black spots were located adjacent to normal black spots. Analysis of these aberrant phenotypes indicated that the spots belonging to the third and fourth arrays are synchronously changeable in position and shape around the discal spot. Thus, these arrays constitute paracore elements of the central symmetry system of the lycaenid butterflies, and the discal spot comprises the core element. These aberrant phenotypes can be explained by the black-inducing signals that propagate from the prospective discal spot, as predicted by the induction model. These results suggest the existence of long-range developmental signals that cover a large area of a wing not only in nymphalid butterflies, but also in lycaenid butterflies.
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Horn, Nikki; Wegmann, Udo; Dertli, Enes; Mulholland, Francis; Collins, Samuel R. A.; Waldron, Keith W.; Bongaerts, Roy J.; Mayer, Melinda J.; Narbad, Arjan
2013-01-01
As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions. PMID:23544114
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Selective chromatid segregation mechanism for Bruchus wings piebald color.
Klar, Amar J S
2015-01-01
The mechanisms of asymmetric organ development have been under intensive investigation for years, yet the proposed mechanisms remain controversial (1-3). The female Bruchus quadrimaculatus beetle insect develops two black-colored spots bilaterally located on each upper elytra wing by an unknown mechanism. Fifty percent of the P (for piebald, two colors) gene homozygous mutant insects, described in 1925, had a normal left elytrum (with two black spots) and an abnormal right elytrum (with two red spots) and the balance supported the converse lateralized pigment arrangement (4). Rather than supporting the conventional morphogen model for the wings pigmentation development, their biological origin is explained here with the somatic strand-specific epigenetic imprinting and selective sister chromatid segregation (SSIS) mechanism (5). We propose that the P gene product performs the selective sister chromatid segregation function to produce symmetric cell division of a specific cell during embryogenesis to result in the bilateral symmetric development of elytra black color spots and that the altered chromatid segregation pattern of the mutant causes asymmetric cell division to confer the piebald phenotype.
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Slavicek, J M; Mercer, M J; Pohlman, D; Kelly, M E; Bischoff, D S
1998-07-01
In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in the polyhedrin or 25K FP gene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in the polyhedrin gene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include the polyhedrin or 25K FP genes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.
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Johnson, Michael E.; Katiyar, Santosh K.; Edlind, Thomas D.
2011-01-01
Echinocandins represent a new antifungal group with potent activity against Candida species. These lipopeptides inhibit the synthesis of β-1,3-glucan, the major cell wall polysaccharide. Acquired resistance or reduced echinocandin susceptibility (RES) is rare and associated with mutations in two “hot spot” regions of Fks1 or Fks2, the probable β-1,3-glucan synthases. In contrast, many fungi demonstrate intrinsic RES for reasons that remain unclear. We are using Saccharomyces cerevisiae to understand the basis for RES by modeling echinocandin-Fks interaction. Previously characterized mutations confer cross-RES; we screened for mutations conferring differential RES, implying direct interaction of that Fks residue with a variable echinocandin side chain. One mutant (in an fks1Δ background) exhibited ≥16-fold micafungin and anidulafungin versus caspofungin RES. Sequencing identified a novel Fks2 mutation, W714L/Y715N. Equivalent W695L/Y696N and related W695L/F/C mutations in Fks1 generated by site-directed mutagenesis and the isolation of a W695L-equivalent mutation in Candida glabrata confirmed the role of the new “hot spot 3” in RES. Further mutagenesis expanded hot spot 3 to Fks1 residues 690 to 700, yielding phenotypes ranging from cross-RES to differential hypersusceptibility. Fks1 sequences from intrinsically RES Scedosporium species revealed W695F-equivalent substitutions; Fks1 hybrids expressing Scedosporium prolificans hot spot 3 confirmed that this substitution imparts RES. PMID:21576441
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Registration of two allelic erect leaf mutants of sorghum
USDA-ARS?s Scientific Manuscript database
Two allelic sorghum [Sorghum bicolor (L.) Moench] erect leaf (erl) mutants were isolated from an Annotated Individually-pedigreed Mutagenized Sorghum (AIMS) mutant library developed at the Plant Stress and Germplasm Development Unit, at Lubbock, Texas. The two mutants, erl1-1 and erl1-2, were isol...
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Pecavar, Verena; Blaschitz, Marion; Hufnagl, Peter; Zeinzinger, Josef; Fiedler, Anita; Allerberger, Franz; Maass, Matthias; Indra, Alexander
2012-06-01
Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.
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Polysaccharide production by a reduced pigmentation mutant of Aureobasidium pullulans NYS-1.
West, T P; Strohfus, B
2001-08-01
To isolate a reduced pigmentation mutant of Aureobasidium pullulans NYS-1 and characterize its cellular pigmentation plus its polysaccharide and biomass production relative to carbon source. Cellular pigmentation, polysaccharide levels and biomass production by the isolated mutant NYSRP-1 were analysed relative to carbon source. Cellular pigmentation of the mutant was lower than its parent strain using either carbon source. The mutant elaborated higher polysaccharide levels on sucrose than on corn syrup. The pullulan content of the polysaccharide synthesized and biomass production by the mutant rose as the carbon source concentration was increased. It is feasible to isolate a reduced pigmentation mutant from strain NYS-1 that exhibits elevated polysaccharide production using corn syrup as a carbon source. The mutant provides an advantage for commercial pullulan production because of its reduced pigmentation and enhanced polysaccharide synthesis.
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Chiaramonte, Fabrizio; Anglade, Patricia; Baraige, Fabienne; Gratadoux, Jean-Jacques; Langella, Philippe; Champomier-Vergès, Marie-Christine; Zagorec, Monique
2010-01-01
We recently showed that Lactobacillus sakei, a natural meat-borne lactic acid bacterium, can colonize the gastrointestinal tracts (GIT) of axenic mice but that this colonization in the intestinal environment selects L. sakei mutants showing modified colony morphology (small and rough) and cell shape, most probably resulting from the accumulation of various mutations that confer a selective advantage for persistence in the GIT. In the present study, we analyzed such clones, issued from three different L. sakei strains, in order to determine which functions were modified in the mutants. In the elongated filamentous cells of the rough clones, transmission electron microscopy (TEM) analysis showed a septation defect and dotted and slanted black bands, suggesting the presence of a helical structure around the cells. Comparison of the cytoplasmic and cell wall/membrane proteomes of the meat isolate L. sakei 23K and of one of its rough derivatives revealed a modified expression for 38 spots. The expression of six oxidoreductases, several stress proteins, and four ABC transporters was strongly reduced in the GIT-adapted strain, while the actin-like MreB protein responsible for cell shaping was upregulated. In addition, the expression of several enzymes involved in carbohydrate metabolism was modified, which may correlate with the observation of modified growth of mutants on various carbon sources. These results suggest that the modifications leading to a better adaptation to the GIT are pleiotropic and are characterized in a rough mutant by a different stress status, a cell wall modification, and modified use of energy sources, leading to an improved fitness for the colonization of the GIT. PMID:20208026
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Margaria, P; Bosco, L; Vallino, M; Ciuffo, M; Mautino, G C; Tavella, L; Turina, M
2014-05-01
Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. Our work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches.
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Margaria, P.; Bosco, L.; Vallino, M.; Ciuffo, M.; Mautino, G. C.; Tavella, L.
2014-01-01
ABSTRACT Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. IMPORTANCE Our work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches. PMID:24623427
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Santos, Fabiana C. P.; Ogrzewalska, Maria; Nascimento, Elvira M. M.; Colombo, Silvia; Marcili, Arlei; Angerami, Rodrigo N.
2014-01-01
Fifteen bacterial isolates from spotted fever group rickettsiosis in Brazil were genetically identified as Rickettsia rickettsii. In a phylogenetic analysis with other R. rickettsii isolates from GenBank, the Central/South American isolates showed low polymorphism and formed a clade distinct from two North American clades, with the North American clades having greater in-branch polymorphism. PMID:25078908
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Proteomic analysis of protein phosphatase Z1 from Candida albicans
Pfliegler, Walter P.; Petrényi, Katalin; Boros, Enikő; Pócsi, István; Tőzsér, József; Dombrádi, Viktor
2017-01-01
Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for CaPpz1 in biofilm formation, was confirmed experimentally. Thus our unbiased proteomic approach lead to the discovery of a novel function for this phosphatase in C. albicans. PMID:28837603
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Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events
Docherty, D.E.; Meteyer, C.U.; Wang, Jingyuan; Mao, J.; Case, S.T.; Chinchar, V.G.
2003-01-01
In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.
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Genomic amplification of the caprine EDNRA locus might lead to a dose dependent loss of pigmentation
Menzi, Fiona; Keller, Irene; Reber, Irene; Beck, Julia; Brenig, Bertram; Schütz, Ekkehard; Leeb, Tosso; Drögemüller, Cord
2016-01-01
The South African Boer goat displays a characteristic white spotting phenotype, in which the pigment is limited to the head. Exploiting the existing phenotype variation within the breed, we mapped the locus causing this white spotting phenotype to chromosome 17 by genome wide association. Subsequent whole genome sequencing identified a 1 Mb copy number variant (CNV) harboring 5 genes including EDNRA. The analysis of 358 Boer goats revealed 3 alleles with one, two, and three copies of this CNV. The copy number is correlated with the degree of white spotting in goats. We propose a hypothesis that ectopic overexpression of a mutant EDNRA scavenges EDN3 required for EDNRB signaling and normal melanocyte development and thus likely lead to an absence of melanocytes in the non-pigmented body areas of Boer goats. Our findings demonstrate the value of domestic animals as reservoir of unique mutants and for identifying a precisely defined functional CNV. PMID:27329507
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Ito, Hiroya; Takahashi, Sayaka; Asai, Tetsuo; Tamura, Yutaka; Yamamoto, Koshi
2018-01-01
An atypical urease-negative mutant of Actinobacillus pleuropneumoniae serovar 2 was isolated in Japan. Nucleotide sequence analysis of the urease gene cluster revealed that the insertion of a short DNA sequence into the cbiM gene was responsible for the urease-negative activity of the mutant. Veterinary diagnostic laboratories should be watchful for the presence of aberrant urease-negative A. pleuropneumoniae isolates.
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Wiebe, M G; Robson, G D; Shuster, J; Trinci, A P
2001-04-20
Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited chemostat cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 chemostat. The highly branched mutants detected in this chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 chemostat complemented representative strains previously isolated from glucose-limited chemostat cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 chemostat culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. Copyright 2001 John Wiley & Sons, Inc.
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Furi, Leonardo; Ciusa, Maria Laura; Knight, Daniel; Di Lorenzo, Valeria; Tocci, Nadia; Cirasola, Daniela; Aragones, Lluis; Coelho, Joana Rosado; Freitas, Ana Teresa; Marchi, Emmanuela; Moce, Laura; Visa, Pilar; Northwood, John Blackman; Viti, Carlo; Borghi, Elisa; Orefici, Graziella
2013-01-01
The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant. PMID:23669380
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Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus.
Field, H J; Darby, G; Wildy, P
1980-07-01
Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.
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Sant'Anna, Juliane R; Miyamoto, Cláudia T; Rosada, Lúcia J; Franco, Claudinéia C S; Kaneshima, Edilson N; Castro-Prado, Marialba A A
2010-01-01
The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.
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Tinette, Sylvette; Zhang, Lixing; Garnier, Amélie; Engler, Gilbert; Tares, Sophie; Robichon, Alain
2007-01-01
Background Drosophila flies explore the environment very efficiently in order to colonize it. They explore collectively, not individually, so that when a few land on a food spot, they attract the others by signs. This behaviour leads to aggregation of individuals and optimizes the screening of mates and egg-laying on the most favourable food spots. Results Flies perform cycles of exploration/aggregation depending on the resources of the environment. This behavioural ecology constitutes an excellent model for analyzing simultaneous processing of neurosensory information. We reasoned that the decision of flies to land somewhere in order to achieve aggregation is based on simultaneous integration of signals (visual, olfactory, acoustic) during their flight. On the basis of what flies do in nature, we designed laboratory tests to analyze the phenomenon of neuronal coincidence. We screened many mutants of genes involved in neuronal metabolism and the synaptic machinery. Conclusion Mutants of NO-dependent cyclase show a specifically-marked behaviour phenotype, but on the other hand they are associated with moderate biochemical defects. We show that these mutants present errors in integrative and/or coincident processing of signals, which are not reducible to the functions of the peripheral sensory cells. PMID:17683617
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Gresty, Karryn J; Gray, Karen-Ann; Bobogare, Albino; Wini, Lyndes; Taleo, George; Hii, Jeffrey; Cheng, Qin; Waters, Norman C
2014-10-14
Plasmodium falciparum and Plasmodium vivax are endemic in Vanuatu and the Solomon Islands. While both countries have introduced artemether-lumefantrine (AL) as first-line therapy for both P. falciparum and P. vivax since 2008, chloroquine and sulphadoxine-pyrimethamine (SP) were used as first-line therapy for many years prior to the introduction of AL. Limited data are available on the extent of SP resistance at the time of policy change. Blood spots were obtained from epidemiological surveys conducted on Tanna Island, Tafea Province, Vanuatu and Temotu Province, Solomon Islands in 2008. Additional samples from Malaita Province, Solomon Islands were collected as part of an AL therapeutic efficacy study conducted in 2008. Plasmodium vivax and P. falciparum dhfr and dhps genes were sequenced to detect nucleotide polymorphisms. All P. falciparum samples analysed (n=114) possessed a double mutant pfdhfr allele (C59R/S108N). Additionally, mutation A437G in pfhdps was detected in a small number of samples 2/13, 1/17 and 3/26 from Tanna Island, Vanuatu and Temotu and Malaita Provinces Solomon Islands respectively. Mutations were also common in pvdhfr from Tanna Island, Vanuatu, where 33/51 parasites carried the double amino acid substitution S58R/S117N, while in Temotu and Malaita Provinces, Solomon Islands 32/40 and 39/46 isolates carried the quadruple amino acid substitution F57L/S58R/T61M/S117T in DHFR respectively. No mutations in pvdhps (n=108) were detected in these three island groups. Prior to the introduction of AL, there was a moderate level of SP resistance in the P. falciparum population that may cause SP treatment failure in young children. Of the P. vivax isolates, a majority of Solomon Islands isolates carried quadruple mutant pvdhfr alleles while a majority of Vanuatu isolates carried double mutant pvdhfr alleles. This suggests a higher level of SP resistance in the P. vivax population in Solomon Islands compared to the sympatric P. falciparum population and there is a higher level of SP resistance in P. vivax parasites from Solomon Islands than Vanuatu. This study demonstrates that the change of treatment policy in these countries from SP to ACT was timely. The information also provides a baseline for future monitoring.
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Labruna, Marcelo B; Santos, Fabiana C P; Ogrzewalska, Maria; Nascimento, Elvira M M; Colombo, Silvia; Marcili, Arlei; Angerami, Rodrigo N
2014-10-01
Fifteen bacterial isolates from spotted fever group rickettsiosis in Brazil were genetically identified as Rickettsia rickettsii. In a phylogenetic analysis with other R. rickettsii isolates from GenBank, the Central/South American isolates showed low polymorphism and formed a clade distinct from two North American clades, with the North American clades having greater in-branch polymorphism. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Gehrke, Flávia S; Angerami, Rodrigo N; Marrelli, Mauro T; de Souza, Eliana R; do Nascimento, Elvira M M; Colombo, Silvia; da Silva, Luiz J; Schumaker, Teresinha T S
2013-01-01
Rickettsial spotted fever is common in southeastern Brazil. Differential diagnosis of pathogens can be performed with proper laboratory methods. A traveler arriving from Portugal developed a fatal febrile hemorrhagic syndrome diagnosed as spotted fever rickettsiosis. We isolated the agent, which was identified as Rickettsia conorii conorii by sequencing rickettsial genes. © 2013 International Society of Travel Medicine.
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Lamason, Rebecca L; Kafai, Natasha M; Welch, Matthew D
2018-01-01
The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, rickettsiae are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improved mariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group species Rickettsia parkeri. Transposon insertions disrupted genes whose products are implicated in a variety of pathways, including bacterial replication and metabolism, the type IV secretion system, factors with previously established roles in host cell interactions and pathogenesis, or are of unknown function. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.
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Molecular characterization of baculovirus Bombyx mori nucleopolyhedrovirus polyhedron mutants.
Katsuma, S; Noguchi, Y; Shimada, T; Nagata, M; Kobayashi, M; Maeda, S
1999-01-01
Four newly isolated and two previously isolated polyhedron mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Two polyhedron deficient mutants, #126 and #136, produced small uncrystallized particles of polyhedrin in the nuclei and cytoplasm of infected cells. Mutant #211 produced a large number of variably sized polyhedra in the nucleus and #220 produced a few large cuboidal polyhedra in the nucleus. Mutant #24 and #128 were previously isolated BmNPV mutants. Mutant #24 could not produce polyhedrin mRNA and polyhedra produced by mutant #128 lacked oral infectivity. Nucleotide sequence analysis indicated that five mutants (#126, #136, #211, #220 and #128) had amino acid substitutions in polyhedrin and mutant #24 had a point mutation only in the promoter region of the polyhedrin gene. Cotransfection experiments showed that the altered phenotypes were due to the mutations found in the polyhedrin gene regions. In mutants #126 and #136, amino acid sequences of the nuclear localization signal of polyhedrin were identical to those of wild-type BmNPV, suggesting that this sequence was necessary but not sufficient for nuclear localization of polyhedrin. Electron microscopic observation revealed that fewer occluded virions were contained in polyhedra of #128 and #220.
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Iwata, Masaki; Hiyama, Atsuki; Otaki, Joji M.
2013-01-01
Developmental studies on wing colour patterns have been performed in nymphalid butterflies, but efficient genetic manipulations, including mutagenesis, have not been well established. Here, we have performed mutagenesis experiments in a lycaenid butterfly, the pale grass blue Zizeeria maha, to produce colour-pattern mutants. We fed the P-generation larvae an artificial diet containing the mutagen ethyl methane sulfonate (EMS), and the F1- and F2-generation adults showed various aberrant colour patterns: dorsoventral transformation, anterioposterior background colouration gap, weak contrast, disarrangement of spots, reduction of the size of spots, loss of spots, fusion of spots, and ectopic spots. Among them, the disarrangement, reduction, and loss of spots were likely produced by the coordinated changes of many spots of a single wing around the discal spot in a system-dependent manner, demonstrating the existence of the central symmetry system. The present study revealed multiple genetic regulations for system-dependent and wing-wide colour-pattern determination in lycaenid butterflies. PMID:23917124
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Isolation of ntrA-like mutants of Azotobacter vinelandii.
Santero, E; Luque, F; Medina, J R; Tortolero, M
1986-01-01
A number of chlorate-resistant mutants of Azotobacter vinelandii affected in a general control of nitrogen metabolism were isolated. These mutants could not utilize dinitrogen, nitrate, or nitrite as a nitrogen source. The reason for this inability is that they were simultaneously deficient in nitrogenase and nitrate and nitrite reductase activities. They were complemented by a cosmid carrying a DNA fragment of A. vinelandii able to complement ntrA mutants of Escherichia coli, so they seemed to be ntrA-like mutants. PMID:3009406
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Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants
Vagasi, Alexandra S.; Rahman, Mohammad M.; Chaudhari, Snehal N.; Kipreos, Edward T.
2017-01-01
The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016. PMID:28868332
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Colot, V.; Rossignol, J. L.
1995-01-01
The ascomycete Ascobolus immersus has been extensively used as a model system for the genetic study of meiotic recombination. More recently, an epigenetic process, known as methylation induced premeiotically (MIP), that acts on duplicated sequences has been discovered in A. immersus and has raised a new interest in this fungus. To try and extend these studies, we have now cloned the A. immersus spore color gene b2, a well characterized recombination hot-spot. Isolation of the whole gene was verified by physical mapping of four large b2 alterations, followed by transformation and mutant rescue of a null b2 allele. Transformation was also used to duplicate b2 and subject it to MIP. As a result, we were able for the first time to observe gene silencing as early as just after meiosis and in single cells. Furthermore, we have found evidence for a modulating effect of MIP on b2 expression, depending on the region of the gene that is duplicated and hence subjected to MIP. PMID:8601475
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Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE
Prakash, Louise; Prakash, Satya
1977-01-01
We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev ) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups. PMID:195865
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Vega-Palas, M A; Madueño, F; Herrero, A; Flores, E
1990-01-01
Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp. PMID:1967601
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USDA-ARS?s Scientific Manuscript database
The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacter...
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Cengiz, M; Sahinturk, P; Sonal, S; Buyukcangaz, E; Sen, A; Arslan, E
2013-05-04
The objective of this work was to investigate the bactericidal activity of enrofloxacin against gyrA mutant and qnr-containing Escherichia coli isolates from animals. The minimum inhibitory concentrations (MICs) of gyrA mutant and qnr-containing E coli isolates ranged from 1 µg/ml to 32 µg/ml for enrofloxacin. Time-kill experiments were performed using selected E coli isolates. For the time-kill experiments, the colony counts were determined by plating each diluted sample onto plate count agar and an integrated pharmacokinetic/pharmacodynamics area measure (log ratio area) was applied to the colony-forming units (cfu) data. In general, enrofloxacin exhibited bactericidal activity against all the gyrA mutant E coli isolates at all concentrations greater than four times the MIC. However, the bactericidal activity of enrofloxacin for all the qnr-containing E coli isolates was less dependent on concentration. The results of the present study indicated that the genetic mechanism of resistance might account for the different bactericidal activities of enrofloxacin observed for the gyrA mutant and the qnr-containing E coli isolates. Therefore, in addition to MIC assays, genetic mechanism-based pharmacodynamic models should be used to provide accurate predictions of the effects of drugs on resistant bacteria.
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Genotoxicity testing of two lead-compounds in somatic cells of Drosophila melanogaster.
Carmona, Erico R; Creus, Amadeu; Marcos, Ricard
2011-09-18
The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogaster by measurement of two different genetic endpoints. We used the wing-spot test and the comet assay. The comet assay was conducted with larval haemocytes. The results from the wing-spot test showed that neither lead chloride, PbCl(2), nor lead nitrate, Pb(NO(3))(2), were able to induce significant increases in the frequency of mutant spots. In addition, the combined treatments with gamma-radiation and PbCl(2) or Pb(NO(3))(2) did not show significant variations in the frequency of the three categories of mutant spots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indicate that the lead compounds tested do not interact with the repair of the genetic damage induced by ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay with haemocytes, Pb(NO(3))(2) was effective in inducing significant increases of DNA damage with a direct dose-response pattern. These results confirm the usefulness of the comet assay with haemocytes as an in vivo model and support the assumption that there is a genotoxic risk associated with lead exposure. Copyright © 2011 Elsevier B.V. All rights reserved.
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Use of Heme Compounds as Iron Sources by Pathogenic Neisseriae Requires the Product of the hemO Gene
Zhu, Wenming; Hunt, Desiree J.; Richardson, Anthony R.; Stojiljkovic, Igor
2000-01-01
Heme compounds are an important source of iron for neisseriae. We have identified a neisserial gene, hemO, that is essential for heme, hemoglobin (Hb), and haptoglobin-Hb utilization. The hemO gene is located 178 bp upstream of the hmbR Hb receptor gene in Neisseria meningitidis isolates. The product of the hemO gene is homologous to enzymes that degrade heme; 21% of its amino acid residues are identical, and 44% are similar, to those of the human heme oxygenase-1. DNA sequences homologous to hemO were ubiquitous in commensal and pathogenic neisseriae. HemO genetic knockout strains of Neisseria gonorrhoeae and N. meningitidis were unable to use any heme source, while the assimilation of transferrin-iron and iron-citrate complexes was unaffected. A phenotypic characterization of a conditional hemO mutant, constructed by inserting an isopropyl-β-d-thiogalactopyranoside (IPTG)-regulated promoter upstream of the ribosomal binding site of hemO, confirmed the indispensability of the HemO protein in heme utilization. The expression of HemO also protected N. meningitidis cells against heme toxicity. hemO mutants were still able to transport heme into the cell, since both heme and Hb could complement an N. meningitidis hemA hemO double mutant for growth. The expression of the HmbR receptor was reduced significantly by the inactivation of the hemO gene, suggesting that hemO and hmbR are transcriptionally linked. The expression of the unlinked Hb receptor, HpuAB, was not altered. Comparison of the polypeptide patterns of the wild type and the hemO mutant led to detection of six protein spots with an altered expression pattern, suggesting a more general role of HemO in the regulation of gene expression in Neisseriae. PMID:10629191
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Kohatsu, Soh; Yamamoto, Daisuke
2015-03-06
The courtship ritual of male Drosophila represents an innate behaviour that is initiated by female-derived sensory stimuli. Here we report that moving light spots can induce courtship-like following pursuit in tethered wild-type male flies provided the fly is primed by optogenetic stimulation of specific dsx-expressing neuronal clusters in the lateral protocerebrum (LPR). Namely, stimulation of the pC1 neuronal cluster initiates unilateral wing extension and vibration of both sides, whereas stimulation of the pC2l cluster initiates only contralateral wing displays. In addition, stimulation of pC2l but not pC1 neurons induced abdominal bending and proboscis extension. Ca(2+) imaging of the pC1 cluster revealed periodic Ca(2+) rises, each corresponding to a turn of the male fly during courtship. In contrast, group-reared fru mutant males exhibit light spot-induced courtship pursuit without optogenetic priming. Ca(2+) imaging revealed enhanced responses of LPR neurons to visual stimuli in the mutants, suggesting a neural correlate of the light spot-induced courtship behaviour.
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Lee, Mei-Feng; Chen, Yen-Hsu; Hsu, Hui-Jine; Peng, Chien-Fang
2010-10-01
In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.
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Rocky Mountain spotted fever, Panama.
Estripeaut, Dora; Aramburú, María Gabriela; Sáez-Llorens, Xavier; Thompson, Herbert A; Dasch, Gregory A; Paddock, Christopher D; Zaki, Sherif; Eremeeva, Marina E
2007-11-01
We describe a fatal pediatric case of Rocky Mountain spotted fever in Panama, the first, to our knowledge, since the 1950s. Diagnosis was established by immunohistochemistry, PCR, and isolation of Rickettsia rickettsii from postmortem tissues. Molecular typing demonstrated strong relatedness of the isolate to strains of R. rickettsii from Central and South America.
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Structurally altered capsular polysaccharides produced by mutant bacteria
NASA Technical Reports Server (NTRS)
Petersen, Gene R. (Inventor); Kern, Roger G. (Inventor); Richards, Gil F. (Inventor)
1995-01-01
Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.
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Harris, S L; Elliott, D A; Blake, M C; Must, L M; Messenger, M; Orndorff, P E
1990-01-01
The product of the pilE (also called fimH) gene is a minor component of type 1 pili in Escherichia coli. Mutants that have insertions in the pilE gene are fully piliated but unable to bind to and agglutinate guinea pig erythrocytes, a characteristic of wild-type type 1 piliated E. coli. In this paper we describe the isolation of 48 mutants with point lesions that map to the pilE gene. Such mutants were isolated by using mutT mutagenesis and an enrichment procedure devised to favor the growth of individuals that could form a pellicle in static broth containing alpha-methylmannoside, an inhibitor of erythrocyte binding and pellicle formation. Results indicated that the enrichment favored mutants expressing pilE gene products that were defective in mediating erythrocyte binding. Characterization of 12 of the mutants in greater detail revealed that certain lesions affected pilus number and length. In addition, a mutant that was temperature sensitive for erythrocyte binding was isolated and used to provide evidence that pellicle formation relies on the intercellular interaction of pilE gene products. Our results suggest a molecular explanation for the old and paradoxical observations connecting pellicle formation and erythrocyte agglutination by type 1 piliated E. coli. Images PMID:1977736
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Fachin, A L; Maffei, C M; Martinez-Rossi, N M
1996-01-01
The in vitro susceptibility of three clinical Trichophyton rubrum isolates to griseofulvin and tioconazole, determined by the minimal inhibitory concentration (MIC), was 2 and 0.5 to 1.0 micrograms/ml, respectively. One mutant (gril) obtained after mutagenic treatment of one of these isolates was selected and showed simultaneous resistance to griseofulvin (MIC > 2000 micrograms/ml) and tioconazole (MIC = 1.0 microgram/ml). The clinical importance and the possibility of a multidrug resistance (MDR)-type mechanism being involved in this event is discussed.
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Rocky Mountain Spotted Fever, Panama
Estripeaut, Dora; Aramburú, María Gabriela; Sáez-Llorens, Xavier; Thompson, Herbert A.; Dasch, Gregory A.; Paddock, Christopher D.; Zaki, Sherif
2007-01-01
We describe a fatal pediatric case of Rocky Mountain spotted fever in Panama, the first, to our knowledge, since the 1950s. Diagnosis was established by immunohistochemistry, PCR, and isolation of Rickettsia rickettsii from postmortem tissues. Molecular typing demonstrated strong relatedness of the isolate to strains of R. rickettsii from Central and South America. PMID:18217566
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Wu, Xiaodong; Wu, Xiaoyun; Li, Wenbin; Cheng, Xiaofei
2018-05-01
Through sequencing and assembly of small RNAs, an orthotospovirus was identified from a celtuce plant (Lactuca sativa var. augustana) showing vein clearing and chlorotic spots in the Zhejiang province of China. The S, M, and L RNAs of this orthotospovirus were determined to be 3146, 4734, and 8934 nt, respectively, and shared 30.4-72.5%, 43.4-80.8%, and 29.84-82.9% nucleotide sequence identities with that of known orthotospoviruses. The full length nucleoprotein (N) of this orthotospovirus shared highest amino acid sequence identity (90.25%) with that of calla lily chlorotic spot virus isolated from calla lily (CCSV-calla) [China: Taiwan: 2001] and tobacco (CCSV-LJ1) [China: Lijiang: 2014]. Phylogenetic analyses showed that this orthotospovirus is phylogenetically associated with CCSV isolates and clustered with CCSV, tomato zonate spot virus (TZSV), and tomato necrotic spot-associated virus (TNSaV) in a separate sub-branch. These results suggest that this orthotospovirus is a divergent isolate of CCSV and was thus named CCSV-Cel [China: Zhejiang: 2017].
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Komitopoulou, Katia; Gans, Madeleine; Margaritis, Lukas H.; Kafatos, Fotis C.; Masson, Michele
1983-01-01
To study genes that function mainly or exclusively during oogenesis, we have isolated and analyzed female-sterile mutations, with special emphasis on those that affect eggshell formation. Following treatment that induced 61 to 66% lethals, 8.1% of the 1071 X chromosomes tested carried recessive female sterility mutations (87 isolates), and 8.0% carried partial female-sterile mutations (86 isolates), respectively. In addition, three dominant female steriles were recovered. Some of the mutants had very low fecundity, and others laid morphologically normal eggs that failed to develop. A third category included 29 mutants that laid eggs with morphological abnormalities: 26 were female steriles, two were partial female steriles and one was fertile. Mutants of this third category were characterized in some detail and compared with 40 previously isolated mutants that laid similarly abnormal eggs. Approximately 28–31 complementation groups with morphological abnormalities were detected, some of which were large allelic series (11, 9, 7, 6 and 5 alleles). Twenty-four groups were mapped genetically or cytogenetically, and 21 were partially characterized by ultrastructural and biochemical procedures. Of the latter, one group showed clear deficiency of yolk proteins, and nine showed prominent ultrastructural defects in the chorion (at least eight accompanied by deficiencies in characterized chorion proteins). At least six groups with clear-cut effects were found at loci not previously identified with known chorion structural genes. PMID:17246182
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Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development
Zhang, Ning; Chen, Feng; Huo, Wang; Cui, Dangqun
2015-01-01
Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS) mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression patterns displayed a dynamic description of middle and late grain formation. Those identified protein species participated in eight biochemical processes: stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction. Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns, and six cultivar-specific protein spots including serpin, small heat shock protein, β-amylase, α-amylase inhibitor, dimeric α-amylase inhibitor precursor, and cold regulated protein. These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars. Our results provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat and the proteomic characterization in this study could also provide insights in the biology of middle and late grain development. PMID:26442048
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The Transcription Factors Ets1 and Sox10 Interact During Murine Melanocyte Development
Saldana-Caboverde, Amy; Perera, Erasmo M.; Watkins-Chow, Dawn; Hansen, Nancy F.; Vemulapalli, Meghana; Mullikin, James C; Pavan, William J.; Kos, Lidia
2015-01-01
Melanocytes, the pigment-producing cells, arise from multipotent neural crest (NC) cells during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The variable spotting mouse pigmentation mutant arose spontaneously at the Jackson Laboratory. We identified a G-to-A nucleotide transition in exon 3 of the Ets1 gene in variable spotting, which results in a missense G102E mutation. Homozygous variable spotting mice exhibit sporadic white spotting. Similarly, mice carrying a targeted deletion of Ets1 exhibit hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The transcription factor Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of various NC derivatives, including melanocytes. We show that Ets1 is required early for murine NC cell and melanocyte precursor survival in vivo. Given the importance of Ets1 for Sox10 expression in the chick, we investigated a potential genetic interaction between these genes by comparing the hypopigmentation phenotypes of single and double heterozygous mice. The incidence of hypopigmentation in double heterozygotes was significantly greater than in single heterozygotes. The area of hypopigmentation in double heterozygotes was significantly larger than would be expected from the addition of the areas of hypopigmentation of single heterozygotes, suggesting that Ets1 and Sox10 interact synergistically in melanocyte development. Since Sox10 is also essential for enteric ganglia development, we examined the distal colons of Ets1 null mutants and found a significant decrease in enteric innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate an enhancer critical for Sox10 expression in NC-derived structures. Furthermore, enhancer activation was significantly inhibited by the variable spotting mutation. Together, these results suggest that Ets1 and Sox10 interact to promote proper melanocyte and enteric ganglia development from the NC. PMID:25912689
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James M. Slavicek; Nancy Hayes-Plazolles; Mary Ellen Kelly
2001-01-01
During Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) replication in cell culture, few-polyhedra (FP) mutants arise at a high frequency and become predominant after only a few serial passages. The formation of LdMNPV FP mutants was an impediment to successful production of polyhedra in cell culture bioreactors. We have isolated and...
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Cagnon, Caroline; Mirabella, Boris; Nguyen, Hoa Mai; Beyly-Adriano, Audrey; Bouvet, Séverine; Cuiné, Stéphan; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua
2013-12-02
Oils produced by microalgae are precursors to biodiesel. To achieve a profitable production of biodiesel from microalgae, identification of factors governing oil synthesis and turnover is desirable. The green microalga Chlamydomonas reinhardtii is amenable to genetic analyses and has recently emerged as a model to study oil metabolism. However, a detailed method to isolate various types of oil mutants that is adapted to Chlamydomonas has not been reported. We describe here a forward genetic approach to isolate mutants altered in oil synthesis and turnover from C. reinhardtii. It consists of a three-step screening procedure: a primary screen by flow cytometry of Nile red stained transformants grown in 96-deep-well plates under three sequential conditions (presence of nitrogen, then absence of nitrogen, followed by oil remobilization); a confirmation step using Nile red stained biological triplicates; and a validation step consisting of the quantification by thin layer chromatography of oil content of selected strains. Thirty-one mutants were isolated by screening 1,800 transformants generated by random insertional mutagenesis (1.7%). Five showed increased oil accumulation under the nitrogen-replete condition and 13 had altered oil content under nitrogen-depletion. All mutants were affected in oil remobilization. This study demonstrates that various types of oil mutants can be isolated in Chlamydomonas based on the method set-up here, including mutants accumulating oil under optimal biomass growth. The strategy conceived and the protocol set-up should be applicable to other microalgal species such as Nannochloropsis and Chlorella, thus serving as a useful tool in Chlamydomonas oil research and algal biotechnology.
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Saad, Maged M; Kobayashi, Hajime; Marie, Corinne; Brown, Ian R; Mansfield, John W; Broughton, William J; Deakin, William J
2005-02-01
Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.
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Saad, Maged M.; Kobayashi, Hajime; Marie, Corinne; Brown, Ian R.; Mansfield, John W.; Broughton, William J.; Deakin, William J.
2005-01-01
Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGRΩrhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB. PMID:15659692
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Marcos-Zambrano, Laura Judith; Gómez-Perosanz, Marta; Escribano, Pilar; Zaragoza, Oscar; Bouza, Emilio
2016-01-01
The echinocandins and liposomal amphotericin B are active against biofilm produced by echinocandin-susceptible Candida strains. However, few data have been reported on the production of biofilm by echinocandin-resistant isolates and their antifungal susceptibility. We studied the production of biofilm by fks mutant Candida strains and intrinsically echinocandin-resistant non-Candida isolates and the susceptibility of both entities to liposomal amphotericin B and echinocandins. We analyzed the production of biofilm by isolates from patients with fungemia (fks mutant Candida, n = 5; intrinsically echinocandin-resistant non-Candida, n = 12; and Candida wild type, n = 10). Biofilm formation was measured to classify strains according to biomass (crystal violet assay) and metabolic activity (XTT reduction assay). Preformed biofilms were tested against liposomal amphotericin B, caspofungin, micafungin, and anidulafungin. The sessile MIC was defined as the antifungal concentration yielding a 50% or 80% reduction in the metabolic activity of the biofilm compared to that of the growth control (SMIC50 and SMIC80, respectively). fks mutant Candida isolates formed biofilms in a fashion similar to that of Candida wild-type strains. The echinocandins had the highest activity against biofilms formed by wild-type Candida isolates, followed by fks mutant Candida isolates and non-Candida isolates. Liposomal amphotericin B had the highest activity against fks mutant Candida biofilms. The formation of biofilm by echinocandin-resistant strains was similar to that of wild-type strains, although resistance to echinocandins remained high. PMID:27021323
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USDA-ARS?s Scientific Manuscript database
Frogeye leaf spot (FLS), caused by Cercospora sojina, causes significant damage to soybean in the US. In this study, C. sojina isolates collected from Jackson and Milan, TN in 2015 and historical isolates collected before 2015 were included. Fifty novel single nucleotide polymorphism (SNP) markers, ...
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USDA-ARS?s Scientific Manuscript database
Frogeye leaf spot (FLS), caused by Cercospora sojina, is a yearly foliar disease in Tennessee and causes substantial economic losses if not properly managed. Quinone outside inhibitor (QoI) fungicides are often used to manage FLS, but C. sojina isolates have developed resistance to this class of fun...
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Isolation and preliminary characterization of temperature-sensitive mutants of influenza virus.
Sugiura, A; Tobita, K; Kilbourne, E D
1972-10-01
Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10(-3) or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.
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Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G
2009-07-01
The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.
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Beard, C B; Benedict, M Q; Primus, J P; Finnerty, V; Collins, F H
1995-01-01
Chromatographic analysis of pigments extracted from wild-type eyes of the mosquito Anopheles gambiae reveals the presence of the ommatin precursor 3-hydroxykynurenine, its transamination derivative xanthurenic acid, and a dark, red-brown pigment spot that probably is composed of two or more low mobility xanthommatins. No colored or fluorescent pteridines are evident. Mosquitoes homozygous for an autosomal recessive mutation at the red-eye (r) locus have a brick-red eye color in larvae, pupae, and young adults, in contrast to the almost black color of the wild eye. Mosquitoes homozygous for this mutant allele have levels of ommochrome precursors that are indistinguishable from the wild-type, but the low-mobility xanthommatin spot is ochre-brown in color rather than red-brown as in the wild-type. Mosquitoes with two different mutant alleles at the X-linked pink-eye locus (p, which confers a pink eye color, and pw, which confers a white eye phenotype in homozygotes or hemizygous males) have normal levels of ommochrome precursors but no detectable xanthommatins. Mosquitoes homozygous for both the r and p mutant alleles have apricot-colored eyes and show no detectable xanthommatins. Both the pink-eye and red-eye mutations appear to involve defects in the transport into or assembly of pigments in the membrane-bound pigment granules rather then defects in ommochrome synthesis.
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USDA-ARS?s Scientific Manuscript database
Frogeye leaf spot (FLS), caused by Cercospora sojina, is a yearly foliar disease of soybean in Tennessee and causes substantial economic losses if not properly managed. Quinone outside inhibitor (QoI) fungicides are often used to manage FLS, but C. sojina isolates have developed resistance to this c...
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Golkhoo, Shokufeh; Barantalab, Fatemeh; Ahmadi, Ali Reza; Hassan, Zuhair Muhammad
2007-03-01
Astaxanthin have been extracted and purified from mutant isolate of Phaffia rhodozyma JH-82. Purified astaxanthin was identified by spectrophotometric, TLC and HPLC analysis and were compared with synthetic astaxanthin. Results of TLC analysis indicated that isolate of P. rhodozyma JH-82 were able to produce nine different carotenoids and high level of carotenoids was belong to astaxanthin. Results of this study for pure astaxanthin production indicated that mutant of JH-82 of P. rhodozyma (230 microg g(-1)(dried yeast)) produced more astaxanthin than natural isolate JH-80 (140 microg g(-1)(dried yeast)). The HPLC spectrum showed retention time 11 min for both purified and synthetic astaxanthin and solvent was CDCl3.
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ERIC Educational Resources Information Center
Vink, Gregory E.; And Others
1985-01-01
Hot spots are isolated areas of geologic activity where volcanic eruptions, earthquakes, and upwelling currents occur far from plate boundaries. These mantle plumes are relatively stable and crustal plates drift over them. The nature and location of hot spots (with particular attention to the Hawaiian Islands and Iceland) are discussed. (DH)
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Pearson, Selina; Brooker, Rachael H.; Spiden, Sarah; Kiernan, Amy E.; Guénet, Jean-Louis; Steel, Karen P.
2012-01-01
Mutanlallemand (mtl) and Belly Spot and Deafness (bsd) are two new spontaneous alleles of the Lmx1a gene in mice. Homozygous mutants show head tossing and circling behaviour, indicative of vestibular defects, and they have short tails and white belly patches of variable size. The analysis of auditory brainstem responses (ABR) showed that mtl and bsd homozygotes are deaf, whereas heterozygous and wildtype littermates have normal hearing. Paint-filled inner ears at E16.5 revealed that mtl and bsd homozygotes lack endolymphatic ducts and semicircular canals and have short cochlear ducts. These new alleles show similarities with dreher (Lmx1a) mutants. Complementation tests between mtl and dreher and between mtl and bsd suggest that mtl and bsd are new mutant alleles of the Lmx1a gene. To determine the Lmx1a mutation in mtl and bsd mutant mice we performed PCR followed by sequencing of genomic DNA and cDNA. The mtl mutation is a single point mutation in the 3′ splice site of exon 4 leading to an exon extension and the activation of a cryptic splice site 44 base pairs downstream, whereas the bsd mutation is a genomic deletion that includes exon 3. Both mutations lead to a truncated LMX1A protein affecting the homeodomain (mtl) or LIM2-domain (bsd), which is critical for LMX1A protein function. Moreover, the levels of Lmx1a transcript in mtl and bsd mutants are significantly down-regulated. Hmx2/3 and Pax2 expression are also down-regulated in mtl and bsd mutants, suggesting a role of Lmx1a upstream of these transcription factors in early inner ear morphogenesis. We have found that these mutants develop sensory patches although they are misshapen. The characterization of these two new Lmx1a alleles highlights the critical role of this gene in the development of the cochlea and vestibular system. PMID:23226461
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Akbar, Sabika; Prasuna, R Gyana; Khanam, Rasheeda
2014-04-01
Aspergillus flavipes, a slow growing pectinase producing ascomycete, was isolated from soil identified and characterised in the previously done preliminary studies. Optimisation studies revealed that Citrus peel--groundnut oil cake [CG] production media is the best media for production of high levels of pectinase up to 39 U/ml using wild strain of A. flavipes. Strain improvement of this isolated strain for enhancement of pectinase production using multistep mutagenesis procedure is the endeavour of this project. For this, the wild strain of A. flavipes was treated with both physical (UV irradiation) and chemical [Colchicine, Ethidium bromide, H2O2] mutagens to obtain Ist generation mutants. The obtained mutants were assayed and differentiated basing on pectinase productivity. The better pectinase producing strains were further subjected to multistep mutagenesis to attain stability in mutants. The goal of this project was achieved by obtaining the best pectinase secreting mutant, UV80 of 45 U/ml compared to wild strain and sister mutants. This fact was confirmed by quantitatively analysing 3rd generation mutants obtained after multistep mutagenesis.
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Interaction of caffeine with the SOS response pathway in Escherichia coli.
Whitney, Alyssa K; Weir, Tiffany L
2015-01-01
Previous studies have highlighted the antimicrobial activity of caffeine, both individually and in combination with other compounds. A proposed mechanism for caffeine's antimicrobial effects is inhibition of bacterial DNA repair pathways. The current study examines the influence of sub-lethal caffeine levels on the growth and morphology of SOS response pathway mutants of Escherichia coli. Growth inhibition after treatment with caffeine and methyl methane sulfonate (MMS), a mutagenic agent, was determined for E. coli mutants lacking key genes in the SOS response pathway. The persistence of caffeine's effects was explored by examining growth and morphology of caffeine and MMS-treated bacterial isolates in the absence of selective pressure. Caffeine significantly reduced growth of E. coli recA- and uvrA-mutants treated with MMS. However, there was no significant difference in growth between umuC-isolates treated with MMS alone and MMS in combination with caffeine after 48 h of incubation. When recA-isolates from each treatment group were grown in untreated medium, bacterial isolates that had been exposed to MMS or MMS with caffeine showed increased growth relative to controls and caffeine-treated isolates. Morphologically, recA-isolates that had been treated with caffeine and both caffeine and MMS together had begun to display filamentous growth. Caffeine treatment further reduced growth of recA- and uvrA-mutants treated with MMS, despite a non-functional SOS response pathway. However, addition of caffeine had very little effect on MMS inhibition of umuC-mutants. Thus, growth inhibition of E. coli with caffeine treatment may be driven by caffeine interaction with UmuC, but also appears to induce damage by additional mechanisms as evidenced by the additive effects of caffeine in recA- and uvrA-mutants.
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Remy, Joan M; Tow-Keogh, Cheryl A; McConnell, Timothy S; Dalton, James M; Devito, Joseph A
2012-12-01
To determine the potential for delafloxacin to select for resistant mutants in methicillin-resistant Staphylococcus aureus (MRSA), including isolates with existing mutations in the quinolone resistance determining region (QRDR). Susceptibility testing by broth microdilution was performed on 30 MRSA clinical isolates. For four of these isolates, the presence or absence of mutations in the QRDR was characterized. Resistance selection was performed on these four isolates by spreading cells on drug-containing agar plates followed by incubation for 48 h. Resistance frequencies and mutant prevention concentrations (MPCs) were calculated for each; PCR amplification and sequencing were performed using standard methods to characterize mutations in the QRDR. Growth rate analysis was performed and relative fitness was determined. Delafloxacin demonstrated potent in vitro activity against this set of MRSA isolates, with MICs of 0.008-1 mg/L and an MIC(50) and MIC(90) of 0.03 and 0.5 mg/L, respectively. Spontaneous delafloxacin resistance frequencies for the MRSA strains were 2 × 10(-9) to <9.5 × 10(-11). Delafloxacin MPCs were one to four times the MIC for any isolate, lower than those of comparator quinolones. Some delafloxacin-selected mutants showed a fitness cost when co-cultured with the parent strain. Delafloxacin demonstrates excellent antibacterial potency and exhibits a low probability for the selection of resistant mutants in MRSA. Although mutants can be selected at low frequencies in vitro from quinolone-resistant isolates, delafloxacin MICs and MPCs remain low and a fitness cost can be observed. Consequently delafloxacin warrants further investigation for the potential treatment of drug-resistant MRSA infections.
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First Report of Black Spot Disease Caused by Alternaria alternata on Sweet Persimmon Fruits
Lee, Jung Han; Kim, Jinwoo
2013-01-01
Black spot of sweet persimmon, caused by Alternaria alternata, occurred in an orchard in Gyeongnam province, Korea in 2012. The symptom was appearance of 0.5 to 4 cm black spots on the surface of fruit. The pathogen was isolated from flesh of disease lesions. The causal agent was identified as A. alternata by morphological characteristics and sequencers of the internal transcribed spacer (ITS) 1 and ITS4 regions of rRNA. Artificial inoculation of the pathogen resulted in development of disease symptoms and the re-isolated pathogen showed characteristics of A. alternata. PMID:24198674
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Adaptation to copper stress influences biofilm formation in Alteromonas macleodii.
Cusick, Kathleen D; Dale, Jason R; Fitzgerald, Lisa A; Little, Brenda J; Biffinger, Justin C
2017-07-01
An Alteromonas macleodii strain was isolated from copper-containing coupons incubated in surface seawater (Key West, FL, USA). In addition to the original isolate, a copper-adapted mutant was created and maintained with 0.78 mM Cu 2+ . Biofilm formation was compared between the two strains under copper-amended and low-nutrient conditions. Biofilm formation was significantly increased in the original isolate under copper amendment, while biofilm formation was significantly higher in the mutant under low-nutrient conditions. Biofilm expression profiles of diguanylate cyclase (DGC) genes, as well as genes involved in secretion, differed between the strains. Comparative genomic analysis demonstrated that both strains possessed a large number of gene attachment harboring cyclic di-GMP synthesis and/or degradation domains. One of the DGC genes, induced at very high levels in the mutant, possessed a degradation domain in the original isolate that was lacking in the mutant. The genetic and transcriptional mechanisms contributing to biofilm formation are discussed.
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Improved Medium for Selecting Nitrate-Nonutilizing (nit) Mutants of Verticillium dahliae.
Korolev, N; Katan, T
1997-10-01
ABSTRACT Nitrate-nonutilizing (nit) mutants are commonly used to determine vegetative compatibility between isolates of Verticillium dahliae by complementation (heterokaryon) testing. These mutants emerge spontaneously as chlorate-resistant sectors growing out of partially restricted, wild-type colonies on chlorate-amended media. The commonly used chlorate media are based on minimal medium (MMC) or cornmeal agar (CMC), amended with potassium chlorate. nit mutants recovered on these media constituted 10 to 36%(on MMC) and 25 to 45%(on CMC) of the apparently resistant sectors. An improved water agar chlorate medium (WAC) is described that is more effective for selecting chlorate-resistant nit mutants. WAC medium consists of agar (2%), glucose (0.02%), and potassium chlorate (2 to 5%). On WAC, growth of most V. dahliae isolates was strongly inhibited, and 66 to 100%(average >80%) of the chlorate-resistant sectors formed were nit mutants. Most mutants were characterized as nit1, and about 6% as NitM.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Cuskey, S.M.; Schamhart, D.H.J.; Chase, T. Jr.
A simple technique, using an esculin-ferric salt visualization system, for selective isolation of mutants of Trichoderma reesei was employed. The ..beta..-glucosidase crude enzyme complexes of the 17 mutants isolated from some 66,000 colonies screened were characterized. Type of inhibition (competitive, noncompetitive) by glucose, and kinetic parameters K/sub m/ (mM pNPG), V/sub max/ (units/mg extracellular protein), and K/sub i/ slopes (mM glucose) were determined for the mutants using p-nitrophenyl ..beta..-D-glucoside (pNPG) as substrate. All the isolates were inhibited competitively by glucose, but certain of them were less sensitive than parent and wild-type to inhibition by glucose. 5 figures, 1 table.
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Favor, Jack; Bradley, Alan; Conte, Nathalie; Janik, Dirk; Pretsch, Walter; Reitmeir, Peter; Rosemann, Michael; Schmahl, Wolfgang; Wienberg, Johannes; Zaus, Irmgard
2009-08-01
In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.
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Wee, Jung-In; Park, Jong-Han; Back, Chang-Gi; You, Young-Hyun; Chang, Taehyun
2016-09-01
In July 2015, diseased leaves of black chokeberry ( Aronia melanocarpa ) were observed in Danyang and Gochang, Korea. The symptoms appeared as circular or irregular brown leaf spots, from which Alternaria tenuissima was isolated. The isolates were cultured on potato dextrose agar, and their morphological characteristics were observed under a light microscope. The colonies were whitish to ash colored. The pathogenicity test on healthy black chokeberry leaves produced circular brown spots, in line with the original symptoms. Molecular analyses of the ITS, GPD, RPB2, and TEF genes were conducted to confirm the identity of the pathogen. The phylogeny of the multi-gene sequences indicated that the causal agent was A. tenuissima . This study is the first report of A. tenuissima leaf spot on black chokeberry ( A. melanocarpa ).
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Brewer, Marin Talbot; Turner, Ashley N; Brannen, Phillip M; Cline, William O; Richardson, Elizabeth A
2014-01-01
Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species. © 2014 by The Mycological Society of America.
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Mishra, S S; Shekhar, M S
2005-07-01
Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.
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Sclerotinia homoeocarpa Overwinters in Turfgrass and Is Present in Commercial Seed
Rioux, Renée A.; Shultz, Jeanette; Garcia, Michelle; Willis, David Kyle; Casler, Michael; Bonos, Stacy; Smith, Damon; Kerns, James
2014-01-01
Dollar spot is the most economically important disease of amenity turfgrasses in the United States, yet little is known about the source of primary inoculum for this disease. With the exception of a few isolates from the United Kingdom, Sclerotinia homoeocarpa, the causal agent of dollar spot, does not produce spores. Consequently, it was assumed that overwintering of this organism in soil, thatch, and plant debris provides primary inoculum for dollar spot epidemics. Overwintering of S. homoeocarpa in roots and shoots of symptomatic and asymptomatic creeping bentgrass turfgrass was quantified over the course of a three-year field experiment. Roots did not consistently harbor S. homoeocarpa, whereas S. homoeocarpa was isolated from 30% of symptomatic shoots and 10% of asymptomatic shoots in the spring of two out of three years. The presence of stroma-like pathogen material on leaf blades was associated with an increase in S. homoeocarpa isolation and colony diameter at 48 hpi. Commercial seed has also been hypothesized to be a potential source of initial inoculum for S. homoeocarpa. Two or more commercial seed lots of six creeping bentgrass cultivars were tested for contamination with S. homoeocarpa using culture-based and molecular detection methods. A viable, pathogenic isolate of S. homoeocarpa was isolated from one commercial seed lot and contamination of this lot was confirmed with nested PCR using S. homoeocarpa specific primers. A sensitive nested PCR assay detected S. homoeocarpa contamination in eight of twelve (75%) commercial seed lots. Seed source, but not cultivar or resistance to dollar spot, influenced contamination by S. homoeocarpa. Overall, this research suggests that seeds are a potential source of initial inoculum for dollar spot epidemics and presents the need for further research in this area. PMID:25333928
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Geryk, J; Mazo, A; Svoboda, J; Hlozánek, I
1980-01-01
The replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus subgroup C was studied using roller cultures. Under such conditions, 10(5)--10(6) infectous units of virus per 0.2 ml were produced, as revealed in both the reverse transcriptase and 16Q complementation tests. A new td daPR-RSV-C mutant was isolated from duck-adapted PR-RSV-C. This mutant replicated in roller cultures with equal efficiency as the original td PR-RSV-C. It was verified that td daPR-RSV-C does not transform chicken fibroblasts, is not oncogenic for 3-week-old chickens and has subgroup C host-range specificity. Both td mutants replicate in duck cells and reach the same titres.
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Walker, C; Muniz, M F B; Rolim, J M; Martins, R R O; Rosenthal, V C; Maciel, C G; Mezzomo, R; Reiniger, L R S
2016-09-16
The objective of this study was to characterize species of the Cladosporium cladosporioides complex isolated from pecan trees (Carya illinoinensis) with symptoms of leaf spot, based on morphological and molecular approaches. Morphological attributes were assessed using monosporic cultures on potato dextrose agar medium, which were examined for mycelial growth, sporulation, color, and conidia and ramoconidia size. Molecular characterization comprised isolation of DNA and subsequent amplification of the translation elongation factor 1α (TEF-1α) region. Three species of the C. cladosporioides complex were identified: C. cladosporioides, Cladosporium pseudocladosporioides, and Cladosporium subuliforme. Sporulation was the most important characteristic differentiating species of this genus. However, morphological features must be considered together with molecular analysis, as certain characters are indistinguishable between species. TEF-1αcan be effectively used to identify and group isolates belonging to the C. cladosporioides complex. The present study provides an important example of a methodology to ascertain similarity between isolates of this complex causing leaf spot in pecan trees, which should facilitate future pathogenicity studies.
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USDA-ARS?s Scientific Manuscript database
Genomes from fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolates were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200...
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Isolation of New Gravitropic Mutants under Hypergravity Conditions.
Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T
2016-01-01
Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 ( eal1 ) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene ( enhancer of eal1 ) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis .
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Isolation of New Gravitropic Mutants under Hypergravity Conditions
Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T.
2016-01-01
Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis. PMID:27746791
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Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA.
Zhao, Carolyn Y; Grinkevich, Vera V; Nikulenkov, Fedor; Bao, Wenjie; Selivanova, Galina
2010-05-01
Expression of mutant p53 correlates with poor prognosis in many tumors, therefore strategies aimed at reactivation of mutant p53 are likely to provide important benefits for treatment of tumors that are resistant to chemotherapy and radiotherapy. We have previously identified and characterized a small molecule RITA which binds p53 and induces a conformational change which prevents the binding of p53 to several inhibitors, including its own destructor MDM2. In this way, RITA rescues the tumor suppression function of wild type p53. Here, we demonstrate that RITA suppressed the growth and induced apoptosis in human tumor cell lines of a diverse origin carrying mutant p53 proteins. RITA restored transcriptional transactivation and transrepression function of several hot spot p53 mutants. The ability of RITA to rescue the activity of different p53 mutants suggests its generic mechanism of action. Thus, RITA is a promising lead for the development of anti-cancer drugs that reactivate the tumor suppressor function of p53 in cancer cells irrespective whether they express mutant or wild type p53.
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Effects of genotype and isolate on expression of dollar spot in seashore paspalum
USDA-ARS?s Scientific Manuscript database
Seashore paspalum (Paspalum vaginatum Swartz) is a warm-season turfgrass species primarily utilized on golf courses and athletic fields and is often impacted by dollar spot disease. Dollar spot, caused by Sclerotinia homoeocarpa F.T. Bennett, is a major fungal disease and the most common turfgrass p...
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Nastasiak, I N; Fedorenko, V A; Danilenko, V N
1997-01-01
The formation of thiostreptone resistant spontaneous and nitrosoguanidine-induced mutants in the erythromycin-producing organism Saccharopolyspora erythraea was investigated. The investigated collection of the mutants was heterogeneous by the level of the thiostreptone resistance (2.5 to 20 micrograms/ml). The thiostreptone resistance mutations had a pleiotropic effect: 17 per cent of the mutants was characterized by the growth thermosensitivity and 26 and 5.8 per cent of the mutants were characterized by loss of the ability to form melanine and aerial mycelium respectively. Such phenotypes were most frequent in the mutants resistant to low concentrations of thiostreptone (2 to 5 micrograms/ml). The absolute majority of the isolated thiostreptone resistant mutants was unstable and formed both the antibiotic resistant and the antibiotic sensitive clones. The greatest portion of the strains with high antibiotic activity (20 per cent) was detected among the S. erythraea spontaneous mutants on the medium with 2.5 micrograms/ ml of thiostreptone. It was shown that the instability of the high antibiotic activity in the mutants was associated with loss of the thiostreptone resistance property.
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Maculosin, a host-specific phytotoxin for spotted knapweed from Alternaria alternata
Stierle, Andrea C.; Cardellina, John H.; Strobel, Gary A.
1988-01-01
Several diketopiperazines have been isolated from liquid cultures of Alternaria alternata, the causal agent of black leaf blight of spotted knapweed, Centaurea maculosa Lam. One of these compounds, maculosin [the diketopiperazine cyclo(-L-Pro-L-Tyr-)], was active in the nicked-leaf bioassay at 10-5 M; synthetic maculosin possessed chemical and biological activities identical to those of the natural product. Other diketopiperazines isolated from the fungus possessed either less activity or none at all. In tests against 19 plant species, maculosin was phytotoxic only to spotted knapweed. Thus maculosin is a host-specific phytotoxin from a weed pathogen. PMID:16593989
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Tani, Tatsunori; Taguchi, Hisataka; Fujimori, Kazuhiro E; Sahara, Takehiko; Ohgiya, Satoru; Kamagata, Yoichi; Akamatsu, Takashi
2016-10-01
To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but <30% of the xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Garita-Cambronero, Jerson; Palacio-Bielsa, Ana; López, María M.
2016-01-01
The draft genome sequences of two strains of Xanthomonas arboricola, isolated from asymptomatic peach trees in Spain, are reported here. These strains are avirulent and do not belong to the same phylogroup as X. arboricola pv. pruni, a causal agent of bacterial spot disease of stone fruits and almonds. PMID:27609931
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Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase.
Green, G N; Gennis, R B
1983-01-01
A screening procedure was devised which permitted the isolation of a cytochrome d-deficient mutant by its failure to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Cytochrome a1 and probably cytochrome b558 were also missing in the mutant. Growth and oxygen uptake rates were similar for both parent and mutant strains. However, the strain lacking cytochrome d had an increased sensitivity to cyanide, indicating that cytochrome d confers some resistance to this respiratory inhibitor. The gene responsible for these phenotypes has been named cyd and maps between tolA and sucB. PMID:6304009
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Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER
Gans, Madeleine; Audit, Claudie; Masson, Michele
1975-01-01
The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed. PMID:814037
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Reimann, Sven; Smits, Sander H. J.; Schmitt, Lutz; Groth, Georg; Gohlke, Holger
2014-01-01
Human heat shock protein of 90 kDa (hHsp90) is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD) should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScorePPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization. PMID:24760083
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Wee, Jung-In; Park, Jong-Han; Back, Chang-Gi; You, Young-Hyun
2016-01-01
In July 2015, diseased leaves of black chokeberry (Aronia melanocarpa) were observed in Danyang and Gochang, Korea. The symptoms appeared as circular or irregular brown leaf spots, from which Alternaria tenuissima was isolated. The isolates were cultured on potato dextrose agar, and their morphological characteristics were observed under a light microscope. The colonies were whitish to ash colored. The pathogenicity test on healthy black chokeberry leaves produced circular brown spots, in line with the original symptoms. Molecular analyses of the ITS, GPD, RPB2, and TEF genes were conducted to confirm the identity of the pathogen. The phylogeny of the multi-gene sequences indicated that the causal agent was A. tenuissima. This study is the first report of A. tenuissima leaf spot on black chokeberry (A. melanocarpa). PMID:27790070
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Cohort analysis of a single nucleotide polymorphism on DNA chips.
Schwonbeck, Susanne; Krause-Griep, Andrea; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Meinl, Walter; Glatt, Hansrüdi; Bier, Frank F
2004-11-15
A method has been developed to determine SNPs on DNA chips by applying a flow-through bioscanner. As a practical application we demonstrated the fast and simple SNP analysis of 24 genotypes in an array of 96 spots with a single hybridisation and dissociation experiment. The main advantage of this methodical concept is the parallel and fast analysis without any need of enzymatic digestion. Additionally, the DNA chip format used is appropriate for parallel analysis up to 400 spots. The polymorphism in the gene of the human phenol sulfotransferase SULT1A1 was studied as a model SNP. Biotinylated PCR products containing the SNP (The SNP summary web site: ) (mutant) and those containing no mutation (wild-type) were brought onto the chips coated with NeutrAvidin using non-contact spotting. This was followed by an analysis which was carried out in a flow-through biochip scanner while constantly rinsing with buffer. After removing the non-biotinylated strand a fluorescent probe was hybridised, which is complementary to the wild-type sequence. If this probe binds to a mutant sequence, then one single base is not fully matching. Thereby, the mismatched hybrid (mutant) is less stable than the full-matched hybrid (wild-type). The final step after hybridisation on the chip involves rinsing with a buffer to start dissociation of the fluorescent probe from the immobilised DNA strand. The online measurement of the fluorescence intensity by the biochip scanner provides the possibility to follow the kinetics of the hybridisation and dissociation processes. According to the different stability of the full-match and the mismatch, either visual discrimination or kinetic analysis is possible to distinguish SNP-containing sequence from the wild-type sequence.
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Method for producing capsular polysaccharides
NASA Technical Reports Server (NTRS)
Richards, Gil F. (Inventor); Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor)
1994-01-01
Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.
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Li, Yongqiang; Deng, Congliang; Bian, Yong; Zhao, Xiaoli; Zhou, Qi
2017-04-01
Apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and prunus necrotic ringspot virus (PNRSV) were identified in a crab apple tree by small RNA deep sequencing. The complete genome sequence of ACLSV isolate BJ (ACLSV-BJ) was 7554 nucleotides and shared 67.0%-83.0% nucleotide sequence identity with other ACLSV isolates. A phylogenetic tree based on the complete genome sequence of all available ACLSV isolates showed that ACLSV-BJ clustered with the isolates SY01 from hawthorn, MO5 from apple, and JB, KMS and YH from pear. The complete nucleotide sequence of ASGV-BJ was 6509 nucleotides (nt) long and shared 78.2%-80.7% nucleotide sequence identity with other isolates. ASGV-BJ and the isolate ASGV_kfp clustered together in the phylogenetic tree as an independent clade. Recombination analysis showed that isolate ASGV-BJ was a naturally occurring recombinant.
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Vassilara, Foula; Galani, Irene; Souli, Maria; Papanikolaou, Konstantinos; Giamarellou, Helen; Papadopoulos, Antonios
2017-07-01
The aim of this study was to determine the propensities of imipenem to select for resistant Pseudomonas aeruginosa mutants by determining the mutant prevention concentrations (MPCs) for 9 unrelated clinical isolates and the accession of any relationship with mechanisms of resistance development. The MPC/MIC ratios ranged from 4 to 16. Detection of resistance mechanisms in the mutant derivatives of the nine isolates mainly revealed inactivating mutations in the gene coding for outer membrane protein OprD. Point mutations leading to premature stop codons or amino acid substitution S278P, ≥1bp deletion leading to frameshift mutations and interruption of the oprD by an insertion sequence, were observed. MPC and mutant selection window (MSW) are unique parameters that may guide the implementation of antimicrobial treatment, providing useful information about the necessary imipenem concentration needed in the infection area, in order to avoid the emergence of resistance, especially in clinical situations with high bacterial load. Copyright © 2017 Elsevier Inc. All rights reserved.
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McGrann, Graham R. D.; Brown, James K. M.
2014-01-01
Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, is a serious, recently emerged disease of barley in Europe and other temperate regions. This study investigated the trade off between strong resistance to powdery mildew conferred by mlo mutant alleles and increased susceptibility to RLS. In field trials and seedling tests, the presence of mlo alleles increased severity of RLS. Genetic analysis of a doubled-haploid population identified one quantitative trait locus for susceptibility to RLS, colocalizing with the mlo-11 allele for mildew resistance. The effect of mlo-11 on RLS severity was environmentally sensitive. Analysis of near-isogenic lines of different mlo mutations in various genetic backgrounds confirmed that mlo alleles increased RLS severity in seedlings and adult plants. For mlo resistance to mildew to be fully effective, the genes ROR1 and ROR2 are required. RLS symptoms were significantly reduced on mlo-5 ror double mutants but fungal DNA levels remained as high as in mlo-5 single mutants, implying that ror alleles modify the transition of the fungus from endophytism to necrotrophy. These results indicate that the widespread use of mlo resistance to control mildew may have inadvertently stimulated the emergence of RLS as a major disease of barley. PMID:24399175
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Phenotypic Characterization of pncA Mutants of Mycobacterium tuberculosis
Morlock, Glenn P.; Crawford, Jack T.; Butler, W. Ray; Brim, Suzanne E.; Sikes, David; Mazurek, Gerald H.; Woodley, Charles L.; Cooksey, Robert C.
2000-01-01
We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147–151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were ≥100 μg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 μg/ml, 3 had the same mutation (Thr47→Ala) and 18 had the wild-type sequence. For the three Thr47→Ala mutants PZA MICs were 12.5 μg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 μg of PZA per ml and the third was resistant to 800 μg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to ≥100 μg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47→Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to ≥100 μg of PZA per ml. PMID:10952570
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Bialek-Davenet, Suzanne; Marcon, Estelle; Leflon-Guibout, Véronique; Lavigne, Jean-Philippe; Bert, Frédéric; Moreau, Richard; Nicolas-Chanoine, Marie-Hélène
2011-01-01
The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression. PMID:21464248
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Thomas, E. V.; Phillippy, K. H.; Brahamsha, B.; Haaland, D. M.; Timlin, J. A.; Elbourne, L. D. H.; Palenik, B.; Paulsen, I. T.
2009-01-01
Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in part to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition. PMID:19404483
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Thomas, E. V.; Phillippy, K. H.; Brahamsha, B.; ...
2009-01-01
Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in partmore » to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition.« less
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Ghosh, Upasana; Chakraborty, Somnath; Balasubramanian, Thangavel; Das, Punyabrata
2014-01-01
Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug. PMID:25183066
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Rick, P. D.; Osborn, M. J.
1972-01-01
A new type of auxotrophic mutant of Salmonella typhimurium has been isolated that is defective in the synthesis of the 3-deoxy-D-mannooctulosonate (ketodeoxyoctonate) region of the lipopolysaccharide and requires D-arabinose-5-phosphate for growth. Genetic and biochemical evidence indicated that the mutant defect was due to an altered ketodeoxyoctonate-8-phosphate synthetase with an apparent Km for D-arabinose-5-phosphate 35-fold higher than that of the parental enzyme. As a result of this enzymatic lesion, the mutant strain was dependent on exogenous D-arabinose-5-phosphate both for growth and for synthesis of a complete lipopolysaccharide. PMID:4566459
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Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae.
Erdmann, R; Veenhuis, M; Mertens, D; Kunau, W H
1989-01-01
Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized. Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iii) absence of detectable peroxisomes at the ultrastructural level. These lesions (pas1-1 and pas2) are shown to be nonallelic and recessive. Crossing of pas1-1 and pas2 strains resulted in diploid cells that had regained the ability to grow on oleic acid as sole carbon source and to form peroxisomes. These pas mutants may provide useful tools for future studies on the molecular mechanisms involved in peroxisomal assembly. Images PMID:2568633
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Omar, Nurul Husna; Mohd, Masratulhawa; Mohamed Nor, Nik Mohd Izham; Zakaria, Latiffah
2018-01-01
Leaf spot diseases are mainly caused by fungi including Fusarium. In the present study several species of Fusarium were isolated from the leaf spot lesion of mango (Mangifera indica L.) Based on morphological characteristics, TEF-1α sequences and phylogenetic analysis, five species were identified as F. proliferatum, F. semitectum, F. mangiferae, F. solani and F. chlamydosporum. Pathogenicity test indicated that representative isolates of F. proliferatum, F. semitectum and F. chlamydosporum were pathogenic on mango leaves causing leaf spot with low to moderate virulence. Nevertheless, abundance of spots on the leaf can disrupt photosynthesis which in turn reduced growth, and lead to susceptibility to infection by opportunistic pathogens due to weakening of the plant. Fusarium solani and F. mangiferae were non-pathogenic and it is possible that both species are saprophyte which associated with nutrient availability on the surface of the leaf through decaying leave tissues. The occurrence of Fusarium spp. on the leaf spot lesion and the effect from the disease needs to be considered when developing disease management method of mango cultivation as numerous spot on the leaves could effect the photosynthesis process and finally giving low yield and less quality of mango. Copyright © 2017. Published by Elsevier Ltd.
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Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells
Brázdová, Marie; Navrátilová, Lucie; Tichý, Vlastimil; Němcová, Kateřina; Lexa, Matej; Hrstka, Roman; Pečinka, Petr; Adámik, Matej; Vojtesek, Borivoj; Paleček, Emil; Deppert, Wolfgang; Fojta, Miroslav
2013-01-01
Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed. PMID:23555710
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Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica
Robbe-Saule, Véronique; Algorta, Gabriela; Rouilhac, Isabelle; Norel, Françoise
2003-01-01
The stationary-phase-inducible sigma factor, σS (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σS protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σS, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease. PMID:12902215
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Isolation and characterization of bioactive components from Mirabilis jalapa L. radix
Gogoi, Jyotchna; Nakhuru, Khonamai Sewa; Policegoudra, Rudragoud S.; Chattopadhyay, Pronobesh; Rai, Ashok Kumar; Veer, Vijay
2015-01-01
The present investigation was carried out to isolate and characterize bioactive components from Mirabilis jalapa L. radix (紫茉莉根 zǐ mò lì gēn). Thin-layer chromatography was used for the separation of spots from fractions of the crude extract. Separated spots were collected for identification of their activities. Free-radical scavenging activity was evaluated by spraying thin-layer chromatography plates (spotted with fractions) with 0.2% of 2,2-diphenyl-1-picrylhydrazyl solution. Activity against human pathogens such as Staphylococcus aureus and Candida albicans were determined using the agar diffusion method. Potential spots were subjected to infrared (IR) analysis and gas chromatography for characterization. Two spots (5F1 and 1F3) showed free-radical scavenging activity. The 1F3 spot was active against both S. aureus and C. albicans, whereas the 5F1 spot was active against S. aureus only. IR spectral analysis indicated that 5F1 spot to be a triterpenoid. Using IR spectral analysis and an IR library search, the 1F3 spot was identified to be a flavone, which may have a hydroxyl group in ring “A” of the flavone nucleus. Our results indicated that the 1F3 and 5F1 spots are potential free-radical scavengers. Both 1F3 and 5F1 exhibited antimicrobial activity. IR spectral analysis coupled with an IR library search indicated 1F3 and 5F1 to be a flavone and a triterpenoid, respectively. PMID:26870679
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NASA Technical Reports Server (NTRS)
Asato, Y.
1972-01-01
Three independently isolated ultraviolet light sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 showed the highest sensitivity to UV by its greatly reduced photoreactivation capacity following irradiation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, thus indicating the presence of excision enzymes involved in dark repair. Under 'black' and 'white' illumination, strain uvs-1 shows photorecovery properties comparable with wild-type cultures. Results indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.
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Mutations and Misconceptions: The Isolation and Study of Mutant Bacteria.
ERIC Educational Resources Information Center
Corner, Thomas R.
1992-01-01
Describes simple, inexpensive activities for teaching students about mutants and mutations in bacteria. Explains how to isolate bacteria from soil and leaves and how to grow bacteria on agar or in broth. Describes how to construct a gradient plate for finding the minimum inhibitory concentration of a substance and how to use this set up to find…
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USDA-ARS?s Scientific Manuscript database
This paper describes the isolation of Wheat ABA-responsive mutants (Warm) in Chinese spring background of allohexaploid Triticum aestivum. The plant hormone abscisic acid (ABA) is required for the induction of seed dormancy, the induction of stomatal closure and drought tolerance, and is associated...
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Dose modification in e.coli by a constitutive radioprotective agent isolated from m. radiodurans
DOE Office of Scientific and Technical Information (OSTI.GOV)
Goldstein, Lawrence S.
1972-01-01
A constitutive low molecular weight radioprotective agent has been isolated from a colorless mutant of Micrococcus radiodurans. The effect of radioprotective extract was also investigated in three mutant strains of E.coli B/r differing from one another at a given locus concerned with the repair of radiation induced damage/
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USDA-ARS?s Scientific Manuscript database
Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits synthesis of type I interferons (IFNs) in infected pigs and in cultured cells. Here we report that one PRRSV mutant A2MC2 induces type I IFNs in cultured cells and has no effect on IFN downstream signaling. The mutant isolate was p...
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Emmenegger, Eveline J.; Troyer, Ryan M.; Kurath, Gael
2003-01-01
Infectious hematopoietic necrosis virus (IHNV) is an RNA virus that causes significant mortalities of salmonids in the Pacific Northwest of North America. RNA virus populations typically contain genetic variants that form a heterogeneous virus pool, referred to as a quasispecies or mutant spectrum. This study characterized the mutant spectra of IHNV populations within individual fish reared in different environmental settings by RT–PCR of genomic viral RNA and determination of partial glycoprotein gene sequences of molecular clones. The diversity of the mutant spectra from ten in vivo populations was low and the average mutation frequencies of duplicate populations did not significantly exceed the background mutation level expected from the methodology. In contrast, two in vitro populations contained variants with an identical mutational hot spot. These results indicated that the mutant spectra of natural IHNV populations is very homogeneous, and does not explain the different magnitudes of genetic diversity observed between the different IHNV genogroups. Overall the mutant frequency of IHNV within its host is one of the lowest reported for RNA viruses.
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New hosts of Myrothecium SPP. In brazil and a preliminary In Vitro assay of fungicides
Quezado Duval, A.M.; Henz, G.P.; Paz-Lima, M.L.; Medeiros, A.R.; Miranda, B.E.C.; Pfenning, L.H.; Reis, A.
2010-01-01
Myrothecium roridum and M. verrucaria are two plant pathogenic species causing foliar spots in a large number of cultivated plants. This paper aims to study the causal agents of foliar spots in vegetable crops (sweet pepper, tomato, cucumber), ornamental plants (Spathiphyllum, Solidago canadensis, Anthurium, Dieffenbachia) and a solanaceous weed plant (Nicandra physalodes). Most of the isolates were identified as M. roridum; only the isolate ‘Myr-02’ from S. canadensis was identified as M. verrucaria. All the isolates were pathogenic to their original plant hosts and also to some other plants. Some fungicides were tested in vitro against an isolate of M. roridum and the mycelial growth recorded after seven days. Fungicides with quartenary ammonium, Tebuconzole and copper were highly effective in inhibiting the mycelial growth of M. roridum. This paper confirms the first record of M. roridum causing leaf spots in sweet pepper, tomato, Spathiphyllum, Anthurium, Dieffenbachia and N. physalodes. We also report M. roridum as causal agent of cucumber fruit rot and also M. verrucaria in tango plants. PMID:24031487
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Lee, Seung Gyu; Lee, Kang Wook; Park, Tae Heung; Park, Ji Yeong; Han, Nam Soo; Kim, Jeong Hwan
2012-04-01
LAB were isolated from makgeolli locally produced around Jinju, Gyeongnam, S. Korea during spring of 2011. Randomly selected 11 isolates from MRS agar plates were identified first by API CHL 50 kits and then 16S rRNA gene sequencing. All 11 isolates were identified as Lactobacillus plantarum. Among them, ST4 grew in MRS broth with ethanol up to 10%, showing the highest alcohol resistance. L. plantarum ST4 was moderately resistant against acid and bile salts. When cellular proteins of L. plantarum ST4 under ethanol stress were analyzed by two-dimensional gel electrophoresis (2DE), the intensities of 6 spots increased, whereas 22 spots decreased at least 2-fold. Those 28 spots were identified by peptide mass fingerprinting (PMF). FusA2 (elongation factor G) increased 18.8-fold (6% ethanol) compared with control. Other proteins were AtpD (ATP synthase subunit beta), DnaK, GroEL, Tuf (elongation factor Tu), and Npr2 (NADH peroxidase), respectively. Among the 22 proteins decreased in intensities, lactate dehydrogenases (LdhD and LdhL1) were included.
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Gechev, Tsanko; Mehterov, Nikolay; Denev, Iliya; Hille, Jacques
2013-01-01
A genetic approach is described to isolate mutants more tolerant to oxidative stress. A collection of T-DNA activation tag Arabidopsis thaliana mutant lines was screened for survivors under conditions that trigger H2O2-induced cell death. Oxidative stress was induced by applying the catalase (CAT) inhibitor aminotriazole (AT) in the growth media, which results in decrease in CAT enzyme activity, H2O2 accumulation, and subsequent plant death. One mutant was recovered from the screening and named oxr1 (oxidative stress resistant 1). The location of the T-DNA insertion was identified by TAIL-PCR. Oxr1 exhibited lack of cell death symptoms and more fresh weight and chlorophyll content compared to wild type. The lack of cell death correlated with more prominent induction of anthocyanins synthesis in oxr1. These results demonstrate the feasibility of AT as a screening agent for the isolation of oxidative stress-tolerant mutants and indicate a possible protective role for anthocyanins against AT-induced cell death. The chapter includes protocols for ethyl methanesulfonate mutagenesis, mutant screening using AT, T-DNA identification by TAIL-PCR, CAT activity measurements, and determination of malondialdehyde, chlorophyll, and anthocyanins. Copyright © 2013 Elsevier Inc. All rights reserved.
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USDA-ARS?s Scientific Manuscript database
The dek18 mutant of maize has decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology in maize seed development. Seeds of the dek18 mutants are smaller compared to wild type seeds and the vegetative d...
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A Novel Two-Step Method for Screening Shade Tolerant Mutant Plants via Dwarfism
Li, Wei; Katin-Grazzini, Lorenzo; Krishnan, Sanalkumar; Thammina, Chandra; El-Tanbouly, Rania; Yer, Huseyin; Merewitz, Emily; Guillard, Karl; Inguagiato, John; McAvoy, Richard J.; Liu, Zongrang; Li, Yi
2016-01-01
When subjected to shade, plants undergo rapid shoot elongation, which often makes them more prone to disease and mechanical damage. Shade-tolerant plants can be difficult to breed; however, they offer a substantial benefit over other varieties in low-light areas. Although perennial ryegrass (Lolium perenne L.) is a popular species of turf grasses because of their good appearance and fast establishment, the plant normally does not perform well under shade conditions. It has been reported that, in turfgrass, induced dwarfism can enhance shade tolerance. Here we describe a two-step procedure for isolating shade tolerant mutants of perennial ryegrass by first screening for dominant dwarf mutants, and then screening dwarf plants for shade tolerance. The two-step screening process to isolate shade tolerant mutants can be done efficiently with limited space at early seedling stages, which enables quick and efficient isolation of shade tolerant mutants, and thus facilitates development of shade tolerant new cultivars of turfgrasses. Using the method, we isolated 136 dwarf mutants from 300,000 mutagenized seeds, with 65 being shade tolerant (0.022%). When screened directly for shade tolerance, we recovered only four mutants from a population of 150,000 (0.003%) mutagenized seeds. One shade tolerant mutant, shadow-1, was characterized in detail. In addition to dwarfism, shadow-1 and its sexual progeny displayed high degrees of tolerance to both natural and artificial shade. We showed that endogenous gibberellin (GA) content in shadow-1 was higher than wild-type controls, and shadow-1 was also partially GA insensitive. Our novel, simple and effective two-step screening method should be applicable to breeding shade tolerant cultivars of turfgrasses, ground covers, and other economically important crop plants that can be used under canopies of existing vegetation to increase productivity per unit area of land. PMID:27752260
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Isolation and characterization of acid-sensitive mutants of Pediococcus acidilactici.
Kurdi, Peter; Smitinont, Thitapha; Valyasevi, Ruud
2009-02-01
Acid-sensitive mutants of Pediococcus acidilactici BCC 9545, a starter culture of the Thai fermented pork sausage nham, were isolated as spontaneous neomycin resistant mutants. The mutants generally produced less acid and acidified the culture media less than the parent strain in a 72 h culturing period. Interestingly, the ATPase activities of the mutants did not differ considerably from that of the parent strain in acidic conditions. It was also found that the internal pH values of the mutant strains were somewhat lower in neutral environment, while at pH 5.0 their internal pHs were significantly lower compared to the parent's. Inhibiting the H(+)-ATPase activities in energized cells by N,N'-dicyclohexyl carbodiimide also revealed that protons were leaking from the mutants at neutral pH, which increased under acidic conditions. In contrast, the parent strain exhibited a smaller proton leak and only under acidic conditions. The membrane fatty acid analysis of the mutants indicated that under acidic conditions the mutants had a significantly smaller major unsaturated/saturated fatty acids ratio ((C(18:1)+C(18:3n6))/(C(16:0)+C(18:0))) compared to the parent strain's membrane. Taken together, these observations suggest there is a reasonable possibility that the membrane fatty acid profile differences in the mutants resulted in their acid-sensitivity.
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Dmytruk, Kostyantyn V; Smutok, Oleh V; Ryabova, Olena B; Gayda, Galyna Z; Sibirny, Volodymyr A; Schuhmann, Wolfgang; Gonchar, Mykhailo V; Sibirny, Andriy A
2007-01-01
Background Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution. Results Hansenula polymorpha (Pichia angusta) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the KM values for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas Vmax values are increased or remain unaffected. The mutant AOX alleles from H. polymorpha mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX. Conclusion The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids. PMID:17567895
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Wu, J R; Yeh, Y C
1975-05-01
Suppressors of gene 59-defective mutants were isolated by screening spontaneous, temperature-sensitive (ts) revertants of the amber mutant, amC5, in gene 59. Six ts revertants were isolated. No gene 59-defective ts recombinant was obtained by crossing each ts revertant with the wild type, T4D. However, suppressors of gene 59-defective mutants were obtained from two of these ts revertants. These suppressor mutants are referred to as dar (DNA arrested restoration). dar mutants specifically restored the abnormalities, both in DNA synthesis and burst size, caused by gene 59-defective mutants to normal levels. It is unlikely that dar mutants are nonsense suppressors since theý failed to suppress amber mutations in 11 other genes investigated. The genetic expression of dar is controlled by gene 55; therefore, dar is a late gene. The genetic location of dar has been mapped between genes 24 and 25, a region contiguous to late genes. dar appears to be another nonessential gene of T4 since burst sizes of dar were almost identical to those of the wild type. Mutations in dar did not affect genetic recombination and repair of UV-damaged DNA, but caused a sensitivity to hydroxyurea in progeny formation. The effect of the dar mutation on host DNA degradation cannot account for its hydroxyurea sensitivity. dar mutant alleles were recessive to the wild-type allele as judged by restoration of arrested DNA synthesis. The possible mechanisms for the suppression of defects in gene 59 are discussed.
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Yerly, S; Rakik, A; Kinloch-de-Loes, S; Erb, P; Vernazza, P; Hirschel, B; Perrin, L
1996-10-26
Zidovudine (ZDV) was the most widely used anti-HIV drug between 1987 and 1995, and, as already reported, transmission of ZDV-resistant viruses occurs. Several mutations of the reverse transcriptase gene have been identified; one of them affects the 215 codon and is associated with a high degree of resistance. We have determined, using selective PCR, the prevalence of transmission of 215 mutant isolates in 134 patients with primary HIV infection (PHI) and have identified 8 patients with 215 mutant virus between 1989 and 1995 in Switzerland. Mutant resistant viruses have been isolated from patients treated with most antiviral drugs. A systematic search for mutant viruses may provide useful information for the adaptation of treatment strategies.
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1992-01-01
We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771
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Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale
Brayton, Kelly A.; Magunda, Forgivemore; Munderloh, Ulrike G.; Kelley, Karen L.; Barbet, Anthony F.
2015-01-01
Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species. PMID:25595772
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Rodriguez-Anaya, Libia Zulema; Gonzalez-Galaviz, Jose Reyes; Casillas-Hernandez, Ramón; Lares-Villa, Fernando; Estrada, Karel
2016-01-01
The first genome sequence of a Mexican white spot syndrome virus is presented here. White spot syndrome is a shrimp pandemic virus that has devastated production in Mexico for more than 10 years. The availability of this genome will greatly aid epidemiological studies worldwide, contributing to the molecular diagnostic and disease prevention in shrimp farming. PMID:26966222
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Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki
2015-08-01
Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants. Copyright © 2015 John Wiley & Sons, Ltd.
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Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei
2006-01-01
A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592
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Yasutani, I.; Ozawa, S.; Nishida, T.; Sugiyama, M.; Komamine, A.
1994-01-01
Three temperature-sensitive mutants of Arabidopsis thaliana that were defective in the redifferentiation of shoots were isolated as tools for the study of organogenesis. M3 lines were constructed by harvesting M3 seeds separately from each M2 plant. Comparative examination of shoot redifferentiation in root explants of 2700 M3 lines at 22[deg]C (permissive temperature) and at 27[deg]C (restrictive temperature) led to the identification of seven temperature-sensitive mutant lines. Genetic tests of three of the seven mutant lines indicated that temperature-sensitive redifferentiation of shoots in these three lines resulted from single, nuclear, recessive mutations in three different genes, designated SRD1, SRD2, and SRD3. The morphology of root explants of srd mutants cultured at the restrictive temperature suggests that the products of these SRD genes function at different stages of the redifferentiation of shoots. PMID:12232244
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Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N
1989-01-01
The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum. PMID:2543291
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Lipopolysaccharide and Aldoheptose Biosynthesis in Transketolase Mutants of Salmonella typhimurium
Eidels, L.; Osborn, M. J.
1971-01-01
Genetic and biochemical evidence that sedoheptulose-7-phosphate is an obligatory precursor of the L-glycero-D-mannoheptose residues of the lipopolysaccharide of Salmonella was obtained by isolation and characterization of transketolase-negative mutants of Salmonella typhimurium. These mutants, which are defective in synthesis of sedoheptulose-7-phosphate, were found to produce an incomplete heptose-deficient lipopolysaccharide, and were also sensitive to bile salts, a characteristic property of heptose-deficient mutants. Phenotypic repair of the defect in lipopolysaccharide synthesis was obtained by addition of exogenous sedoheptulose-7-phosphate to growing cultures of the mutant strains. Characterization of revertants isolated either as transketolase-positive or heptose-positive provided further evidence that the heptose deficiency resulted from mutation at the transketolase locus. On the basis of these findings a possible pathway for conversion of sedoheptulose-7-phosphate to L-glycero-D-mannoheptose is proposed. PMID:4942911
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Description of a novel allelic “thick leafed” mutant of sorghum
USDA-ARS?s Scientific Manuscript database
An allelic sorghum [Sorghum bicolor (L.) Moench] mutant with thick and narrow erect leaves (thl) and reduced adaxial stomatal density was isolated from the Annotated Individually pedigreed Mutagenized Sorghum (AIMS) mutant library developed at the Plant Stress and Germplasm Development Unit at Lubbo...
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Cheng, Zhongyi; Peplowski, Lukasz; Cui, Wenjing; Xia, Yuanyuan; Liu, Zhongmei; Zhang, Jialei; Kobayashi, Michihiko; Zhou, Zhemin
2018-03-01
Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money-, and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity toward rac-mandelonitrile (MAN). All the four selected residues had numerous collisions with rac-MAN. Five mutants significantly shifting stereoselectivity towards (S)-MAN were obtained from site-saturation mutagenesis, one of them, at position βPhe37, exhibiting efficient production of (S)-MAN with 96.8% ee p , was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)-MAN and residue βHis37. (R)-MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi-rational design significantly reduced the select range of hot-spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity. © 2017 Wiley Periodicals, Inc.
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2012-01-01
Background Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. Results We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. Conclusions We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin. PMID:22272603
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kuila, R.K.; Ranganathan, B.
1978-04-01
A strain of Streptococcus lactis subspecies diacetylactis S/sub 1/ isolated from fresh milk was exposed to 7200 ergs/mm/sup 2/ of ultraviolet radiation. Over 8100 colonies surviving from 7.4 x 10/sup 6/ cells exposed to radiation were screened on citrate agar for detection and isolation of mutants with increased flavor and/or acid production. Of the survivors, 960 were type-I mutants that exhibited clear zone on citrate agar after 18 h (presumed to be high diacetyl producers), and 288 were type-II mutants which did not exhibit clear zones on citrate agar for up to 72 h (high acid producers). Type-II mutants producedmore » an average .93 percent titratable acidity which was 34 percent more than the .69 percent of the parent. Reduction in titratable acidity (56 percent less) was considerable in type-I mutants, compared with the parent culture. Diacetyl + acetoin production by type-I mutants was 137.9 ppM which has 4.5 times more than that of the parental strain. Acetaldehyde production in the mutants varied from 1.5 to 34.5 ppM (parent culture 3.0 ppM). The mutants with increased acid and high acetoin plus diacetyl production were stable after 50 subcultures in milk.« less
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Nordhues, André; Schöttler, Mark Aurel; Unger, Ann-Katrin; Geimer, Stefan; Schönfelder, Stephanie; Schmollinger, Stefan; Rütgers, Mark; Finazzi, Giovanni; Soppa, Barbara; Sommer, Frederik; Mühlhaus, Timo; Roach, Thomas; Krieger-Liszkay, Anja; Lokstein, Heiko; Crespo, José Luis; Schroda, Michael
2012-01-01
The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b6f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the QA/QA− redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids. PMID:22307852
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Demir, Eşref; Turna, Fatma; Vales, Gerard; Kaya, Bülent; Creus, Amadeu; Marcos, Ricard
2013-11-01
As in vivo system, we propose Drosophila melanogaster as a useful model for study the genotoxic risks associated with nanoparticle exposure. In this study we have carried out a genotoxic evaluation of titanium dioxide (TiO2), zirconium oxide (ZrO2) and aluminium oxide (Al2O3) nanoparticles and their microparticulated forms in D. melanogaster by using the wing somatic mutation and recombination assay. This assay is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in treated larvae, which are expressed as mutant spots on the wings of adult flies. Third instar larvae were feed with TiO2, ZrO2 and Al2O3 nanoparticles, and their microparticulated forms, at concentrations ranging from 0.1 to 10mM. Although a certain level of aggregation/agglomeration was observed in solution, it must be noted than the constant digging activity of larvae ensures that treated medium pass constantly through the digestive tract ensuring exposure. The results showed that no significant increases in the frequency of all spots (e.g. small single, large single, twin, total mwh and total spots) were observed, indicating that these nanoparticles were not able to induce genotoxic activity in the wing spot assay of D. melanogaster. Negative data were also obtained with the microparticulated forms. This indicates that the nanoparticulated form of the selected nanomaterials does not modify the potential genotoxicity of their microparticulated versions. These in vivo results contribute to increase the genotoxicity database on the TiO2, ZrO2 and Al2O3 nanoparticles. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Isolation, characterization, and expression analyses of tryptophan
USDA-ARS?s Scientific Manuscript database
The dek18 mutant of maize has decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology in maize seed development. Seeds of the dek18 mutants are smaller compared to wild type seeds and the vegetative d...
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A Laboratory Exercise for Isolation and Characterizing Microbial Mutants with Metabolic Defects.
ERIC Educational Resources Information Center
Doe, Frank J.; Leslie, John F.
1993-01-01
Describes science experiments for undergraduate biology instruction on the concepts of mutation and characterization of the resulting mutant strains. The filamentous fungi "Fusarium moniliforme" is used to illustrate the induction of mutants (mutagenesis), identification of the mutated gene, construction of a biochemical pathway, and…
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Chemotaxis-defective mutants of the nematode Caenorhabditis elegans.
Dusenbery, D B; Sheridan, R E; Russell, R L
1975-06-01
The technique of countercurrent separation has been used to isolate 17 independent chemotaxis-defective mutants of the nematode Caenorhabditis elegans. The mutants, selected to be relatively insensitive to the normally attractive salt NaCl, show varying degrees of residual sensitivity; some are actually weakly repelled by NaCl. The mutants are due to single gene defects, are autosomal and recessive, and identify at least five complementation groups.
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Rapid strain improvement through optimized evolution in the cytostat.
Gilbert, Alan; Sangurdekar, Dipen P; Srienc, Friedrich
2009-06-15
Acetate is present in lignocellulosic hydrolysates at growth inhibiting concentrations. Industrial processes based on such feedstock require strains that are tolerant of this and other inhibitors present. We investigated the effect of acetate on Saccharomyces cerevisiae and show that elevated acetate concentrations result in a decreased specific growth rate, an accumulation of cells in the G1 phase of the cell cycle, and an increased cell size. With the cytostat cultivation technology under previously derived optimal operating conditions, several acetate resistant mutants were enriched and isolated in the shortest possible time. In each case, the isolation time was less than 5 days. The independently isolated mutant strains have increased specific growth rates under conditions of high acetate concentrations, high ethanol concentrations, and high temperature. In the presence of high acetate concentrations, the isolated mutants produce ethanol at higher rates and titers than the parental strain and a commercial ethanol producing strain that has been analyzed for comparison. Whole genome microarray analysis revealed gene amplifications in each mutant. In one case, the LPP1 gene, coding for lipid phosphate phosphatase, was amplified. Two mutants contained amplified ENA1, ENA2, and ENA5 genes, which code for P-type ATPase sodium pumps. LPP1 was overexpressed on a plasmid, and the growth data at elevated acetate concentrations suggest that LPP1 likely contributes to the phenotype of acetate tolerance. A diploid cross of the two mutants with the amplified ENA genes grew faster than either individual haploid parent strain when 20 g/L acetate was supplemented to the medium, which suggests that these genes contribute to acetate tolerance in a gene dosage dependent manner. 2009 Wiley Periodicals, Inc.
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Nakase, Keisuke; Sakuma, Yui; Nakaminami, Hidemasa; Noguchi, Norihisa
2016-12-01
With the aim of elucidating the mechanisms of fluoroquinolones resistance in Propionibacterium acnes, we determined the susceptibility of fluoroquinolones in 211 isolates from patients with acne vulgaris. We identified five isolates (2.4%) with reduced susceptibility to nadifloxacin (minimum inhibitory concentration ≥ 4 μg/ml). Determination of the sequences of the DNA gyrase (gyrA and gyrB) and DNA topoisomerase (parC and parE) genes showed the amino acid substitutions Ser101Leu and Asp105Gly of GyrA in four and one of the isolates, respectively. In vitro mutation experiments showed that low-level fluoroquinolone-resistant mutants with the Ser101Leu or Asp105Gly substitution in GyrA could be obtained from selection with ciprofloxacin and levofloxacin. The pattern of substitution (Ser101Trp in GyrA) caused by nadifloxacin selection was different from that induced by the other fluoroquinolones. In the isolation of further high-level resistant mutants, acquisition of another amino acid substitution of GyrB in addition to those of GyrA was detected, but there were no substitutions of ParC and ParE. In addition, the mutant prevention concentration and mutation frequency of nadifloxacin were lowest among the tested fluoroquinolones. The growth of the Ser101Trp mutant was lower than that of the other mutants. Our findings suggest that the Ser101Trp mutant of P. acnes emerges rarely and disappears immediately, and the risk for the prevalence of fluoroquinolones-resistant P. acnes differs according to the GyrA mutation type. To our knowledge, this study is the first to demonstrate the mechanisms of resistance to fluoroquinolones in P. acnes. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Imidazopyridine Compounds Inhibit Mycobacterial Growth by Depleting ATP Levels.
O'Malley, Theresa; Alling, Torey; Early, Julie V; Wescott, Heather A; Kumar, Anuradha; Moraski, Garrett C; Miller, Marvin J; Masquelin, Thierry; Hipskind, Philip A; Parish, Tanya
2018-06-01
The imidazopyridines are a promising new class of antitubercular agents with potent activity in vitro and in vivo We isolated mutants of Mycobacterium tuberculosis resistant to a representative imidazopyridine; the mutants had large shifts (>20-fold) in MIC. Whole-genome sequencing revealed mutations in Rv1339, a hypothetical protein of unknown function. We isolated mutants resistant to three further compounds from the series; resistant mutants isolated from two of the compounds had single nucleotide polymorphisms in Rv1339 and resistant mutants isolated from the third compound had single nucleotide polymorphisms in QcrB, the proposed target for the series. All the strains were resistant to two compounds, regardless of the mutation, and a strain carrying the QcrB T313I mutation was resistant to all of the imidazopyridine derivatives tested, confirming cross-resistance. By monitoring pH homeostasis and ATP generation, we confirmed that compounds from the series were targeting QcrB; imidazopyridines disrupted pH homeostasis and depleted ATP, providing further evidence of an effect on the electron transport chain. A representative compound was bacteriostatic against replicating bacteria, consistent with a mode of action against QcrB. The series had a narrow inhibitory spectrum, with no activity against other bacterial species. No synergy or antagonism was seen with other antituberculosis drugs under development. In conclusion, our data support the hypothesis that the imidazopyridine series functions by reducing ATP generation via inhibition of QcrB. Copyright © 2018 O'Malley et al.
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Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis
Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J.; Xiang, Cheng-Bin
2010-01-01
Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a ‘sulphur-free’ solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance. PMID:20547563
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A new type of gene-disruption cassette with a rescue gene for Pichia pastoris.
Shibui, Tatsuro; Hara, Hiroyoshi
2017-09-01
Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017. © 2017 American Institute of Chemical Engineers.
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Vogel, G; Thilo, L; Schwarz, H; Steinhart, R
1980-01-01
The recognition step in the phagocytotic process of the unicellular amoeba dictyostelium discoideum was examined by analysis of mutants defective in phagocytosis, Reliable and simple assays were developed to measure endocytotic uptake. For pinocytosis, FITC-dextran was found to be a suitable fluid-phase marker; FITC-bacteria, latex beads, and erythrocytes were used as phagocytotic substrates. Ingested material was isolated in one step by centrifuging through highly viscous poly(ethyleneglycol) solutions and was analyzed optically. A selection procedure for isolating mutants defective in phagocytosis was devised using tungsten beads as particulate prey. Nonphagocytosing cells were isolated on the basis of their lower density. Three mutant strains were found exhibiting a clear-cut phenotype directly related to the phagocytotic event. In contrast to the situation in wild-type cells, uptake of E. coli B/r by mutant cells is specifically and competitively inhibited by glucose. Mutant amoeba phagocytose latex beads normally but not protein-coated latex, nonglucosylated bacteria, or erythrocytes. Cohesive properties of mutant cells are altered: they do not form EDTA-sensitive aggregates, and adhesiveness to glass or plastic surfaces is greatly reduced. Based upon these findings, a model for recognition in phagocytosis is proposed: (a) A lectin-type receptor specifically mediates binding of particles containing terminal glucose (E. coli B/r). (b) A second class of "nonspecific" receptors mediate binding of a variety of particles by hydrophobic interaction. Nonspecific binding is affected by mutation in such a way that only strongly hydrophobic (latex) but not more hydrophilic particles (e.g., protein-coated latex, bacteria, erythrocytes) can be phagocytosed by mutant amoebae. PMID:6995464
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Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis.
Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J; Xiang, Cheng-Bin
2010-07-01
Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a 'sulphur-free' solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance.
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Ando, Hiroki; Kondo, Yuji; Suetake, Toshinori; Toyota, Emiko; Kato, Seiya; Mori, Toru; Kirikae, Teruo
2010-01-01
Isoniazid (INH) is an effective first-line antituberculosis drug. KatG, a catalase-peroxidase, converts INH to an active form in Mycobacterium tuberculosis, and katG mutations are major causes of INH resistance. In the present study, we sequenced katG of 108 INH-resistant M. tuberculosis clinical isolates. Consequently, 9 novel KatG mutants with a single-amino-acid substitution were found. All of these mutants had significantly lower INH oxidase activities than the wild type, and each mutant showed various levels of activity. Isolates having mutations with relatively low activities showed high-level INH resistance. On the basis of our results and known mutations associated with INH resistance, we developed a new hybridization-based line probe assay for rapid detection of INH-resistant M. tuberculosis isolates. PMID:20211896
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Caws, Maxine; Tho, Dau Quang; Duy, Phan Minh; Lan, Nguyen Thi Ngoc; Hoa, Dai Viet; Torok, Mili Estee; Chau, Tran Thi Hong; Van Vinh Chau, Nguyen; Chinh, Nguyen Tran; Farrar, Jeremy
2007-01-01
PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates. PMID:17428939
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Genetic Analysis of Collective Motility of Paenibacillus sp. NAIST15-1
Kobayashi, Kazuo; Kanesaki, Yu
2016-01-01
Bacteria have developed various motility mechanisms to adapt to a variety of solid surfaces. A rhizosphere isolate, Paenibacillus sp. NAIST15-1, exhibited unusual motility behavior. When spotted onto 1.5% agar media, Paenibacillus sp. formed many colonies, each of which moved around actively at a speed of 3.6 μm/sec. As their density increased, each moving colony began to spiral, finally forming a static round colony. Despite its unusual motility behavior, draft genome sequencing revealed that both the composition and organization of flagellar genes in Paenibacillus sp. were very similar to those in Bacillus subtilis. Disruption of flagellar genes and flagellar stator operons resulted in loss of motility. Paenibacillus sp. showed increased transcription of flagellar genes and hyperflagellation on hard agar media. Thus, increased flagella and their rotation drive Paenibacillus sp. motility. We also identified a large extracellular protein, CmoA, which is conserved only in several Paenibacillus and related species. A cmoA mutant could neither form moving colonies nor move on hard agar media; however, motility was restored by exogenous CmoA. CmoA was located around cells and enveloped cell clusters. Comparison of cellular behavior between the wild type and cmoA mutant indicated that extracellular CmoA is involved in drawing water out of agar media and/or smoothing the cell surface interface. This function of CmoA probably enables Paenibacillus sp. to move on hard agar media. PMID:27764113
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McGrann, Graham R. D.; Steed, , Andrew; Burt, Christopher; Nicholson, Paul; Brown, James K. M.
2015-01-01
Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. PMID:25873675
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Wang, G; Rahman, M S; Humayun, M Z; Taylor, D E
1999-03-01
Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.
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Wang, Ge; Rahman, M. Sayeedur; Humayun, M. Zafri; Taylor, Diane E.
1999-01-01
Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates. PMID:10049289
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Suppression of mutants aberrant in light intensity responses of complementary chromatic adaptation.
Casey, E S; Kehoe, D M; Grossman, A R
1997-01-01
Complementary chromatic adaptation is a process in which cyanobacteria alter the pigment protein (phycocyanin and phycoerythrin) composition of their light-harvesting complexes, the phycobilisomes, to help optimize the absorbance of prevalent wavelengths of light in the environment. Several classes of mutants that display aberrant complementary chromatic adaptation have been isolated. One of the mutant classes, designated "blue" or FdB, accumulates high levels of the blue chromoprotein phycocyanin in low-intensity green light, a condition that normally suppresses phycocyanin synthesis. We demonstrate here that the synthesis of the phycocyanin protein and mRNA in the FdB mutants can be suppressed by increasing the intensity of green light. Hence, these mutants have a decreased sensitivity to green light with respect to suppression of phycocyanin synthesis. Although we were unable to complement the blue mutants, we did isolate genes that could suppress the mutant phenotype. These genes, which have been identified previously, encode a histidine kinase sensor and response regulator protein that play key roles in controlling complementary chromatic adaptation. These findings are discussed with respect to the mechanism by which light quality and quantity control the biosynthesis of the phycobilisome. PMID:9226271
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An efficient screen for peroxisome-deficient mutants of Pichia pastoris.
Liu, H; Tan, X; Veenhuis, M; McCollum, D; Cregg, J M
1992-01-01
We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis. Images PMID:1629154
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HTLV-1 Tax Effects on Cellular Mitotic Regulation
2007-04-12
39 2 MutagenesisTax mutagenesis and selection for tax mutants...43 Isolation of tax mutants that do not cause growth arrest in S. cerevisiae. .......... 43 Tax mutants selected in W303a are functional in...114). One currently-approved therapeutic agent , Indinavir, is an effective inhibitor of both proteases. New protease inhibitors are currently being
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Isolation and characterization of OmpC porin mutants with altered pore properties
DOE Office of Scientific and Technical Information (OSTI.GOV)
Misra, R.; Benson, S.A.
1988-02-01
The LamB protien is normally required for the uptake of maltodextrins. Starting with a LamB/sup -/ OmpF/sup -/ strain, we have isolated mutants that will grow on maltodextrins. The mutation conferring the Dex/sup +/ phenotype in the majority of these mutants has been mapped to the ompC locus. These mutants, unlike LamB/sup -/ OmpF/sup -/ strains, grew on maltotriose and maltotetraose, but not on maltopentaose, and showed a significantly higher rate of (/sup 14/C) maltose uptake than the parent strain did. In addition, these mutants showed increased sensitivity to certain ..beta..-lactam antibiotics and sodium dodecyl sulfate, but did not exhibitmore » an increase in sensitivity to other antibiotics and detergents. The nucleotide sequence of these mutants has been determined. In all cases, residue 74 (arginine) of the mature OmpC protein was affected. The results suggest that this region of the OmpC protein is involved in the pore domain and that the alterations lead to an increased pore size.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Dantzig, A.H.; Slayman, C.W.; Adelberg, E.A.
A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of (/sup 3/H)proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increasemore » in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment.« less
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Regulatory Mutants at the his1 Locus of Yeast
Lax, Carol; Fogel, Seymour; Cramer, Carole
1979-01-01
The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1–7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auoxtrophic parental alleles. The double mutants, HIS1–7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1–7 background, the mutant regulatory site (HIS1–e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine. PMID:385447
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Refinetti, Paulo; Arstad, Christian; Thilly, William G; Morgenthaler, Stephan; Ekstrøm, Per Olaf
2017-01-01
The growth of tumor cells is accompanied by mutations in nuclear and mitochondrial genomes creating marked genetic heterogeneity. Tumors also contain non-tumor cells of various origins. An observed somatic mitochondrial mutation would have occurred in a founding cell and spread through cell division. Micro-anatomical dissection of a tumor coupled with assays for mitochondrial point mutations permits new insights into this growth process. More generally, the ability to detect and trace, at a histological level, somatic mitochondrial mutations in human tissues and tumors, makes these mutations into markers for lineage tracing. A tumor was first sampled by a large punch biopsy and scanned for any significant degree of heteroplasmy in a set of sequences containing known mutational hotspots of the mitochondrial genome. A heteroplasmic tumor was sliced at a 12 μm thickness and placed on membranes. Laser capture micro-dissection was used to take 25000 μm 2 subsamples or spots. After DNA amplification, cycling temperature capillary electrophoresis (CTCE) was used on the laser captured samples to quantify mitochondrial mutant fractions. Of six testicular tumors studied, one, a Leydig tumor, was discovered to carry a detectable degree of heteroplasmy for two separate point mutations: a C → T mutation at bp 64 and a T → C mutation found at bp 152. From this tumor, 381 spots were sampled with laser capture micro-dissection. The ordered distribution of spots exhibited a wide range of fractions of the mutant sequences from 0 to 100% mutant copies. The two mutations co-distributed in the growing tumor indicating they were present on the same genome copies in the founding cell. Laser capture microdissection of sliced tumor samples coupled with CTCE-based point mutation assays provides an effective and practical means to obtain maps of mitochondrial mutational heteroplasmy within human tumors.
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SHARIFI-SARASIABI, Khojasteh; HAGHIGHI, Ali; KAZEMI, Bahram; TAGHIPOUR, Niloofar; MOJARAD, Ehsan Nazemalhosseini; GACHKAR, Latif
2016-01-01
In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment. PMID:27007559
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Phytotoxic properties of cnicin, a sesquiterpene lactone fromcentaurea maculosa (spotted knapweed).
Kelsey, R G; Locken, L J
1987-01-01
Water and solvent extracts from the aerial tissues ofCentaurea maculosa, spotted knapweed, inhibited the root growth of lettuce. Column chromatography and lettuce bioassay of a chloroform extract led to the isolation of cnicin, a sesquiterpene lactone. Pure cnicin was bioassayed at 0, 1, 2, 4, 6, 8, and 10 mg/5 ml water with lettuce, created wheatgrass, bluebunch wheatgrass, rough fescue, western larch, lodgepole pine, and spotted knapweed. Germination was inhibited at one or more concentrations for all species except lodgepole pine and spotted knapweed. Growth, particularly of the roots, was retarded between 1 and 4 mg of cnicin. Lettuce, bluebunch wheatgrass, and spotted knapweed were inhibited significantly at all concentrations tested.
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Analysis of the intergenic region of tomato spotted wilt Tospovirus medium RNA segment.
Bhat, A I; Pappu, S S; Pappu, H R; Deom, C M; Culbreath, A K
1999-06-01
The intergenic region (IGR) of the medium (M) RNA of tomato spotted wilt Tospovirus (TSWV) isolates naturally infecting peanut (groundnut), pepper, potato, stokesia, tobacco and watermelon in Georgia (GA) and a peanut isolate from Florida (FL) was cloned and sequenced. The IGR sequences were compared with one another and with respective M RNA IGRs of TSWV isolates from Brazil and Japan and other tospoviruses. The length of M IGR of GA and FL isolates varied from 271 to 277 nucleotides. The M IGRs of TSWV from potato and stokesia, and tobacco and watermelon were identical with each other in their length and sequence. IGR sequences were more conserved (95-100%) among the populations of TSWV from GA and FL, than when compared with those of TSWV isolates from other countries (83-94%). The conserved motif (CAAACTTTGG) present in the IGRs of both M and small (S) RNAs of a Brazilian isolate of TSWV was also conserved in the isolates studied. Cluster analysis of the IGR sequences showed that all GA and FL isolates are closely clustered and are distinct from the TSWV isolates from other countries as well as from other tospoviruses.
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Mutants of Neurospora crassa that alter gene expression and conidia development.
Madi, L; Ebbole, D J; White, B T; Yanofsky, C
1994-01-01
Several genes have been identified that are highly expressed during conidiation. Inactivation of these genes has no observable phenotypic effect. Transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. To identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. Mutants were then selected that were resistant to the drug during mycelial growth. Mutations in several of the isolates had trans effects; they activated transcription of the corresponding intact gene and, in most isolates, one or more of the other con genes. Most interestingly, resistant mutants were obtained that were defective at different stages of conidiation. One mutant conidiated under conditions that do not permit conidiation in wild type. Images PMID:8016143
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Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing
Lepage, Étienne; Zampini, Éric; Boyle, Brian; Brisson, Normand
2013-01-01
Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity. PMID:23951038
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Li, Yafei; Zhang, Yanan; Ding, Huanzhong; Mei, Xian; Liu, Wei; Zeng, Jiaxiong; Zeng, Zhenling
2016-11-09
Mutant prevention concentration (MPC) is an alternative pharmacodynamic parameter that has been used to measure antimicrobial activity and represents the propensities of antimicrobial agents to select resistant mutants. The concentration range between minimum inhibitory concentration (MIC) and MPC is defined as mutant selection window (MSW). The MPC and MSW parameters represent the ability of antimicrobial agents to inhibit the bacterial mutants selected. This study was conducted to determine the MIC and MPC values of four antimicrobials including ceftiofur, cefquinome, florfenicol and tilmicosin against 105 Riemerella anatipestifer isolates. The MIC 50 /MIC 90 values of clinical isolates tested in our study for ceftiofur, cefquinome, florfenicol and tilmicosin were 0.063/0.5、0.031/0.5、1/4、1/4 μg/mL, respectively; MPC 50 / MPC 90 values were 4/64、8/64、4/32、16/256 μg/mL, respectively. These results provided information on the use of these compounds in treating the R. anatipestifer infection; however, additional studies are needed to demonstrate their therapeutic efficacy. Based on the MSW theory, the hierarchy of these tested antimicrobial agents with respect to selecting resistant subpopulations was as follows: cefquinome > ceftiofur > tilmicosin > florfenicol. Cefquinome was the drug that presented the highest risk of selecting resistant mutant among the four antimicrobial agents.
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Degli-Innocenti, F; Russo, V E
1984-01-01
White collar (wc) mutants of Neurospora crassa are thought to be regulatory mutants blocked in the photoinduction of carotenogenesis. Eight new wc mutants have been isolated after UV mutagenesis; their morphology and linear growth rate are not altered, although blue light-induced carotenogenesis is completely blocked. All of the wc mutations fall into two complementation groups corresponding to the already-known wc-1 and wc-2 loci. It is shown that the wc mutations impair another blue light effect, the photoinduction of protoperithecia formation, as well as the low constitutive production of protoperithecia in the dark. These effects are not due to the lack of carotenoids since the albino mutants show a normal sexual development. The pleiotropic effects of the mutations in the wc genes indicate that they play a key role in the mechanisms of regulation of the blue light-induced responses of N. crassa. PMID:6235211
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Alternaria leaf spot in Michigan and fungicide sensitivity issues
USDA-ARS?s Scientific Manuscript database
Since 2010 there has been an increase in identification of Alternaria leaf spot on sugar beet in Michigan and other growing regions in the US and Canada. In 2016, the disease was severe enough to cause economic losses in the Michigan growing region. Michigan isolates from sugar beet were examined ...
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Reaction of the BASE 120 lines to angular leaf spot in Puerto Rico
USDA-ARS?s Scientific Manuscript database
Common bean (Phaseolus vulgaris L.) is limited by diseases such as Angular leaf spot (ALS), caused by Phaeoisariopsis griseola (Sacc.) Ferraris sin. Pseudocercospora griseola (Sacc.) Crous & U. Braun. The virulence of Phaeoisariopis griseola isolate ALS-9029-JD2 from Juana Diaz, PR was determined by...
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Stemphylium leaf spot of parsley in California caused by Stemphylium vesicarium
USDA-ARS?s Scientific Manuscript database
From 2009 through 2011, a previously undescribed disease occurred on commercial parsley in coastal (Ventura County) California. Symptoms of the disease consisted of circular to oval, tan to brown leaf spots and resulted in loss of crop quality and hence reduced yields. A fungus was consistently isol...
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Dublan, Maria de los Angeles; Ortiz-Marquez, Juan Cesar Federico; Lett, Lina; Curatti, Leonardo
2014-01-01
Background Escherichia coli is a widespread gut commensal and often a versatile pathogen of public health concern. E. coli are also frequently found in different environments and/or alternative secondary hosts, such as plant tissues. The lifestyle of E. coli in plants is poorly understood and has potential implications for food safety. Methods/Principal Findings This work shows that a human commensal strain of E. coli K12 readily colonizes lettuce seedlings and produces large microcolony-like cell aggregates in leaves, especially in young leaves, in proximity to the vascular tissue. Our observations strongly suggest that those cell aggregates arise from multiplication of single bacterial cells that reach those spots. We showed that E. coli isolated from colonized leaves progressively colonize lettuce seedlings to higher titers, suggesting a fast adaptation process. E. coli cells isolated from leaves presented a dramatic rise in tolerance to oxidative stress and became more chemotactic responsive towards lettuce leaf extracts. Mutant strains impaired in their chemotactic response were less efficient lettuce colonizers than the chemotactic isogenic strain. However, acclimation to oxidative stress and/or minimal medium alone failed to prime E. coli cells for enhanced lettuce colonization efficiency. Conclusion/Significance These findings help to understand the physiological adaptation during the alternative lifestyle of E. coli in/on plant tissues. PMID:25313845
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Benomyl-resistant mutant strain of Trichoderma sp. with increased mycoparasitic activity.
Olejníková, P; Ondrusová, Z; Krystofová, S; Hudecová, D
2010-01-01
Application of UV radiation to the strain Trichoderma sp. T-bt (isolated from lignite) resulted in the T-brm mutant which was resistant to the systemic fungicide benomyl. The tub2 gene sequence in the T-brm mutant differed from the parent as well as the collection strain (replacing tyrosine with histidine in the TUB2 protein). Under in vitro conditions this mutant exhibited a higher mycoparasitic activity toward phytopathogenic fungi.
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Sign language spotting with a threshold model based on conditional random fields.
Yang, Hee-Deok; Sclaroff, Stan; Lee, Seong-Whan
2009-07-01
Sign language spotting is the task of detecting and recognizing signs in a signed utterance, in a set vocabulary. The difficulty of sign language spotting is that instances of signs vary in both motion and appearance. Moreover, signs appear within a continuous gesture stream, interspersed with transitional movements between signs in a vocabulary and nonsign patterns (which include out-of-vocabulary signs, epentheses, and other movements that do not correspond to signs). In this paper, a novel method for designing threshold models in a conditional random field (CRF) model is proposed which performs an adaptive threshold for distinguishing between signs in a vocabulary and nonsign patterns. A short-sign detector, a hand appearance-based sign verification method, and a subsign reasoning method are included to further improve sign language spotting accuracy. Experiments demonstrate that our system can spot signs from continuous data with an 87.0 percent spotting rate and can recognize signs from isolated data with a 93.5 percent recognition rate versus 73.5 percent and 85.4 percent, respectively, for CRFs without a threshold model, short-sign detection, subsign reasoning, and hand appearance-based sign verification. Our system can also achieve a 15.0 percent sign error rate (SER) from continuous data and a 6.4 percent SER from isolated data versus 76.2 percent and 14.5 percent, respectively, for conventional CRFs.
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Wechsler, Cindy; Meyer, Danilo; Loschonsky, Sabrina; Funk, Lisa-Marie; Neumann, Piotr; Ficner, Ralf; Brodhun, Florian; Müller, Michael; Tittmann, Kai
2015-12-01
Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild-type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism-inspired and structure-guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Mutagenesis in human cells with accelerated H and Fe ions
NASA Technical Reports Server (NTRS)
Kronenberg, Amy
1994-01-01
The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants.
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Singh, Shweta; Fatima, Zeeshan; Hameed, Saif
2016-07-01
The anticandidal potential of Geraniol (Ger) against Candida albicans has already been established. The present study reveals deeper insights into the mechanisms of action of Ger. We observed that the repertoire of antifungal activity was not only limited to C. albicans and its clinical isolates but also against non-albicans species of Candida. The membrane tampering effect was visualized through transmission electron micrographs, depleted ergosterol levels and altered plasma membrane ATPase activity. Ger also affects cell wall as revealed by spot assays with cell wall-perturbing agents and scanning electron micrographs. Functional calcineurin pathway seems to be indispensable for the antifungal effect of Ger as calcineurin signaling mutant was hypersensitive to Ger while calcineurin overexpressing strain remained resistant. Ger also causes mitochondrial dysfunction, impaired iron homeostasis and genotoxicity. Furthermore, Ger inhibits both virulence attributes of hyphal morphogenesis and biofilm formation. Taken together, our results suggest that Ger is potential antifungal agent that warrants further investigation in clinical applications so that it could be competently employed in therapeutic strategies to treat Candida infections.
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Du, Yu; Xie, Guizhen; Yang, Chunfa; Fang, Baishan; Chen, Hongwen
2014-06-01
pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
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García, Katherine; Gavilán, Ronnie G.; Höfle, Manfred G.; Martínez-Urtaza, Jaime; Espejo, Romilio T.
2012-01-01
The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10−4 mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered. PMID:22292049
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Tolerance of an Antarctic Bacterium to Multiple Environmental Stressors.
Sengupta, Dipanwita; Sangu, Kavya; Shivaji, Sisinthy; Chattopadhyay, Madhab K
2015-10-01
A population of cold-tolerant Antarctic bacteria was screened for their ability to tolerate other environmental stress factors. Besides low temperature, they were predominantly found to be tolerant to alkali. Attempt was also made to postulate a genetic basis of their multistress-tolerance. Transposon mutagenesis of an isolate Pseudomonas syringae Lz4W was performed, and mutants with delayed growth at low temperature were further screened for sensitivity to some other stress factors. A number of multistress-sensitive mutants were isolated. The mutated gene in one of the mutants sensitive to low temperature, acid and alkali was found to encode citrate synthase. Possible role of citrate synthase in conferring multistress-tolerance was postulated.
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A Candida guilliermondii lysine hyperproducer capable of elevated citric acid production.
West, Thomas P
2016-05-01
A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Guard-Petter, J.; Parker, C.T.; Asokan, K.
1999-05-01
Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and themore » O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, the authors analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.« less
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Inverse gene-for-gene interactions contribute additively to tan spot susceptibility in wheat.
Liu, Zhaohui; Zurn, Jason D; Kariyawasam, Gayan; Faris, Justin D; Shi, Gongjun; Hansen, Jana; Rasmussen, Jack B; Acevedo, Maricelis
2017-06-01
Tan spot susceptibility is conferred by multiple interactions of necrotrophic effector and host sensitivity genes. Tan spot of wheat, caused by Pyrenophora tritici-repentis, is an important disease in almost all wheat-growing areas of the world. The disease system is known to involve at least three fungal-produced necrotrophic effectors (NEs) that interact with the corresponding host sensitivity (S) genes in an inverse gene-for-gene manner to induce disease. However, it is unknown if the effects of these NE-S gene interactions contribute additively to the development of tan spot. In this work, we conducted disease evaluations using different races and quantitative trait loci (QTL) analysis in a wheat recombinant inbred line (RIL) population derived from a cross between two susceptible genotypes, LMPG-6 and PI 626573. The two parental lines each harbored a single known NE sensitivity gene with LMPG-6 having the Ptr ToxC sensitivity gene Tsc1 and PI 626573 having the Ptr ToxA sensitivity gene Tsn1. Transgressive segregation was observed in the population for all races. QTL mapping revealed that both loci (Tsn1 and Tsc1) were significantly associated with susceptibility to race 1 isolates, which produce both Ptr ToxA and Ptr ToxC, and the two genes contributed additively to tan spot susceptibility. For isolates of races 2 and 3, which produce only Ptr ToxA and Ptr ToxC, only Tsn1 and Tsc1 were associated with tan spot susceptibility, respectively. This work clearly demonstrates that tan spot susceptibility in this population is due primarily to two NE-S interactions. Breeders should remove both sensitivity genes from wheat lines to obtain high levels of tan spot resistance.
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Snelders, Eveline; Zwaan, Bas J.; Schoustra, Sijmen E.; van Dijk, Karin; Hagen, Ferry; van der Beek, Martha T.; Kampinga, Greetje A.; Zoll, Jan; Melchers, Willem J. G.; Verweij, Paul E.; Debets, Alfons J. M.
2017-01-01
ABSTRACT This study investigated the dynamics of Aspergillus fumigatus azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR46/Y121F/T289A genotype, 2% (1/45) harbored the TR46/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR463/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR463 mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR46 repeats. Importantly, in the laboratory, we recovered the TR463 mutation from a sexual cross between two TR46 isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases. PMID:28655821
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NASA Technical Reports Server (NTRS)
Vogel, J. P.; Schuerman, P.; Woeste, K.; Brandstatter, I.; Kieber, J. J.; Evans, M. L. (Principal Investigator)
1998-01-01
Cytokinins elevate ethylene biosynthesis in etiolated Arabidopsis seedlings via a post-transcriptional modification of one isoform of the key biosynthetic enzyme ACC synthase. In order to begin to dissect the signaling events leading from cytokinin perception to this modification, we have isolated a series of mutants that lack the ethylene-mediated triple response in the presence of cytokinin due to their failure to increase ethylene biosynthesis. Analysis of genetic complementation and mapping revealed that these Cin mutants (cytokinin-insensitive) represent four distinct complementation groups, one of which, cin4, is allelic to the constitutive photomorphogenic mutant fus9/cop10. The Cin mutants have subtle effects on the morphology of adult plants. We further characterized the Cin mutants by analyzing ethylene biosynthesis in response to various other inducers and in adult tissues, as well as by assaying additional cytokinin responses. The cin3 mutant did not disrupt ethylene biosynthesis under any other conditions, nor did it disrupt any other cytokinin responses. Only cin2 disrupted ethylene biosynthesis in multiple circumstances. cin1 and cin2 made less anthocyanin in response to cytokinin. cin1 also displayed reduced shoot initiation in tissue culture in response to cytokinin, suggesting that it affects a cytokinin signaling element.
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Vigeolas, Hélène; Duby, Francéline; Kaymak, Esra; Niessen, Guillaume; Motte, Patrick; Franck, Fabrice; Remacle, Claire
2012-11-30
This paper describes the isolation and partial biomass characterization of high triacylglycerol (TAG) mutants of Chlorella sorokiniana and Scenedesmus obliquus, two algal species considered as potential source of biodiesel. Following UV mutagenesis, 2000 Chlorella and 2800 Scenedesmus colonies were screened with a method based on Nile Red fluorescence. Several mutants with high Nile Red fluorescence were selected by this high-throughput method in both species. Growth and biomass parameters of the strongest mutants were analyzed in detail. All of the four Chlorella mutants showed no significant changes in growth rate, cell weight, cell size, protein and chlorophyll contents on a per cell basis. Whereas all contained elevated total lipid and TAG content per unit of dry weight, two of them were also affected for starch metabolism, suggesting a change in biomass/storage carbohydrate composition. Two Scenedesmus mutants showed a 1.5 and 2-fold increased cell weight and larger cells compared to the wild type, which led to a general increase of biomass including total lipid and TAG content on a per cell basis. Such mutants could subsequently be used as commercial oleaginous algae and serve as an alternative to conventional petrol. Copyright © 2012 Elsevier B.V. All rights reserved.
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Autolytic defective mutant of Streptococcus faecalis.
Cornett, J B; Redman, B E; Shockman, G D
1978-01-01
Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme. PMID:415045
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FTIR Spectroscopy of Protein Isolates of Salt-Tolerant Soybean Mutants
NASA Astrophysics Data System (ADS)
Akyuz, S.; Akyuz, T.; Celik, O.; Atak, C.
2018-01-01
The effect of salinity on the conformation of proteins of four salt-tolerant M2 generation mutants of soybean plants (S04-05/150-2, S04-05/150-8, S04-05/150-106, and S04-05/150-114) was investigated using Fourier transform infrared (FTIR) spectroscopy. Salinity is one of the important abiotic stress factors that limits growth and productivity of plants. The mutants belonging to the M2 generation were determined as tolerant to 90 mM NaCl. The relative contents of α-helix, β-sheet, turn, and irregular conformations for the soybean protein isolates were determined depending on the analysis of the amide I region. The comparison of the secondary structures of soybean proteins of the mutants with those of the control group indicated that the α-helix structure percentage was diminished while β-turn and disordered structures were increased as a result of the salt stress.
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RESISTANCE AND CROSS-RESISTANCE OF ESCHERICHIA COLI S MUTANTS TO THE RADIOMIMETIC AGENT PROFLAVINE
Woody-Karrer, Pearl; Greenberg, Joseph
1964-01-01
Woody-Karrer, Pearl (Palo Alto Medical Research Foundation, Palo Alto, Calif.), and Joseph Greenberg. Resistance and cross-resistance of Escherichia coli S mutants to the radiomimetic agent proflavine. J. Bacteriol. 87:536–542. 1964.—All 50 of the first-step mutants of Escherichia coli S selected for resistance to proflavine were resistant to ultraviolet light and each of five different radiomimetic chemicals. The mutants were classified into eight types on the basis of their relative resistance to six different radiomimetic drugs and on the basis of the shape of their ultraviolet survival curves. Three of these types are identical to types previously isolated with other radiomimetic drugs; five of the types are new. A high proportion of the clones surviving proflavine treatment were phenotypically but not genetically resistant, and no strains were isolated which were resistant to proflavine but were not resistant to radiation. PMID:14129667
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USDA-ARS?s Scientific Manuscript database
Cercospora leaf spot caused by Cercospora beticola is the major foliar disease effecting sugar beet (Beta vulgaris L.) production in North Dakota and Minnesota. The sterol demethylation inhibitor (DMI) fungicide tetraconazole is widely-used to manage Cercospora leaf spot. However, there has been an ...
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Characterization of two biologically distinct variants of Tomato spotted wilt virus
USDA-ARS?s Scientific Manuscript database
Significant economic losses result on a wide range of crops due to infection with Tomato spotted wilt virus (TSWV). In this study, two TSWV isolates, one from basil and a second from tomato, were established in a common plant host. Viral proteins were monitored over time, plant host ranges were comp...
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USDA-ARS?s Scientific Manuscript database
Cercospora beticola causes Cercospora leaf spot of sugarbeet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to th...
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Molecular genetic analysis of plant gravitropism
NASA Technical Reports Server (NTRS)
Lomax, T. L.
1997-01-01
The analysis of mutants is a powerful approach for elucidating the components of complex biological processes. A growing number of mutants have been isolated which affect plant gravitropism and the classes of mutants found thus far provide important information about the gravity response mechanism. The wide variety of mutants isolated, especially in Arabidopsis, indicates that gravitropism is a complex, multi-step process. The existence of mutants altered in either root gravitropism alone, shoot gravitropism alone, or both indicates that the root and shoot gravitropic mechanisms have both separate and common steps. Reduced starch mutants have confirmed the role of amyloplasts in sensing the gravity signal. The hormone auxin is thought to act as the transducing signal between the sites of gravity perception (the starch parenchyma cells surrounding the vascular tissue in shoots and the columella cells of root caps) and asymmetric growth (the epidermal cells of the elongation zone(s) of each organ). To date, all mutants that are resistant to high concentrations of auxin have also been found to exhibit a reduced gravitropic response, thus supporting the role of auxin. Not all gravitropic mutants are auxin-resistant, however, indicating that there are additional steps which do not involve auxin. Studies with mutants of tomato which exhibit either reduced or reversed gravitropic responses further support the role of auxin redistribution in gravitropism and suggest that both red light and cytokinin interact with gravitropism through controlling lateral auxin transport. Plant responses to gravity thus likely involve changes in both auxin transport and sensitivity.
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Ali, Sikander; Nawaz, Wajeeha
2016-08-01
The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 μg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 μg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 μg/ml) than did wild-type I-2 (21 μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.
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Nicholson, Tracy L.; Brockmeier, Susan L.; Loving, Crystal L.
2009-01-01
Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract. PMID:19237531
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Petit-spot geology reveals melts in upper-most asthenosphere dragged by lithosphere
NASA Astrophysics Data System (ADS)
Machida, Shiki; Hirano, Naoto; Sumino, Hirochika; Hirata, Takafumi; Yoneda, Shigekazu; Kato, Yasuhiro
2015-09-01
Petit-spot volcanism is a phenomenon ubiquitous on Earth. It originates from melt in the upper-most mantle asthenosphere, occurring where the plate flexes and fractures before subduction. Recent geochemical and petrological studies of petit-spot volcanism lava have shown that understanding this form of volcanism can contribute to the investigation of mantle dynamics and CO2 degassing of Earth. However, geological information constraining the magma source of petit-spot remains limited. Here, we present a comprehensive dataset of geochemistry (major and trace elements, and Sr and Nd isotopic compositions) and 40Ar/39Ar ages of alkaline basaltic rocks and glasses to define the geological characteristics of petit-spot volcanoes in the northwestern Pacific. The geochemical and geochronological variations of the basalts indicate that petit-spot volcanism is characterized by a petrogenetically and temporally isolated magma system for each volcano. The basalt geochemistry further indicates that the magmas at the volcanoes were derived from the melting of a heterogeneous regional-scale source under a range of conditions. In addition, slight temporal intra-field migration of petit-spot vent fields against the plate motion was detected. These features indicate that the magma originates from isolated melt ponds at the lithosphere-asthenosphere boundary, and that the speed at which the melt ponds are dragged by the plate is only slightly slower than that of the plate motion. Our results provide detailed insight into eruption processes of asthenosphere melts induced by plate-flexure, and also suggest the complete coupling of the lithosphere to the upper-most asthenosphere in the case of large plate subduction.
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Kattah, Andrea; Milic, Natasa; White, Wendy; Garovic, Vesna
2017-10-01
We performed a prospective, longitudinal study of pregnant women presenting to their first obstetrics visits to characterize the changes in spot urine protein-to-creatinine (UPCR) and albumin-to-creatinine ratios (UACR) in normotensive pregnancies, as well as identify clinical characteristics associated with isolated proteinuria and preeclampsia. We measured spot urinary albumin, protein, and creatinine at the first prenatal visit, end of the second trimester, and at delivery. In the normotensive pregnancies ( n = 142), we found that from the beginning of pregnancy to delivery, UACR increased by a median [interquartile range (IQR)] of 14.7 mg/g Cr (3.74-51.8) and UPCR by 60 mg/g Cr (30-130) ( P < 0.001 for both changes). Isolated proteinuria (defined as UPCR > 300 mg/g Cr in the absence of hypertension) was identified in 19/142 (13.4%) normotensive pregnancies. Increases in systolic and diastolic blood pressure from early pregnancy to delivery and increases in UACR from early to midpregnancy were associated with isolated proteinuria at delivery. Twelve women developed preeclampsia. Nulliparity, early, and midpregnancy diastolic blood pressures were strongly associated with the development of preeclampsia, but early changes in UACR were not. In conclusion, women who develop isolated proteinuria at delivery have a larger increase in blood pressure than women without proteinuria and have a "microalbuminuric" phase earlier in gestation, unlike women who develop preeclampsia. These findings suggest a different mechanism of urine protein excretion in women with isolated proteinuria as compared with women with preeclampsia, where proteinuria has a more abrupt onset. Copyright © 2017 the American Physiological Society.
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R Factor-Controlled Restriction and Modification of Deoxyribonucleic Acid: Restriction Mutants
Yoshimori, Robert; Roulland-Dussoix, Daisy; Boyer, Herbert W.
1972-01-01
Restriction mutants of two different R factor-controlled host specificities (RI and RII) were isolated. All of the restriction mutants examined had a normal modification phenotype. No complementation was observed between the RI and RII host specificities. It is concluded that for each host specificity no protein subunit is shared by the restriction endonuclease and modification methylase. PMID:4565538
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A Developmental Study of Photosystem I Peripheral Chlorophyll Proteins 1
Mullet, John E.; Burke, John J.; Arntzen, Charles J.
1980-01-01
An isolated “native” photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae (≃45 Chl/P700) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association. Chloroplasts were isolated from a Chl b-less barley mutant and from partially-developed cucumber cotyledons (greened under intermittent illumination [ImL] chloroplasts) and were compared to control chloroplasts isolated from wild-type barley and mature cucumber. Both the mutant and ImL chloroplasts exhibited a long wavelength fluorescence maximum at 724 nanometers at 77 K as compared to 735 to 738 nanometers emission maximum in the respective controls. Both the mutant and ImL chloroplasts were deficient in polypeptides of 21,500 to 24,500 daltons which were present in control membranes and in PSI fractions isolated from control membranes. In light-induced maturation of the ImL cucumbers, the synthesis of polypeptides in the 21,500 to 24,500 molecular weight range paralleled the appearance of PSI Chl species fluorescing at long wavelength (≃735 nm). The PSI spectral properties of the control membranes were retained in isolated PSI particles containing 100 to 120 Chl/P700 (PSI-110). Detergent extraction of PSI-110 removed polypeptides of 21,500 to 24,500 daltons plus ≃ 45 Chl/P700. The antennae-depleted PSI particle mimics PSI properties exhibited by incompletely differentiated mutant or ImL chloroplasts. Images PMID:16661289
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Jones, S; Yu, B; Bainton, N J; Birdsall, M; Bycroft, B W; Chhabra, S R; Cox, A J; Golby, P; Reeves, P J; Stephens, S
1993-01-01
Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes. Images PMID:8508773
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Miller, K W; Schamber, R; Osmanagaoglu, O; Ray, B
1998-06-01
A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.
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Pham, Quang Toan; Veillette, Maxime; Brandariz-Nuñez, Alberto; Pawlica, Paulina; Thibert-Lefebvre, Caroline; Chandonnet, Nadia; Diaz-Griffero, Felipe; Berthoux, Lionel
2013-05-01
Human-derived antiretroviral transgenes are of great biomedical interest and are actively pursued. HIV-1 is efficiently inhibited at post-entry, pre-integration replication stages by point mutations in the variable region 1 (v1) of the human restriction factor TRIM5α. Here we use a mutated megaprimer approach to create a mutant library of TRIM5αHu v1 and to isolate a mutation at Gly330 (G330E) that inhibits transduction of an HIV-1 vector as efficiently as the previously described mutants at positions Arg332 and Arg335. As was the case for these other mutations, modification of the local v1 charge toward increased acidity was key to inhibiting HIV-1. G330E TRIM5αHu also disrupted replication-competent HIV-1 propagation in a human T cell line. Interestingly, G330E did not enhance restriction of HIV-1 when combined with mutations at Arg332 or Arg335. Accordingly, the triple mutant G330E-R332G-R335G bound purified recombinant HIV-1 capsid tubes less efficiently than the double mutant R332G-R335G did. In a structural model of the TRIM5αHu PRYSPRY domain, the addition of G330E to the double mutant R332G-R335G caused extensive changes to the capsid-binding surface, which may explain why the triple mutant was no more restrictive than the double mutant. The HIV-1 inhibitory potential of Gly330 mutants was not predicted by examination of natural TRIM5α orthologs that are known to strongly inhibit HIV-1. This work underlines the potential of random mutagenesis to isolate novel variants of human proteins with antiviral properties. Copyright © 2013 Elsevier B.V. All rights reserved.
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Gopal, Pooja; Tasneen, Rokeya; Yee, Michelle; Lanoix, Jean-Philippe; Sarathy, Jansy; Rasic, George; Li, Liping; Dartois, Véronique; Nuermberger, Eric; Dick, Thomas
2017-07-14
Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.
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Grandin, Nathalie; Corset, Laetitia; Charbonneau, Michel
2012-01-01
Background In budding yeast, the highly conserved Tel2 protein is part of several complexes and its main function is now believed to be in the biogenesis of phosphatidyl inositol 3-kinase related kinases. Principal Findings To uncover potentially novel functions of Tel2, we set out to isolate temperature-sensitive (ts) mutant alleles of TEL2 in order to perform genetic screenings. MED15/GAL11, a subunit of Mediator, a general regulator of transcription, was isolated as a suppressor of these mutants. The isolated tel2 mutants exhibited a short telomere phenotype that was partially rescued by MED15/GAL11 overexpression. The tel2-15mutant was markedly deficient in the transcription of EST2, coding for the catalytic subunit of telomerase, potentially explaining the short telomere phenotype of this mutant. In parallel, a two-hybrid screen identified an association between Tel2 and Rvb2, a highly conserved member of the AAA+ family of ATPases further found by in vivo co-immunoprecipitation to be tight and constitutive. Transiently overproduced Tel2 and Med15/Gal11 associated together, suggesting a potential role for Tel2 in transcription. Other Mediator subunits, as well as SUA7/TFIIB, also rescued the tel2-ts mutants. Significance Altogether, the present data suggest the existence of a novel role for Tel2, namely in transcription, possibly in cooperation with Rvb2 and involving the existence of physical interactions with the Med15/Gal11 Mediator subunit. PMID:22291956
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Rhaese, H J; Hoch, J A; Groscurth, R
1977-03-01
To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.
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Isolation of Phaffia rhodozyma Mutants with Increased Astaxanthin Content
An, Gil-Hwan; Schuman, Donald B.; Johnson, Eric A.
1989-01-01
Plating of the astaxanthin-producing yeast Phaffia rhodozyma onto yeast-malt agar containing 50 μM antimycin A gave rise to colonies of unusual morphology, characterized by a nonpigmented lower smooth surface that developed highly pigmented vertical papillae after 1 to 2 months. Isolation and purification of the pigmented papillae, followed by testing for pigment production in shake flasks, demonstrated that several antimycin isolates were increased two- to fivefold in astaxanthin content compared with the parental natural isolate (UCD-FST 67-385). One of the antimycin strains (ant-1) and a nitrosoguanidine derivative of ant-1 (ant-1-4) produced considerably more astaxanthin than the parent (ant-1 had 800 to 900 μg/g; ant-1-4 had 900 to 1,300 μg/g; and 67-385 had 300 to 450 μg/g). The mutant strains were compared physiologically with the parent. The antimycin mutants grew slower on ammonia, glutamate, or glutamine as nitrogen sources compared with the natural isolate and also had lower cell yields on several carbon sources. Although isolated on antimycin plates, they were found to be more susceptible to antimycin A, apparently owing to the spatial separation of the papillae from the agar. They were also more susceptible than the parent to the respiratory inhibitor thenoyltrifluoroacetone and were slightly more susceptible to cyanide, but did not differ from the natural isolate in susceptibility to azide. The antimycin-derived strains were also killed faster than the parent by hydrogen peroxide. The carotenoid compositions of the parent and the antimycin-derived strains were similar to those previously determined in the type strain (UCD-FST 67-210) except that two carotenoids not previously found in the type strain were present in increased quantities in the antimycin mutants and phoenicoxanthin was a minor component. The chemical properties of the unknown carotenoids suggested that the strains isolated on antimycin agar tended to oxygenate and desaturate carotene precursors to a greater extent than the parent. The physiology of the antimycin isolates and the known specificity of antimycin for cytochrome b in the respiratory chain suggests that alteration of cytochrome b or cytochrome P-450 components involved in oxygenation and desaturation of carotenes in mitochondria are affected, which results in increased astaxanthin production. These astaxanthin-overproducing mutants and more highly pigmented derivative strains could be useful in providing a natural source of astaxanthin for the pen-reared-salmon industry or for other farmed animals that contain astaxanthin as their principal carotenoid. Images PMID:16347815
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Cushman, John C.; Agarie, Sakae; Albion, Rebecca L.; Elliot, Stewart M.; Taybi, Tahar; Borland, Anne M.
2008-01-01
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM. PMID:18326789
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Disturbance Impacts on Thermal Hot Spots and Hot Moments at the Peatland-Atmosphere Interface
NASA Astrophysics Data System (ADS)
Leonard, R. M.; Kettridge, N.; Devito, K. J.; Petrone, R. M.; Mendoza, C. A.; Waddington, J. M.; Krause, S.
2018-01-01
Soil-surface temperature acts as a master variable driving nonlinear terrestrial ecohydrological, biogeochemical, and micrometeorological processes, inducing short-lived or spatially isolated extremes across heterogeneous landscape surfaces. However, subcanopy soil-surface temperatures have been, to date, characterized through isolated, spatially discrete measurements. Using spatially complex forested northern peatlands as an exemplar ecosystem, we explore the high-resolution spatiotemporal thermal behavior of this critical interface and its response to disturbances by using Fiber-Optic Distributed Temperature Sensing. Soil-surface thermal patterning was identified from 1.9 million temperature measurements under undisturbed, trees removed and vascular subcanopy removed conditions. Removing layers of the structurally diverse vegetation canopy not only increased mean temperatures but it shifted the spatial and temporal distribution, range, and longevity of thermal hot spots and hot moments. We argue that linking hot spots and/or hot moments with spatially variable ecosystem processes and feedbacks is key for predicting ecosystem function and resilience.
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de Freitas, P L; Dias, A C S; Moreira, V R; Monteiro, S G; Pereira, S R F
2015-08-19
The mutagenic and antimutagenic activities of triterpene betulinic acid {3b-3-hydroxy-lup-20(29)-en-28-oic} isolated from the roots of Scoparia dulcis (Scrophulariaceae) were analyzed using the somatic mutation and recombination test (SMART) in the wings of Drosophila melanogaster. The mutagenic potential of betulinic acid was evaluated at 3 different concentrations (1.64, 3.28, and 6.57 mM). Antimutagenic activity evaluation was performed by co-treatment trials in which the flies received betulinic acid at 3 different concentrations in addition to 10 mM pro-mutagenic urethane. The results demonstrated that betulinic acid was not capable of causing DNA damage. However, the frequency of small single spots, large spots, and twin spots was significantly reduced. In the high bioactivation cross, betulinic acid was significantly active and exerted enhanced antimutagenic activity, possibly as a desmutagen.
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Diagnosing and controlling mix in National Ignition Facility implosion experiments a)
NASA Astrophysics Data System (ADS)
Hammel, B. A.; Scott, H. A.; Regan, S. P.; Cerjan, C.; Clark, D. S.; Edwards, M. J.; Epstein, R.; Glenzer, S. H.; Haan, S. W.; Izumi, N.; Koch, J. A.; Kyrala, G. A.; Landen, O. L.; Langer, S. H.; Peterson, K.; Smalyuk, V. A.; Suter, L. J.; Wilson, D. C.
2011-05-01
High mode number instability growth of "isolated defects" on the surfaces of National Ignition Facility [Moses et al., Phys. Plasmas 16, 041006 (2009)] capsules can be large enough for the perturbation to penetrate the imploding shell, and produce a jet of ablator material that enters the hot-spot. Since internal regions of the CH ablator are doped with Ge, mixing of this material into the hot-spot results in a clear signature of Ge K-shell emission. Evidence of jets entering the hot-spot has been recorded in x-ray images and spectra, consistent with simulation predictions [Hammel et al., High Energy Density Phys. 6, 171 (2010)]. Ignition targets have been designed to minimize instability growth, and capsule fabrication improvements are underway to reduce "isolated defects." An experimental strategy has been developed where the final requirements for ignition targets can be adjusted through direct measurements of mix and experimental tuning.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nunn, D.N.; Lidstrom, M.E.
A method has been developed for the direct selection of methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 (formerly Pseudomonas sp. strain AM1). Using this direct selection technique, we have isolated mutants of Methylobacterium sp. strain AM1 that are no longer capable of growth on methanol but retain the ability to grow on methylamine. These methanol oxidation (Mox) mutants were complemented with a genomic clone bank of this organism constructed in the broad-host-range cosmid pVK100, and subcloning and Tn5 mutagenesis experiments have assigned the Mox mutants to 10 distinct complementation groups. Using an open reading frame beta-galactosidasemore » fusion vector and antibodies specific for Methylobacterium sp. strain AM1 methanol dehydrogenase, we have identified the methanol dehydrogenase structural gene and determined the direction of transcription. The results suggest that the synthesis and utilization of an active methanol dehydrogenase in this organism requires at least 10 different gene functions.« less
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Isolation of an essential Schizosaccharomyces pombe gene, prp31+, that links splicing and meiosis
Bishop, Danielle T.; McDonald, W. Hayes; Gould, Kathleen L.; Forsburg, Susan L.
2000-01-01
We carried out a screen for mutants that arrest prior to premeiotic S phase. One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31+ gene. The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression. It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature. We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype. Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation. PMID:10871341
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RESISTANCE AND CROSS-RESISTANCE OF ESCHERICHIA COLI S MUTANTS TO THE RADIOMIMETIC AGENT PROFLAVINE
DOE Office of Scientific and Technical Information (OSTI.GOV)
Woody-Karrer, P.; Greenberg, J.
1964-03-01
All 50 of the first-step mutants of Escherichia coli S selected for resistance to proflavine were resistant to uv light and each of five different radiomimetic chemicals. The mutants were classified into eight types on the basis of their relative resistance to six different radiomimetic drugs and on the basis of the shape of their uv survival curves. Three of these types are identical to types previously isolated with other radiomimetic drugs; five of the types are new. A high proportion of the clones surviving proflavine treatment were phenotypically but not genetically resistant, and no strains were isolated which weremore » resistant to proflavine but were not resistant to radiation. (auth)« less
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Vaccination of guinea pigs using mce operon mutants of Mycobacterium tuberculosis
Obregón-Henao, Andrés; Shanley, Crystal; Bianco, María Verónica; Cataldi, Angel A; Basaraba, Randall J; Orme, Ian M; Bigi, Fabiana
2011-01-01
The limited efficacy of the BCG vaccine for tuberculosis, coupled with emerging information suggesting that it is poorly protective against newly emerging strains of Mycobacterium tuberculosis such as the W-Beijing isolates, makes it paramount to search for more potent alternatives. One such class of candidates is attenuated mutants derived from M. tuberculosis itself. We demonstrate here, in an initial short term assay, that mutants derived from disruption of the mce genes of the bacillus were highly protective in guinea pigs exposed by low dose aerosol infection with the virulent W-Beijing isolate SA161. This protection was demonstrated by a significant reduction in the numbers of bacilli harvested from the lungs, and dramatic improvements in lung histopathology. PMID:21515327
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Forest stand structure of the northern spotted owl's foraging habitat.
Malcolm P. North; Jerry F. Franklin; Andrew B. Carey; Eric D. Forsman; Tom Hamer
1999-01-01
Although the spotted owl's close association with old growth has been extensively studied, it more difficult to identify and quantify the abundance of particular stand structures associated with preferred owl foraging sites. Old-growth forests have a suite of characteristics that distinguish them from younger forests but which also make it difficult to isolate...
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Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.
Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S
1995-01-01
An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants. PMID:7770517
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Smith, Mark R.; Boenzli, Matthew G.; Hindagolla, Vihangi; Ding, Jun; Miller, John M.; Hutchison, James E.; Greenwood, Jeffrey A.; Abeliovich, Hagai
2013-01-01
Positively charged gold nanoparticles (0.8-nm core diameter) reduced yeast survival, but not growth, at a concentration of 10 to 100 μg/ml. Among 17 resistant deletion mutants isolated in a genome-wide screen, highly significant enrichment was observed for respiration-deficient mutants lacking genes encoding proteins associated with the mitochondrion. PMID:23144132
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Fermentation method producing ethanol
Wang, Daniel I. C.; Dalal, Rajen
1986-01-01
Ethanol is the major end product of an anaerobic, thermophilic fermentation process using a mutant strain of bacterium Clostridium thermosaccharolyticum. This organism is capable of converting hexose and pentose carbohydrates to ethanol, acetic and lactic acids. Mutants of Clostridium thermosaccharolyticum are capable of converting these substrates to ethanol in exceptionally high yield and with increased productivity. Both the mutant organism and the technique for its isolation are provided.
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Chandra, Saurabh; Kapur, Reuben; Chuzhanova, Nadia; Summey, Victoria; Prentice, David; Barker, Jane; Cooper, David N; Williams, David A
2003-11-15
Kit ligand (Kitl), encoded by the Steel (Sl) locus, plays an essential role in hematopoiesis, gametogenesis, and melanogenesis during both embryonic and adult life. We have characterized a new spontaneous mutant of the Sl locus in mice designated KitlSl-20J that arose in the breeding colony at Jackson Laboratories. Heterozygous KitlSl-20J mice display a white belly spot and intercrossing results in an embryonic lethal phenotype in the homozygous state. Analysis of homozygous embryos demonstrated a significant reduction in fetal liver cellularity, colony forming unit-erythroid (CFU-E) progenitors, and a total absence of germ cells. Although expressed in vivo, recombinant mutant protein demonstrated loss of bioactivity that was correlated with lack of receptor binding. Analysis of the Sl gene transcripts in heterozygous KitlSl-20J mice revealed an in-frame tandem duplication of exon 3. A long-range polymerase chain reaction (PCR) strategy using overlapping primers in exon 3 amplified an approximately 7-kilobase (kb) product from DNA isolated from heterozygous KitlSl-20J mice but not from wild-type DNA that contained sequences from both introns 2 and 3 and an inverted intron 2 sequence, suggesting a complex rearrangement as the mechanism of the mutation. "Complexity analysis" of the sequence of the amplified product strongly suggests that local DNA motifs may have contributed to the generation of this spontaneous KitlSl-20J allele, likely mediated by a 2-step process. The KitlSl-20J mutation is a unique KitlSl allele and represents an unusual mechanism of mutation.
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Bhat, Ashwin; Tamuli, Ranjan; Kasbekar, Durgadas P
2004-01-01
The pseudohomothallic fungus Neurospora tetrasperma is naturally resistant to the antibiotic hygromycin. We discovered that mutation of its erg-3 (sterol C-14 reductase) gene confers a hygromycin-sensitive phenotype that can be used to select transformants on hygromycin medium by complementation with the N. crassa erg-3+ and bacterial hph genes. Cotransformation of hph with PCR-amplified DNA of other genes enabled us to construct strains duplicated for the amplified DNA. Using transformation we constructed self-fertile strains that were homoallelic for an ectopic erg-3+ transgene and a mutant erg-3 allele at the endogenous locus. Self-crosses of these strains yielded erg-3 mutant ascospores that produced colonies with the characteristic morphology on Vogel's sorbose agar described previously for erg-3 mutants of N. crassa. The mutants were generated by repeat-induced point mutation (RIP), a genome defense process that causes numerous G:C to A:T mutations in duplicated DNA sequences. Homozygosity for novel recessive RIP-deficient mutations was signaled by self-crosses of erg-3-duplication strains that fail to produce erg-3 mutant progeny. Using this assay we isolated a UV-induced mutant with a putative partial RIP defect. RIP-induced mutants were isolated in rid-1 and sad-1, which are essential genes, respectively, for RIP and another genome defense mechanism called meiotic silencing by unpaired DNA. PMID:15280231
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Cornelis, P; Anjaiah, V; Koedam, N; Delfosse, P; Jacques, P; Thonart, P; Neirinckx, L
1992-07-01
Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.
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A mutational approach for the detection of genetic factors affecting seed size in maize.
Sangiorgio, Stefano; Carabelli, Laura; Gabotti, Damiano; Manzotti, Priscilla Sofia; Persico, Martina; Consonni, Gabriella; Gavazzi, Giuseppe
2016-12-01
Genes influencing seed size. The designation emp (empty pericarp) refers to a group of defective kernel mutants that exhibit a drastic reduction in endosperm tissue production. They allow the isolation of genes controlling seed development and affecting seed size. Nine independently isolated emp mutants have been analyzed in this study and in all cases longitudinal sections of mature seeds revealed the absence of morphogenesis in the embryo proper, an observation that correlates with their failure to germinate. Complementation tests with the nine emp mutants, crossed inter se in all pairwise combinations, identified complementing and non-complementing pairs in the F 1 progenies. Data were then validated in the F 2 /F 3 generations. Mutant chromosomal location was also established. Overall our study has identified two novel emp genes and a novel allele at the previously identified emp4 gene. The introgression of single emp mutants in a different genetic background revealed the existence of a cryptic genetic variation (CGV) recognizable as a variable increase in the endosperm tissue. The unmasking of CGV by introducing single mutants in different genetic backgrounds is the result of the interaction of the emp mutants with a suppressor that has no obvious phenotype of its own and is present in the genetic background of the inbred lines into which the emp mutants were transferred. On the basis of these results, emp mutants could be used as tools for the detection of genetic factors that enhance the amount of endosperm tissue in the maize kernel and which could thus become valuable targets to exploit in future breeding programs.
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Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors
NASA Astrophysics Data System (ADS)
Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.
1990-12-01
Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.
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Henderson, Morgan L; Kreuzer, Kenneth N
2015-01-01
Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.
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Pope, C D; O'Connell, W; Cianciotto, N P
1996-01-01
Legionella pneumophila, a parasite of macrophages and protozoa, requires iron for optimal extracellular and intracellular growth. However, its mechanisms of iron acquisition remain uncharacterized. Using mini-Tn10 mutagenesis, we isolated 17 unique L. pneumophila strains which appeared to be defective for iron acquisition and assimilation. Eleven of these mutants were both sensitive to the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid) and resistant to streptonigrin, an antibiotic whose lethal effect requires high levels of intracellular iron. Six mutants were also defective for the infection of macrophage-like U937 cells. Although none were altered in entry, mutants generally exhibited prolonged lag phases and in some cases replicated at slower rates. Overall, the reduced recoveries of mutants, relative to that of the wild type, ranged from 3- to 1,000-fold. Strain NU216, the mutant displaying the most severe lag phase and the slowest rate of replication, was studied further. Importantly, within U937 cells, NU216 was approximately 100-fold more sensitive than the wild type was to treatment with the Fe3+ chelator deferoxamine, indicating that it is defective for intracellular iron acquisition and assimilation. Furthermore, this strain was unable to mediate any cytopathic effect and was impaired for infectivity of an amoebal host. Taken together, the isolation of these mutants offers genetic proof that iron acquisition and assimilation are critical for intracellular infection by L. pneumophila. PMID:8550218
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Kim, Min Soo; Kim, Young Deuk; Hong, Sung Sik; Park, Kwangseo; Ko, Kwan Soo
2014-01-01
In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria. PMID:25416767
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Zhao, Kaixi; Margaria, Paolo; Rosa, Cristina
2018-05-10
Impatiens necrotic spot orthotospovirus (INSV) can impact economically important ornamental plants and vegetables worldwide. Characterization studies on INSV are limited. For most INSV isolates, there are no complete genome sequences available. This lack of genomic information has a negative impact on the understanding of the INSV genetic diversity and evolution. Here we report the first complete nucleotide sequence of a US INSV isolate. INSV-UP01 was isolated from an impatiens in Pennsylvania, US. RT-PCR was used to clone its full-length genome and Vector NTI to assemble overlapping sequences. Phylogenetic trees were constructed by using MEGA7 software to show the phylogenetic relationships with other available INSV sequences worldwide. This US isolate has genome and biological features classical of INSV species and clusters in the Western Hemisphere clade, but its origin appears to be recent. Furthermore, INSV-UP01 might have been involved in a recombination event with an Italian isolate belonging to the Asian clade. Our analyses support that INSV isolates infect a broad plant-host range they group by geographic origin and not by host, and are subjected to frequent recombination events. These results justify the need to generate and analyze complete genome sequences of orthotospoviruses in general and INSV in particular.
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Single cell isolation process with laser induced forward transfer.
Deng, Yu; Renaud, Philippe; Guo, Zhongning; Huang, Zhigang; Chen, Ying
2017-01-01
A viable single cell is crucial for studies of single cell biology. In this paper, laser-induced forward transfer (LIFT) was used to isolate individual cell with a closed chamber designed to avoid contamination and maintain humidity. Hela cells were used to study the impact of laser pulse energy, laser spot size, sacrificed layer thickness and working distance. The size distribution, number and proliferation ratio of separated cells were statistically evaluated. Glycerol was used to increase the viscosity of the medium and alginate were introduced to soften the landing process. The role of laser pulse energy, the spot size and the thickness of titanium in energy absorption in LIFT process was theoretically analyzed with Lambert-Beer and a thermal conductive model. After comprehensive analysis, mechanical damage was found to be the dominant factor affecting the size and proliferation ratio of the isolated cells. An orthogonal experiment was conducted, and the optimal conditions were determined as: laser pulse energy, 9 μJ; spot size, 60 μm; thickness of titanium, 12 nm; working distance, 700 μm;, glycerol, 2% and alginate depth, greater than 1 μm. With these conditions, along with continuous incubation, a single cell could be transferred by the LIFT with one shot, with limited effect on cell size and viability. LIFT conducted in a closed chamber under optimized condition is a promising method for reliably isolating single cells.
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Liberti, D; Marais, A; Svanella-Dumas, L; Dulucq, M J; Alioto, D; Ragozzino, A; Rodoni, B; Candresse, T
2005-04-01
ABSTRACT A trichovirus closely related to Apple chlorotic leaf spot virus (ACLSV) was detected in symptomatic apricot and Japanese plum from Italy. The Sus2 isolate of this agent cross-reacted with anti-ACLSV polyclonal reagents but was not detected by broad-specificity anti- ACLSV monoclonal antibodies. It had particles with typical trichovirus morphology but, contrary to ACLSV, was unable to infect Chenopodium quinoa and C. amaranticolor. The sequence of its genome (7,494 nucleotides [nt], missing only approximately 30 to 40 nt of the 5' terminal sequence) and the partial sequence of another isolate were determined. The new virus has a genomic organization similar to that of ACLSV, with three open reading frames coding for a replication-associated protein (RNA-dependent RNA polymerase), a movement protein, and a capsid protein, respectively. However, it had only approximately 65 to 67% nucleotide identity with sequenced isolates of ACLSV. The differences in serology, host range, genome sequence, and phylogenetic reconstructions for all viral proteins support the idea that this agent should be considered a new virus, for which the name Apricot pseudo-chlorotic leaf spot virus (APCLSV) is proposed. APCLSV shows substantial sequence variability and has been recovered from various Prunus sources coming from seven countries, an indication that it is likely to have a wide geographical distribution.
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Phenotypic mutant library: potential for gene discovery
USDA-ARS?s Scientific Manuscript database
The rapid development of high throughput and affordable Next- Generation Sequencing (NGS) techniques has renewed interest in gene discovery using forward genetics. The conventional forward genetic approach starts with isolation of mutants with a phenotype of interest, mapping the mutation within a s...
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Djaman, Joseph Allico; Olefongo, Dagnogo; Ako, Aristide Berenger; Roman, Jocelyne; Ngane, Vincent Foumane; Basco, Leonardo K; Tahar, Rachida
2017-07-01
Artemisinin-resistant malaria has not been reported from Africa, but resistance can possibly spread from Asia or arise independently in Africa. The emergence of artemisinin resistance in Africa can be monitored by molecular assay of Kelch 13 (K13) propeller sequences. A total of 251 archived DNA samples of Plasmodium falciparum isolates collected in 2002, 2003, and 2006 in Yaounde, Cameroon, and 47 samples collected in 2006 and 2013 in Abidjan, Côte d'Ivoire, were analyzed for K13-propeller sequence polymorphism. Only one isolate carried a mutant K13-propeller allele (E602D). None of the isolates carried the key mutant alleles (Y493H, R539T, I543T, and C580Y) associated with artemisinin resistance in Cambodia. The presence of the mutant allele was not correlated with in vitro response to dihydroartemisinin determined by the classical hypoxanthine incorporation assay. There was no evidence of K13 mutations associated with artemisinin resistance before and soon after the introduction of artemisinin-based combination therapies in Cameroon and Côte d'Ivoire.
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Roberti, K.A.; Rohovec, J.S.; Winton, J.R.
1998-01-01
A neutralizing monoclonal antibody against infectious hematopoietic necrosis virus (IHNV) was used to select neutralization-resistant mutants from isolates of virus obtained from adult steelhead Oncorhynchus mykiss returning to the Round Butte Hatchery (RB mutants) on the Deschutes River in Oregon, USA, and from rainbow trout (nonanadromous O. mykiss) at a commercial hatchery in the Hagerman Valley of Idaho, USA (193-110 mutants). Two of the mutants, RB-1 and 193-110-4, were significantly (P 0.05) in protection among fish exposed to the RB-1 vaccine strain at a dose of 1 x 105 TCID50/mL for periods of either 1, 12, or 24 h, held for 14 d, and then challenged with the wild-type RB isolate, although the 1-h exposure seemed to be somewhat less effective. Fish were vaccinated with the RB-1 strain at 1 x 103-1 x 105 TCID50/mL for 24 h then challenged after 1, 7, 14, or 21 d with the wild-type RB isolate. No significant (P > 0.1) protection was observed at 1 d postvaccination, but the relative percent survival increased progressively at each subsequent challenge period, becoming statistically significant by day 7 (P < 0.001) and beyond. These results suggested that resistance to challenge with wild-type virus resulted from development of IHNV-specific immunity and not from viral interference or interferon induction, and they reinforce the potential of an attenuated vaccine to control this important disease.
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Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis
1994-01-01
We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning. PMID:7929583
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Apparatus for producing diamond-like carbon flakes
NASA Technical Reports Server (NTRS)
Banks, Bruce A. (Inventor)
1986-01-01
A vacuum arc from a spot at the face of a graphite cathode to a graphite anode produces a beam of carbon ions and atoms. A carbon coating from this beam is deposited on an ion beam sputtered target to produce diamond-like carbon flakes. A graphite tube encloses the cathode, and electrical isolation is provided by an insulating sleeve. The tube forces the vacuum arc spot to be confined to the surface on the outermost end of the cathode. Without the tube the arc spot will wander to the side of the cathode. This spot movement results in low rates of carbon deposition, and the properties of the deposited flakes are more graphite-like than diamond-like.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Staedtler, F.; Locher, F.; Sreenan, G.
1997-10-01
In order to evaluate the in vivo genotoxic potential of three putative genotoxic mouse liver carcinogens, high doses of 4-chloro-o-phenylenediamine, 2-nitro-p-phenylenediamine and 2, 4-diaminotoluene were tested short term in the Big Blue{reg_sign} transgenic mouse mutation assay. Small statistically significant increases in the lacI mutant frequencies in the liver by factors 1.7 to 2.0 were found. A representative number of 347 lacI mutants isolated from liver tissue of male and female animals were analyses by DNA sequencing. The mutational spectra were examined with the Adams-Skopek algorithm. The spontaneous mutational spectra from untreated male and female animals were similar and consistent withmore » spectral Big Blue{reg_sign} control data stored in the lacI database. Most of the background mutations were located in the 5{prime} portion of the coding region of the lacI gene. Single base substitutions were most prominent. G:C to A:T transitions and G:C to T:A transversions occurred predominatly and were preferentially located at CpG sites. Despite the increases observed in the mutant frequencies of the treated animals, the corresponding mutational spectra did not differ from the controls. However, it is possible that certain classes of point mutations were substantially increased but not detected due to the limited number of sequenced mutants. In two animals treated with 2, 4- diaminotoluene unusually high mutant frequencies and the multiple occurrence of certain mutations in the liver was observed. From one of these animals six lacI mutants isolated from colon tissue were all different. Since 2, 4-diaminotoluene was shown to induce liver cell proliferation these results may reflect clonal expansion of single mutated liver cells.« less
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Ohya, Y.; Botstein, D.
1994-01-01
Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe -> Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium, 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl(2) in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks. PMID:7896089
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Inducamides A–C, Chlorinated Alkaloids from an RNA Polymerase Mutant Strain of Streptomyces sp.
2015-01-01
Inducamides A–C (1–3), three new chlorinated alkaloids featuring an amide skeleton generated by a tryptophan fragment and a 6-methylsalicylic acid unit, were isolated from a chemically induced mutant strain of Streptomyces sp. with the inducamides only being produced in the mutant strain. Their structures, including stereochemistry, were determined by spectroscopic analysis, Marfey’s method, and CD spectroscopy. PMID:25338006
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Mutant Enrichment in the Colonial Alga, EUDORINA ELEGANS
Toby, A. L.; Kemp, C. L.
1975-01-01
An enrichment procedure has been developed that results in at least a 200x increase in mutation frequency in the colonial alga, Eudorina elegans. A period of nitrogen starvation followed by treatment with 8-azaguanine results in the death of wild-type cells and the maintenance of mutants. N'-nitro-N-nitro-soguanidine-induced acetate, p-aminobenzoic acid and reduced nitrogen requiring mutants have been isolated by this procedure. PMID:1205128
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McCammon, M. T.
1996-01-01
The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn(-) (``ACetate Nonutilizing'') mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism. PMID:8878673
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Shrestha, Sandesh Kumar; Cochran, Alicia; Mengistu, Alemu; Castro-Rocha, Arturo; Young-Kelly, Heather
2017-01-01
Frogeye leaf spot (FLS), caused by Cercospora sojina, causes significant damage to soybean in the U.S. One control strategy is the use of quinone outside inhibitor (QoI) fungicides. QoI resistant isolates were first reported in Tennessee (TN) in 2010. To investigate the disease dynamics of C. sojina, we collected 437 C. sojina isolates in 2015 from Jackson and Milan, TN and used 40 historical isolates collected from 2006–2009 from TN and ten additional states for comparison. A subset of 186 isolates, including historical isolates, were genotyped for 49 single nucleotide polymorphism (SNP) markers and the QoI resistance locus, revealing 35 unique genotypes. The genotypes clustered into three groups with two groups containing only sensitive isolates and the remaining group containing all resistant isolates and a dominant clonal lineage of 130 isolates. All 477 C. sojina isolates were genotyped for the QoI locus revealing 344 resistant and 133 sensitive isolates. All isolates collected prior to 2015 were QoI sensitive. Both mating type alleles (MAT1-1-1 and MAT1-2) were found in Jackson and Milan, TN and recovered from single lesions suggesting sexual recombination may play a role in the epidemiology of field populations. Analysis of C. sojina isolates using SNP markers proved useful to investigate population diversity and to elaborate on diversity as it relates to QoI resistance and mating type. PMID:28486517
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Dzelzkalns, V A; Bogorad, L
1988-01-01
Photosynthesis-defective mutants of the transformable cyanobacterium Synechocystis 6803 have been isolated following nitrosoguanidine mutagenesis. The photosystem II- phenotype of one of these mutants is shown by DNA sequencing to be attributable to a short deletion in psbC, the gene encoding the 44-kd, chlorophyll-binding protein of photosystem II. Although not a component of the reaction center of photosystem II, the 44-kd protein is none the less shown to be essential in vivo for photosystem II activity. The deletion in psbC also results in greatly diminished levels of D-2 (a component of the reaction center of photosystem II) indicating that the loss of the product of the psbC gene affects the assembly or stability of the photosystem II reaction center. The isolation of a clone capable of restoring both photosystem II activity and photoautotrophy to the mutant cells was aided by the observation that restriction fragments or cloned Synechocystis 6803 DNA applied in liquid or in melted agarose directly onto a lawn of Synechocystis 6803 will lead to the transformation of the cells. This in situ 'dot' transformation procedure provides a convenient method for the rapid identification of fractions or clones containing complementing Synechocystis 6803 DNA. Images PMID:3130247
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Ohashi, Y; Sugimaru, K; Nanamiya, H; Sebata, T; Asai, K; Yoshikawa, H; Kawamura, F
1999-03-18
We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the sigmaH protein, respectively, and located in the highly conserved region, "2", among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43 degrees C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the beta subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the sigmaH-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43 degrees C. Western blot analysis showed that the level of sigmaH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of sigmaH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigmaH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of sigmaH protein showed that the short life of sigmaH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-sigmaH formation may be involved in stabilization of sigmaH at the onset of sporulation.
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Code of Federal Regulations, 2014 CFR
2014-07-01
... NAAQS. Paragraph (d) of this section explains when a hot-spot test is required. Paragraph (e) of this... insignificant motor vehicle emissions. Paragraph (g) of this section addresses isolated rural nonattainment and....5 hot-spots. § 93.117 PM10 and PM2.5 control measures Project (Not From a Conforming Plan and TIP...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... NAAQS. Paragraph (d) of this section explains when a hot-spot test is required. Paragraph (e) of this... insignificant motor vehicle emissions. Paragraph (g) of this section addresses isolated rural nonattainment and....5 hot-spots. § 93.117 PM10 and PM2.5 control measures Project (Not From a Conforming Plan and TIP...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... NAAQS. Paragraph (d) of this section explains when a hot-spot test is required. Paragraph (e) of this... insignificant motor vehicle emissions. Paragraph (g) of this section addresses isolated rural nonattainment and....5 hot-spots. § 93.117 PM10 and PM2.5 control measures Project (Not From a Conforming Plan and TIP...
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A dinoflagellate mutant with higher frequency of multiple fission.
Lam, C M; Chong, C; Wong, J T
2001-01-01
The dinoflagellate Crypthecodinium cohnii Biecheler propagates by both binary and multiple fission. By a newly developed mutagenesis protocol based on using ethyl methanesulfonate and a cell size screening method, a cell cycle mutant, mf2, was isolated with giant cells which predominantly divide by multiple fission. The average cell size of the mutant mf2 is larger than the control C. cohnii. Cell cycle synchronization experiments suggest that mutant mf2, when compared with the control strain, has a prolonged G1 phase with a corresponding delay of the G2 + M phase.
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Isogai, M; Saitou, Y; Takahashi, N; Itabashi, T; Terada, M; Satoh, H; Yoshikawa, N
2003-03-01
To understand why transgenic Nicotiana occidentalis plants expressing a functional movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) show specific resistance to Grapevine berry inner necrosis virus (GINV), the MPs of ACLSV (50KP) and GINV (39KP) were fused to green, yellow, or cyan fluorescent proteins (GFP, YFP, or CFP). These fusion proteins were transiently expressed in leaf cells of both transgenic (50KP) and nontransgenic (NT) plants, and the intracellular and intercellular trafficking and tubule-inducing activity of these proteins were compared. The results indicate that in epidermal cells and protoplasts from 50KP plant leaves, the trafficking and tubule-inducing activities of GINV-39KP were specifically blocked while those of ACLSV-50KP and Apple stem grooving virus MP (36KP) were not affected. Additionally, when 39KP-YFP and 50KP-CFP were coexpressed in the leaf epidermis of NT plants, the fluorescence of both proteins was confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP and vice versa. Mutational analyses of 50KP showed that the deletion mutants that retained the activities described above still blocked cell-to-cell trafficking of 39KP, but the dysfunctional 50KP mutants could no longer impede cell-to-cell movement of 39KP. Transgenic plants expressing the functional 50KP deletion mutants showed specific resistance against GINV. In contrast, transgenic plants expressing the dysfunctional 50KP mutants did not show any resistance to the virus. From these results, we conclude that the specific resistance of 50KP plants to GINV is due to the ability of the 50KP to block intracellular and intercellular trafficking of GINV 39KP.
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Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mineo, C.; Murakami, Y.; Ishimi, Y.
1986-11-01
A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33/sup 0/C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39/sup 0/C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39/sup 0/C for at least 10h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. Thismore » suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.« less
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Juni, E; Heym, G A; Maurer, M J; Miller, M L
1987-01-01
A combined genetic transformation and nutritional assay is described that permits definitive identification of clinically isolated strains of Moraxella nonliquefaciens. Crude DNA preparations of strains of various Moraxella species were used to transform nutritional mutants of a stably competent strain of M. nonliquefaciens for ability to grow on a defined medium (Mn-B). DNA samples from 24 independently isolated strains of M. nonliquefaciens all resulted in massive (4+) transformation of each of two mutant assay strains. DNA samples from strains of M. bovis and M. lacunata frequently gave weak (1+) transformation of one of the mutant assay strains (Mn64) but almost always failed to transform another assay strain (Mn136). DNA samples from eight other Moraxella species failed completely to transform either of the mutant assay strains. When streaked on the defined medium used for the transformation assay (Mn-B), 23 of the 24 strains of M. nonliquefaciens grew well, but all strains of M. bovis and M. lacunata failed to grow on this medium. Images PMID:3654942
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Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase.
Fujita, Y; Freese, E
1981-01-01
A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes. Images PMID:6257649
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Uchida, Takeshi; Koshino, Hiroyuki; Takahashi, Shunya; Shimizu, Eisaku; Takahashi, Honoka; Yoshida, Jun; Shinden, Hisao; Tsujimura, Maiko; Kofujita, Hisayoshi; Uesugi, Shota; Kimura, Ken-Ichi
2018-04-27
A podocarpatriene and a labdatriene derivative, named kujiol A [13-methyl-8,11,13-podocarpatrien-19-ol (1)] and kujigamberol B [15,20-dinor-5,7,9-labdatrien-13-ol (2)], respectively, were isolated from Kuji amber through detection with the aid of their growth-restoring activity against a mutant yeast strain ( zds1Δ erg3Δ pdr1Δ pdr3Δ), which is known to be hypersensitive with respect to Ca 2+ -signal transduction. The structures were elucidated by spectroscopic data analysis. Compounds 1 and 2 are rare organic compounds from Late Cretaceous amber, and the mutant yeast used seems useful for elucidating a variety of new compounds from Kuji amber specimens, produced before the K-Pg boundary.
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Rallos, Lynn Esther E.; Baudoin, Anton B.
2016-01-01
Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970
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Reith, A D; Ellis, C; Maroc, N; Pawson, T; Bernstein, A; Dubreuil, P
1993-01-01
Point mutations in highly conserved amino acid residues in the catalytic domain of the Kit receptor tyrosine kinase (RTK) are responsible for the coat color, fertility and hematopoietic defects of mice bearing mutant alleles at the dominant white-spotting (W) locus. The dominant nature of structural Kit mutations suggests that expression of other kinase-defective RTKs might also specifically interfere with signal transduction by normal receptors. To test this possibility, we have investigated the functional consequences of introducing analogous mutations into the RTK encoded by the c-fms proto-oncogene. Both Fms37 (glu582-->lys) and Fms42 (asp776-->asn) mutant proteins, corresponding to the strongly dominant-negative W37 and W42 mutant c-kit alleles, had undetectable in vitro kinase activity and were unable to transform Rat-2 fibroblasts in the presence of exogenous CSF-1. Moreover, expression of Fms37 or Fms42 proteins in Rat-2 cells specifically inhibited anchorage-independent growth mediated by the normal Fms receptor in the presence of exogenous CSF-1 and conferred a dominant loss of Fms-associated PI3-kinase activity on CSF-1 stimulation. Mutant RTKs, bearing point substitutions identical to those present in mild or severe W mutants, may provide a generally applicable strategy for inducing dominant loss of function defects in RTK-mediated signalling pathways.
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Sineokiĭ, S P; Pogosov, V Z; Iankovskiĭ, N K; Krylov, V N
1976-01-01
123 Amber mutants of lambdoid bacteriophage phi81 are isolated and distributed into 19 complementation groups. Deletion mapping made possible to locate 5 gene groups on the genetic map of bacteriophage phi81 and to determine a region of possible location of mm' sticky ends on the prophage genetic map. A gene of phage phi81 is localized, which controls the adsorption specificity, and which functional similarity to a respective gene of phage phi80 is demonstrated.
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Chaudhari, Aditi; Krumlinde, Daniel; Lundqvist, Annika; Akyürek, Levent M; Bandaru, Sashidhar; Skålén, Kristina; Ståhlman, Marcus; Borén, Jan; Wettergren, Yvonne; Ejeskär, Katarina; Rotter Sopasakis, Victoria
2015-10-01
The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Dong, Yuan; Cui, Cheng-Bin; Li, Chang-Wei; Hua, Wei; Wu, Chang-Jing; Zhu, Tian-Jiao; Gu, Qian-Qun
2014-07-29
A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(D-Pro-D-Phe) (1), cyclo(D-Tyr-D-Pro) (2), phenethyl 5-oxo-L-prolinate (3), cyclo(L-Ile-L-Pro) (4), cyclo(L-Leu-L-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1-6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.
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Wheat ABA-insensitive mutants result in reduced grain dormancy
USDA-ARS?s Scientific Manuscript database
This paper describes the isolation of wheat mutants in the hard red spring Scarlet resulting in reduced sensitivity to the plant hormone abscisic acid (ABA) during seed germination. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature seeds. Wheat sensitivity t...
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Moragrega, Concepció; Puig, Mireia; Ruz, Lídia; Montesinos, Emilio; Llorente, Isidre
2018-02-01
Brown spot of pear, caused by the fungus Stemphylium vesicarium, is an emerging disease of economic importance in several pear-growing areas in Europe. In recent years, new control strategies combining sanitation practices and fungicide applications according to developed forecasting models have been introduced to manage the disease. However, the pathogenic and saprophytic behavior of this pathogen makes it difficult to manage the disease. In addition, climate change can also result in variations in the severity and geographical distribution of the disease. In this study, ecological and epidemiological aspects of brown spot of pear disease related to inoculum characterization and climate change impact were elucidated. The pathogenic variation in S. vesicarium populations from pear orchards and its relationship to inoculum sources (air samples, leaf debris, and infected host and nonhost tissues) was determined using multivariate analysis. In total, six variables related to infection and disease development on cultivar Conference pear detached leaves of 110 S. vesicarium isolates were analyzed. A high proportion of isolates (42%) were nonpathogenic to pear; 85% of these nonpathogenic isolates were recovered from air samples. Most isolates recovered from lesions (93%) and pseudothecia (83%) were pathogenic to pear. A group of pathogenic isolates rapidly infected cultivar Conference pear leaves resulted in disease increase that followed a monomolecular model, whereas some S. vesicarium isolates required a period of time after inoculation to initiate infection and resulted in disease increase that followed a logistic model. The latter group was mainly composed of isolates recovered from pseudothecia on leaf debris, whereas the former group was mainly composed of isolates recovered from lesions on pear fruit and leaves. The relationship between the source of inoculum and pathogenic/aggressiveness profile was confirmed by principal component analysis. The effect of climate change on disease risk was analyzed in two pear-growing areas of Spain under two scenarios (A2 and B1) and for three periods (2005 to 2009, 2041 to 2060, and 2081 to 2100). Simulations showed that the level of risk predicted by BSPcast model increased to high or very high under the two scenarios and was differentially distributed in the two regions. This study is an example of how epidemiological models can be used to predict not only the onset of infections but also how climate change could affect brown spot of pear. [Formula: see text] Copyright © 2018 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .
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Rodríguez, Cristina; Sanz, Pascual; Gancedo, Carlos
2003-03-01
We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation.
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Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion.
Luo, Jing; Lu, Liaoxun; Gu, Yanrong; Huang, Rong; Gui, Lin; Li, Saichao; Qi, Xinhui; Zheng, Wenping; Chao, Tianzhu; Zheng, Qianqian; Liang, Yinming; Zhang, Lichen
2018-06-07
Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection. Copyright © 2018 Elsevier B.V. All rights reserved.
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Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams
Takano, Nao; Takahashi, Yuko; Yamamoto, Mitsuru; Teranishi, Mika; Yamaguchi, Hiroko; Sakamoto, Ayako N.; Hase, Yoshihiro; Fujisawa, Hiroko; Wu, Jianzhong; Matsumoto, Takashi; Toki, Seiichi; Hidema, Jun
2013-01-01
UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar ‘Sasanishiki’) that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined. PMID:23381954
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Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams.
Takano, Nao; Takahashi, Yuko; Yamamoto, Mitsuru; Teranishi, Mika; Yamaguchi, Hiroko; Sakamoto, Ayako N; Hase, Yoshihiro; Fujisawa, Hiroko; Wu, Jianzhong; Matsumoto, Takashi; Toki, Seiichi; Hidema, Jun
2013-07-01
UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar 'Sasanishiki') that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined.
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Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.
Ivanov, E L; Kovaltzova, S V; Korolev, V G
1989-08-01
We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.
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Imaginal Disc Abnormalities in Lethal Mutants of Drosophila
Shearn, Allen; Rice, Thomas; Garen, Alan; Gehring, Walter
1971-01-01
Late lethal mutants of Drosophila melanogaster, dying after the larval stage of development, were isolated. The homozygous mutant larvae were examined for abnormal imaginal disc morphology, and the discs were injected into normal larval hosts to test their capacities to differentiate into adult structures. In about half of the mutants analyzed, disc abnormalities were found. Included among the abnormalities were missing discs, small discs incapable of differentiating, morphologically normal discs with limited capacities for differentiation, and discs with homeotic transformations. In some mutants all discs were affected, and in others only certain discs. The most extreme abnormal phenotype is a class of “discless” mutants. The viability of these mutant larvae indicates that the discs are essential only for the development of an adult and not of a larva. The late lethals are therefore a major source of mutants for studying the genetic control of disc formation. Images PMID:5002822
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Bertolotti, Alicia C; Griffiths, Sarah M; Truelove, Nathan K; Box, Stephen J; Preziosi, Richard F; Salinas de Leon, Pelayo
2015-01-01
The white-spotted sandbass (Paralabrax albomaculatus) is a commercially important species in the Galapagos Marine Reserve, but is classified as endangered in the IUCN Red List. For this study, 10 microsatellite loci were isolated and characterized using Illumina paired-end sequencing. These loci can be used for genetic studies of population structure and connectivity to aid in the management of the white-spotted sandbass and other closely-related species. The 10 characterized loci were polymorphic, with 11-49 alleles per locus, and observed heterozygosity ranged from 0.575 to 0.964. This set of markers is the first to be developed for this species.
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Harner, Nicole K; Bajwa, Paramjit K; Habash, Marc B; Trevors, Jack T; Austin, Glen D; Lee, Hung
2014-01-01
A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.
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Garita-Cambronero, J.; Sena-Vélez, M.; Palacio-Bielsa, A.
2014-01-01
We report the annotated genome sequence of Xanthomonas arboricola pv. pruni strain Xap33, isolated from almond leaves showing bacterial spot disease symptoms in Spain. The availability of this genome sequence will aid our understanding of the infection mechanism of this bacterium as well as its relationship to other species of the same genus. PMID:24903863
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Miras, Manuel; Rodríguez-Hernández, Ana M; Romero-López, Cristina; Berzal-Herranz, Alfredo; Colchero, Jaime; Aranda, Miguel A; Truniger, Verónica
2018-01-01
In eukaryotes, the formation of a 5'-cap and 3'-poly(A) dependent protein-protein bridge is required for translation of its mRNAs. In contrast, several plant virus RNA genomes lack both of these mRNA features, but instead have a 3'-CITE (for cap-independent translation enhancer), a RNA element present in their 3'-untranslated region that recruits translation initiation factors and is able to control its cap-independent translation. For several 3'-CITEs, direct RNA-RNA long-distance interactions based on sequence complementarity between the 5'- and 3'-ends are required for efficient translation, as they bring the translation initiation factors bound to the 3'-CITE to the 5'-end. For the carmovirus melon necrotic spot virus (MNSV), a 3'-CITE has been identified, and the presence of its 5'-end in cis has been shown to be required for its activity. Here, we analyze the secondary structure of the 5'-end of the MNSV RNA genome and identify two highly conserved nucleotide sequence stretches that are complementary to the apical loop of its 3'-CITE. In in vivo cap-independent translation assays with mutant constructs, by disrupting and restoring sequence complementarity, we show that the interaction between the 3'-CITE and at least one complementary sequence in the 5'-end is essential for virus RNA translation, although efficient virus translation and multiplication requires both connections. The complementary sequence stretches are invariant in all MNSV isolates, suggesting that the dual 5'-3' RNA:RNA interactions are required for optimal MNSV cap-independent translation and multiplication.
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He, Guochun; Matsuura, Hideyuki; Yoshihara, Teruhiko
2004-10-01
Lasiodiplodia theobromae is known as a multi-infectious microorganism that causes considerable crop damage, particularly to tropical fruits. When the fruits are infected by L. theobromae, the typical symptom is the appearance of black spots on the surface of the infected fruit. When injected in to the peel of banana, the culture filtrate of L. theobromae induced formation of black spots. The structure of the isolated compound responsible for this effect was determined to be (3S,4R)-3-carboxy-2-methylene-heptan-4-olide on the basis of analysis of MS, IR, and 1H and 13C NMR spectroscopic data, including HMQC, HMBC, and 1H-1H COSY experiments. The active compound was not only isolated from the culture filtrate derived from potato dextrose medium, but also from the extract of infected peels of bananas.
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Gupta, Suruchi; Kaul, Sanjana; Singh, Baljinder; Vishwakarma, Ram A; Dhar, Manoj K
2016-11-01
Endophytes from medicinal plants represent a potential source of bioactive compounds. During the present investigation, fungal endophytes were isolated from turmeric (Curcuma longa), an important medicinal plant. A total of 207 endophytic fungal isolates were obtained from the rhizome of C. longa L. They were grouped into seven genera based on morphological and molecular data. The fungal endophytes of C. longa were evaluated for antifungal activity against Colletotrichum gloeosporioides, the causal organism of leaf spot of turmeric. The disease is a major cause for economic loss in turmeric cultivation. Endophytic Phoma herbarum showed significant activity against C. gloeosporioides and was therefore selected for further studies. A compound gentisyl alcohol was isolated from P. herbarum which showed effective antagonism against C. gloeosporioides. The organism could therefore be used as a biocontrol agent against C. gloeosporioides.
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A novel two-step method for screening shade tolerant mutant plants via dwarfism
USDA-ARS?s Scientific Manuscript database
When subjected to shade, plants undergo rapid shoot elongation, which often makes them more prone to disease and mechanical damage. It has been reported that, in turfgrass, induced dwarfism can enhance shade tolerance. Here, we describe a two-step procedure for isolating shade tolerant mutants of ...
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Deryke, C Andrew; Du, Xiaoli; Nicolau, David P
2006-09-01
The increasingly recognized prevalence of first-step parC mutants in Streptococcus pneumoniae and the development of de novo resistance while on fluoroquinolone therapy are of concern. Previous work by our group demonstrated the ability of moxifloxacin, but not levofloxacin, to eradicate parC mutants. The objective of this experiment was to determine whether these fluoroquinolone antibiotics provided equivalent bacterial kill when similar AUC/MICs were examined. An in vitro pharmacodynamic model was used to simulate the epithelial lining fluid (ELF) concentrations following oral administration of levofloxacin 500 mg once daily and moxifloxacin 400 mg once daily in older adults. In addition, a range of AUC/MICs were also modelled, including levofloxacin 750 mg once daily. Five different S. pneumoniae containing first-step parC mutations and one isolate without mutations were tested for 48 h and time-kill curves were constructed. Samples at 0, 24 and 48 h were collected for phenotypic and genotypic profiling. HPLC was used to verify that target exposures were achieved. The isolate without a parC mutation displayed a 4 log reduction in cfu after treatment with levofloxacin 500 mg and did not select for resistance. In all five isolates containing first-step parC mutations, resistance emerged within 48 h with a > or =16-fold increase in MIC and the acquisition of a gyrA mutant. Increasing the exposure of levofloxacin to approximately 750 mg dose still led to > or =16-fold increase in MIC at 48 h in two of the four isolates containing parC mutations. On the other hand, moxifloxacin 400 mg sustained bacterial killing against the two isolates tested without the selection of resistant mutants. It appears that the critical AUC/MIC necessary to prevent the acquisition of resistance for levofloxacin is 200 and approximately 400 for moxifloxacin. Due to suboptimal exposures, once-daily oral regimens of levofloxacin at both 500 and 750 mg inconsistently led to bactericidal activity and the frequent acquisition of a second-step gyrA mutation in S. pneumoniae isolates already containing a first-step parC mutation. Conversely, once-daily moxifloxacin 400 mg provides exposures that vastly exceed the apparent efficacy breakpoint and did not select for second-step mutants until exposures were decreased 4-fold. As a result of these data and the emerging literature involving mutations in the pneumococcus, caution should be exercised when the respiratory fluoroquinolones are used to treat patients infected with S. pneumoniae suspected of having parC mutations.
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Abdullah, Sidrat; Sehgal, Sunish K; Glover, Karl D; Ali, Shaukat
2017-06-01
Rye ( Secale cereale L.) serves as an alternative host of Pyrenophora tritici-repentis ( PTR ) the cause of tan spot on wheat. Rye is cultivated as a forage or cover crop and overlaps with a significant portion of wheat acreage in the U.S. northern Great Plains; however, it is not known whether the rye crop influences the evolution of PTR races. We evaluated a global collection of 211 rye accessions against tan spot and assessed the diversity in PTR population on rye in South Dakota. All the rye genotypes were inoculated with PTR races 1 and 5, and infiltrated with Ptr ToxA and Ptr ToxB, at seedling stage. We observed 21% of the genotypes exhibited susceptibility to race 1, whereas, 39% were susceptible to race 5. All 211 accessions were insensitive to both the Ptr toxins. It indicates that though rye exhibits diversity in reaction to tan spot, it lacks Ptr ToxA and ToxB sensitivity genes. This suggests that unknown toxins or other factors can lead to PTR establishment in rye. We characterized the race structure of 103 PTR isolates recovered from rye in South Dakota. Only 22% of the isolates amplified Ptr ToxA gene and were identified as race 1 based on their phenotypic reaction on the differential set. The remaining 80 isolates were noted to be race 4. Our results show that races 1 and 4 are prevalent on rye in South Dakota with a higher frequency of race 4, suggesting a minimal role of rye in the disease epidemiology.
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Abdullah, Sidrat; Sehgal, Sunish K.; Glover, Karl D.; Ali, Shaukat
2017-01-01
Rye (Secale cereale L.) serves as an alternative host of Pyrenophora tritici-repentis (PTR) the cause of tan spot on wheat. Rye is cultivated as a forage or cover crop and overlaps with a significant portion of wheat acreage in the U.S. northern Great Plains; however, it is not known whether the rye crop influences the evolution of PTR races. We evaluated a global collection of 211 rye accessions against tan spot and assessed the diversity in PTR population on rye in South Dakota. All the rye genotypes were inoculated with PTR races 1 and 5, and infiltrated with Ptr ToxA and Ptr ToxB, at seedling stage. We observed 21% of the genotypes exhibited susceptibility to race 1, whereas, 39% were susceptible to race 5. All 211 accessions were insensitive to both the Ptr toxins. It indicates that though rye exhibits diversity in reaction to tan spot, it lacks Ptr ToxA and ToxB sensitivity genes. This suggests that unknown toxins or other factors can lead to PTR establishment in rye. We characterized the race structure of 103 PTR isolates recovered from rye in South Dakota. Only 22% of the isolates amplified Ptr ToxA gene and were identified as race 1 based on their phenotypic reaction on the differential set. The remaining 80 isolates were noted to be race 4. Our results show that races 1 and 4 are prevalent on rye in South Dakota with a higher frequency of race 4, suggesting a minimal role of rye in the disease epidemiology. PMID:28592942
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USDA-ARS?s Scientific Manuscript database
Several isolates of a previously unreported disease were discovered on common cocklebur seedlings in Chicot County, Arkansas and Washington County, Mississippi. Diseased plants in nature exhibited angular-shaped leaf spotting symptoms on leaf margins and central leaf areas. The isolates were cultu...
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Global genotype flow in Cercospora beticola populations confirmed through genotyping-by-sequencing
USDA-ARS?s Scientific Manuscript database
Genotyping-by-sequencing (GBS) was conducted on 333 Cercospora isolates collected from Beta vulgaris (sugar beet, table beet and Swiss chard) in the USA and Europe. Cercospora beticola was confirmed as the species predominantly isolated from leaves with Cercospora leaf spot (CLS) symptoms. However, ...
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Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.
Santangelo, J D; Jones, D T; Woods, D R
1991-01-01
An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710
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High-Throughput, Motility-Based Sorter for Microswimmers such as C. elegans
Yuan, Jinzhou; Zhou, Jessie; Raizen, David M.; Bau, Haim H.
2015-01-01
Animal motility varies with genotype, disease, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method is implemented in a simple microfluidic device capable of sorting thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriches for known C. elegans motility mutants. Furthermore, using this device, we isolate low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates C. elegans sleep. By performing genetic complementation tests, we demonstrate that our motility-based sorting device efficiently isolates mutants for the same gene identified by tedious visual inspection of behavior on an agar surface. Therefore, our motility-based sorter is capable of performing high throughput gene discovery approaches to investigate fundamental biological processes. PMID:26008643
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Deng, Yu; Huang, Zhigang; Wang, Wenbing; Chen, Yinghuai; Guo, Zhongning; Chen, Ying
2017-01-01
Aiming to improve the laser-induced forward transfer (LIFT) cell isolation process, a polydimethylsiloxane (PDMS) layer with micro-hole arrays was employed to improve the cell separation precision, and a microchip with heater was developed to maintain the working area at 100% humidity and 37°C with the purpose to preserve the viability of the isolated cells. A series of experiments were conducted to verify the contributions of the optimization to LIFT cell isolation process as well as to study the effect of laser pulse energy, laser spot size and the titanium thickness on cell isolation. With 40µm laser spot size and 40nm thick of titanium, laser energy threshold for 100% single cell isolating succeed ratio is 7µJ. According to the staining images and proliferation ratios, the chip did help to improve the cell availability and the cells can recover from the juries at least a day earlier comparing to the samples processed without the chip. With a Lattice Boltzmann model, the cell isolation process is numerically studied and it turns out that the micro-hole makes the isolation process shift to a micro-syringe injection model leading to the lower laser energy threshold for cell separation and fewer injuries. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O
2017-03-28
Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.
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Sphingolipid-Mediated Apoptosis and Tumor Suppression in Breast Carcinoma.
1996-10-01
diacylglycerol and ceramide. The radioactive spots corresponding to phosphatidic acid and ceramide-phosphate, the phosphorylated products of diacylglycerol (DAG...threonine protein phosphatase (7). This phosphatase is inhibited by okadaic acid , and okadaic acid appears to inhibit the effects of ceramide on...Arg to Thr at amino acid 291 in the reactive site loop. In MCF-7 cells expressing this mutant, there was no significant inhibition of ceramide
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Proteins evolve on the edge of supramolecular self-assembly.
Garcia-Seisdedos, Hector; Empereur-Mot, Charly; Elad, Nadav; Levy, Emmanuel D
2017-08-10
The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils. Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.
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Proteins evolve on the edge of supramolecular self-assembly
NASA Astrophysics Data System (ADS)
Garcia-Seisdedos, Hector; Empereur-Mot, Charly; Elad, Nadav; Levy, Emmanuel D.
2017-08-01
The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils. Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.
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Flores-Rozas, H; Kolodner, R D
1998-10-13
The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.
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Isolation of the Chlamydomonas Regulatory Gene Nit2 by Transposon Tagging
Schnell, R. A.; Lefebvre, P. A.
1993-01-01
Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhardtii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOC1, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent genes are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrate assimilation pathway in Chlamydomonas. PMID:8394263
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Gwon, Hui-Jeong; Baik, Sang-Ho
2010-01-01
Diastereoselectivity-enhanced mutants of L: -threonine aldolase (L: -TA) for L: -threo-3,4-dihydroxyphenylserine (L: -threo-DOPS) synthesis were isolated by error-prone PCR followed by a high-throughput screening. The most improved mutant was achieved from the mutant T3-3mm2, showing a 4-fold increase over the wild-type L: -TA. When aldol condensation activity was examined using whole cells of T3-3mm2, its de was constantly maintained at 55% during the batch reactions for 80 h, yielding 3.8 mg L: -threo-DOPS/ml.
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Density-based kinetics for mesoscale simulations of detonation initiation in energetic materials
NASA Astrophysics Data System (ADS)
Jackson, Thomas Luther; Zhang, Ju
2017-07-01
In this work we present one- and two-dimensional mesoscale simulations of detonation initiation in energetic materials. We solve the reactive Euler equations, with the energy equation augmented by a power deposition term. The reaction rate at the mesoscale is modelled using a density-based kinetics scheme, adapted from standard 'Ignition and Growth' models. The deposition term is based on previous results of simulations of void collapse at the microscale, modelled at the mesoscale as hot spots. For an isolated hot spot in a homogeneous medium, it is found that a critical size of the hot spots exists. If the hot spots exceed the critical size, initiation of detonation can be achieved. For sub-critical hot-spot sizes, we show that it takes a collection of hot spots to achieve detonation. We also carry out two-dimensional mesoscale simulations of random packs of HMX crystals in a binder, and show that the transition between no detonation and detonation depends on the number density of the hot spots, the initial radius of the hot spot, the post-shock pressure of an imposed shock, and the amplitude of the power deposition term.
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Okabe, Yoshihiro; Asamizu, Erika; Saito, Takeshi; Matsukura, Chiaki; Ariizumi, Tohru; Brès, Cécile; Rothan, Christophe; Mizoguchi, Tsuyoshi; Ezura, Hiroshi
2011-01-01
To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1–SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato. PMID:21965606
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Sirikhachornkit, Anchalee; Vuttipongchaikij, Supachai; Suttangkakul, Anongpat; Yokthongwattana, Kittisak; Juntawong, Piyada; Pokethitiyook, Prayad; Kangvansaichol, Kunn; Meetam, Metha
2016-05-28
The production cost of biodiesel from microalgae is still not competitive, compared with that of petroleum fuels. The genetic improvement of microalgal strains to increase triacylglycerol (TAG) accumulation is one way to reduce production costs. One of the most promising approaches is the isolation of starch-deficient mutants, which have been reported to successfully increase TAG yields. To date, such a stable mutant is not available in an oleaginous marine microalga, despite several advantages of using marine species for biodiesel production. Algae in the genus Dunaliella are known to tolerate high salt concentration and other environmental stresses. In addition, the cultivation processes for large-scale outdoor commercialization have been well established for this genus. In this study, Dunaliella tertiolecta was used to screen for starch-deficient mutants, using an iodine vapor-staining method. Four out of 20,016 UV-mutagenized strains showed a substantial reduction of starch content. A significantly higher TAG content, up to 3-fold of the wild-type level, was observed in three of the mutants upon induction by nitrogen depletion. The carotenoid production and growth characteristics of these mutants, under both normal and oxidative stress conditions, were not compromised, suggesting that these processes are not necessarily affected by starch deficiency. The results from this work open up new possibilities for exploring Dunaliella for biodiesel production.
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Vakharia, Hema; German, Greg J.; Misra, Rajeev
2001-01-01
This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed α-helical domain, while the remaining two mapped within the outer membrane-embedded β-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC. PMID:11698380
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Okabe, Yoshihiro; Asamizu, Erika; Saito, Takeshi; Matsukura, Chiaki; Ariizumi, Tohru; Brès, Cécile; Rothan, Christophe; Mizoguchi, Tsuyoshi; Ezura, Hiroshi
2011-11-01
To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.
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Kritikos, A; Neofytos, D; Khanna, N; Schreiber, P W; Boggian, K; Bille, J; Schrenzel, J; Mühlethaler, K; Zbinden, R; Bruderer, T; Goldenberger, D; Pfyffer, G; Conen, A; Van Delden, C; Zimmerli, S; Sanglard, D; Bachmann, D; Marchetti, O; Lamoth, F
2018-06-14
Echinocandins represent the first-line treatment of candidemia. Acquired echinocandin resistance is mainly observed among Candida albicans and glabrata and is associated with FKS hotspot mutations. The commercial Sensititre YeastOne TM (SYO) kit is widely used for antifungal susceptibility testing, but interpretive clinical breakpoints are not well defined. We determined echinocandins epidemiological cut-off values (ECV) for C. albicans/glabrata tested by SYO and assessed their ability to identify FKS mutants in a national survey of candidemia. Bloodstream isolates of C. albicans and C. glabrata were collected in 25 Swiss hospitals from 2004 to 2013 and tested by SYO. FKS hotspot sequencing was performed for isolates with a minimal inhibitory concentration (MIC) ≥ECV for any echinocandin. 1277 C. albicans and 347 C. glabrata were included. ECV 97.5% [μg/ml] of caspofungin, anidulafungin and micafungin were 0.12, 0.06, 0.03 for C. albicans, and 0.25, 0.12, 0.03 for C. glabrata. FKS hotspot sequencing was performed for 70 isolates. No mutation was found in the 52 "limit wild-type" isolates (MIC=ECV for ≥1 echinocandin). Among the 18 "non wild-type" isolates (MIC>ECV for ≥1 echinocandin), FKS mutations were recovered in the only two isolates with MIC>ECV for all 3 echinocandins, but not in those exhibiting a "non wild-type" phenotype for only one or two echinocandins. This 10-year nationwide survey showed that the rate of echinocandin resistance among C. albicans and C. glabrata remains low in Switzerland despite increased echinocandin use. SYO-ECV could discriminate FKS mutants from wild-type isolates tested by SYO in this population. Copyright © 2018. Published by Elsevier Ltd.
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Selection and Characterization of Dunaliella salina Mutants Defective in Haloadaptation 1
Chitlaru, Edith; Pick, Uri
1989-01-01
A technique for selection of Dunaliella mutants defective in their capacity to recover from osmotic shocks has been developed. The selection is based on physical separation of mutants on density gradients. This technique takes advantage of the fact that Dunaliella cells, when exposed to osmotic shocks, initially change volume and density due to water gain or loss and subsequently recover their volume and density by readjusting their intracellular glycerol. Eight mutants that do not recover their original density following hyperosmotic shocks have been isolated. The mutants grow similar to wild type cells in 1 molar NaCl, and recover like the wild type from hypotonic shocks but are defective in recovering from hypertonic shocks. A partial characterization of one of the mutants is described. Images Figure 1 PMID:16667101
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Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yim, L.K.; Stuart, W.D.
This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa. Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C. Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C. Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C. These lines were tested for uptake ofmore » L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations. Cells were tested at 35 and at 39 degrees C. The data were compared to those obtained for the parental HeLa cell line under identical conditions. The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C. Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine. The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system.« less
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Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T
2011-07-01
Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.
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Park, Hye-Jee; Jung, Ho Won; Han, Sang-Wook
2014-09-26
The bacterial envelope possesses diverse functions, including protection against environmental stress and virulence factors for host infection. Here, we report the function of wxcB in Xanthomonas campestris pv. vesicatoria (Xcv), a causal agent of bacterial leaf spot disease in tomato and pepper. To characterize roles of wxcB, we generated a knockout mutant (XcvΔwxcB) and found that the virulence of the mutant was weaker than that of the wild type in tomato plants. To predict the mechanism affected by wxcB, we compared protein expressions between the wild type and the mutant. Expression of 152 proteins showed a greater than 2-fold difference. Proteins involved in motility and cell wall/membrane were the most abundant. Through phenotypic assays, we further demonstrated that the mutant displayed reduced motility and tolerance to treatment, but it showed increased biofilm formation. Interestingly, the LPS profile was unchanged. These results lead to new insights into the functions of wxcB that is associated with cell wall/membrane functions, which contributes to pathogen virulence. Copyright © 2014 Elsevier Inc. All rights reserved.
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Pham, Cau D.; Iqbal, Naureen; Bolden, Carol B.; Kuykendall, Randall J.; Harrison, Lee H.; Farley, Monica M.; Schaffner, William; Beldavs, Zintars G.; Chiller, Tom M.; Park, Benjamin J.; Cleveland, Angela A.
2014-01-01
Candida glabrata is the second leading cause of candidemia in U.S. hospitals. Current guidelines suggest that an echinocandin be used as the primary therapy for the treatment of C. glabrata disease due to the high rate of resistance to fluconazole. Recent case reports indicate that C. glabrata resistance to echinocandins may be increasing. We performed susceptibility testing on 1,380 isolates of C. glabrata collected between 2008 and 2013 from four U.S. cities, Atlanta, Baltimore, Knoxville, and Portland. Our analysis showed that 3.1%, 3.3%, and 3.6% of the isolates were resistant to anidulafungin, caspofungin, and micafungin, respectively. We screened 1,032 of these isolates, including all 77 that had either a resistant or intermediate MIC value with respect to at least one echinocandin, for mutations in the hot spot regions of FKS1 and FKS2, the major mechanism of echinocandin resistance. Fifty-one isolates were identified with hot spot mutations, 16 in FKS1 and 35 in FKS2. All of the isolates with an FKS mutation except one were resistant to at least one echinocandin by susceptibility testing. Of the isolates resistant to at least one echinocandin, 36% were also resistant to fluconazole. Echinocandin resistance among U.S. C. glabrata isolates is a concern, especially in light of the fact that one-third of those isolates may be multidrug resistant. Further monitoring of U.S. C. glabrata isolates for echinocandin resistance is warranted. PMID:24890592
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2014-01-01
Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under photoautotrophic conditions. These results can pave the way towards a more feasible microalgae-driven TAG production platform. PMID:24920957
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Identification and proteomic analysis of a novel gossypol-degrading fungal strain.
Yang, Xia; Sun, Jian-Yi; Guo, Jian-Lin; Weng, Xiao-Yan
2012-03-15
Cottonseed meal, an important source of feed raw materials, has limited use in the feed industry because of the presence of the highly toxic gossypol. The aim of the current work was to isolate the gossypol-degrading fungus from a soil microcosm and investigate the proteins involved in gossypol degradation. A fungal strain, AN-1, that uses gossypol as its sole carbon source was isolated and identified as Aspergillus niger. A large number of intracellular proteins were detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no significant difference was observed between the glucose-containing and gossypol-containing mycelium extracts. Two-dimensional gel electrophoresis results showed that the protein spots were concentrated in the 25.0-66.2 kDa range and distributed in different pI gradients. PDQuest software showed that 51 protein spots in the gels were differentially expressed. Of these, 20 differential protein spots, including six special spots expressed in gossypol, were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The fungus AN-1 biodegraded gossypol and the proteomic analysis results indicate that some proteins were involved in the gossypol biodegradation during fungus survival, using gossypol as its sole carbon source. Copyright © 2011 Society of Chemical Industry.
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Escobar-Tovar, Lina; Guzmán-Quesada, Mauricio; Sandoval-Fernández, Jorge A; Gómez-Lim, Miguel A
2015-06-01
Black Sigatoka, a devastating disease of bananas and plantains worldwide, is caused by the fungus Mycosphaerella fijiensis. Several banana cultivars such as 'Yangambi Km 5' and Calcutta IV, have been known to be resistant to the fungus, but the resistance has been broken in 'Yangambi Km 5' in Costa Rica. Since the resistance of this variety still persists in Mexico, the aim of this study was to compare the in vitro and in planta secretomes from two avirulent and virulent M. fijiensis isolates using proteomics and bioinformatics approaches. We aimed to identify differentially expressed proteins in fungal isolates that differ in pathogenicity and that might be responsible for breaking the resistance in 'Yangambi Km 5'. We were able to identify 90 protein spots in the secretomes of fungal isolates encoding 42 unique proteins and 35 differential spots between them. Proteins involved in carbohydrate transport and metabolism were more prevalent. Several proteases, pathogenicity-related, ROS detoxification and unknown proteins were also highly or specifically expressed by the virulent isolate in vitro or during in planta infection. An unknown protein representing a virulence factor candidate was also identified. These results demonstrated that the secretome reflects major differences between both M. fijiensis isolates. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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Wright, David J.; Tate, Christopher G.
2015-01-01
The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4–5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination. PMID:26143388
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Ji, S H; Gururani, M A; Lee, J W; Ahn, B-O; Chun, S-C
2014-03-01
We have isolated a severe dwarf mutant derived from a Ds (Dissociation) insertion mutant rice (Oryza sativa var. japonica c.v. Dongjin). This severe dwarf phenotype, has short and dark green leaves, reduced shoot growth early in the seedling stage, and later severe dwarfism with failure to initiate flowering. When treated with bioactive GA3 , mutants are restored to the normal wild-type phenotype. Reverse transcription PCR analyses of 22 candidate genes related to the gibberellin (GA) biosynthesis pathway revealed that among 22 candidate genes tested, a dwarf mutant transcript was not expressed only in one OsKS2 gene. Genetic analysis revealed that the severe dwarf phenotype was controlled by recessive mutation of a single nuclear gene. The putative OsKS2 gene was a chromosome 4-located ent-kaurene synthase (KS), encoding the enzyme that catalyses an early step of the GA biosynthesis pathway. Sequence analysis revealed that osks2 carried a 1-bp deletion in the ORF region of OsKS2, which led to a loss-of-function mutation. The expression pattern of OsKS2 in wild-type cv Dongjin, showed that it is expressed in all organs, most prominently in the stem and floral organs. Morphological characteristics of the dwarf mutant showed dramatic modifications in internal structure and external morphology. We propose that dwarfism in this mutant is caused by a point mutation in OsKS2, which plays a significant role in growth and development of higher plants. Further investigation on OsKS2 and other OsKS-like proteins is underway and may yield better understanding of the putative role of OsKS in severe dwarf mutants. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.
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Hermsen, Elizabeth D; Hovde, Laurie B; Konstantinides, George N; Rotschafer, John C
2005-04-01
The purpose of this study was to compare the mutant prevention concentration (MPC) of ABT-492 to those of levofloxacin, moxifloxacin, and gatifloxacin against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The fluoroquinolones had comparable mutation selection windows, which is the ratio of MPC/MIC, for all isolates.
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Potential of multiseeded mutant (msd) to boost sorghum grain yield
USDA-ARS?s Scientific Manuscript database
Seed number per plant is an important determinant of the grain yield in cereal and other crops. We have isolated a class of multiseeded (msd) sorghum (Sorghum bicolor L. Moench) mutants that are capable of producing three times the seed number and twice the seed weight per panicle as compared with t...
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Ali, Sikander; Nawaz, Wajeeha
2017-02-01
The optimisation of nutritional requirements for dopamine (DA) synthesis by calcium alginate-entrapped mutant variant of Aspergillus oryzae EMS-6 using submerged fermentation technique was investigated. A total of 13 strains were isolated from soil. Isolate I-2 was selected as a better producer of DA and improved by exposing with ethyl methylsulphonate (EMS). EMS-6 was selected as it exhibited 43 μg/mL DA activity. The mutant variable was further treated with low levels of l-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of mutant variant were entrapped in calcium alginate beads for stable product formation. EMS-6 gave maximum DA activity (124 μg/mL) when supplemented with 0.1% peptone and 0.2% sucrose, under optimised parameters viz. pH 3, temperature of 55 °C and incubation time of 70 min. The study involves the high profile of DA activity and is needed, as DA is capable to control numerous neurogenic disorders.
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Breidt, F.; Crowley, K. A.; Fleming, H. P.
1993-01-01
Leuconostoc mesenteroides strains that are resistant to high levels of nisin (up to 25,000 IU/ml in broth) were isolated. These nisin-resistant mutants were evaluated to determine their potential use as starter culture strains for cabbage fermentations. We found that some L. mesenteroides strains could be adapted to high levels of nisin resistance, while others could not. The nisin resistance trait was found to be stable for at least 35 generations, in the absence of nisin selection, for all mutants tested. The effects of nisin and salt, separately and in combination, on growth kinetics of the nisin-resistant strains were determined. Salt was the most influential factor on the specific growth rates of the mutants, and no synergistic effect between nisin and salt on specific growth rates was observed. The nisin-resistant strains were unimpaired in their ability to rapidly produce normal heterolactic fermentation end products. The use of these L. mesenteroides mutants as starter cultures in combination with nisin may extend the heterolactic phase of cabbage fermentations. PMID:16349090
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McLean, K M; Gutman, P D; Minton, K W; Clark, E P
1992-06-01
Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).
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Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species▿ ‡
Murray, Gerald L.; Morel, Viviane; Cerqueira, Gustavo M.; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I.; Dellagostin, Odir A.; Bulach, Dieter M.; Sermswan, Rasana W.; Adler, Ben; Picardeau, Mathieu
2009-01-01
Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans. PMID:19047402
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Genome-wide transposon mutagenesis in pathogenic Leptospira species.
Murray, Gerald L; Morel, Viviane; Cerqueira, Gustavo M; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I; Dellagostin, Odir A; Bulach, Dieter M; Sermswan, Rasana W; Adler, Ben; Picardeau, Mathieu
2009-02-01
Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.
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Predicting plasticity with soft vibrational modes: from dislocations to glasses.
Rottler, Jörg; Schoenholz, Samuel S; Liu, Andrea J
2014-04-01
We show that quasilocalized low-frequency modes in the vibrational spectrum can be used to construct soft spots, or regions vulnerable to rearrangement, which serve as a universal tool for the identification of flow defects in solids. We show that soft spots not only encode spatial information, via their location, but also directional information, via directors for particles within each soft spot. Single crystals with isolated dislocations exhibit low-frequency phonon modes that localize at the core, and their polarization pattern predicts the motion of atoms during elementary dislocation glide in two and three dimensions in exquisite detail. Even in polycrystals and disordered solids, we find that the directors associated with particles in soft spots are highly correlated with the direction of particle displacements in rearrangements.
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USDA-ARS?s Scientific Manuscript database
In 2008 fluorescent bacteria were isolated from bacterial leaf spot symptoms on Italian parsley (Petroselinum crispum) in Ceres, California. These isolates were different from the known bacterial pathogens of parsley in California. To determine the etiology of this disease pathogenicity was evaluate...
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Abdominal candidiasis is a hidden reservoir of echinocandin resistance.
Shields, Ryan K; Nguyen, M Hong; Press, Ellen G; Clancy, Cornelius J
2014-12-01
FKS mutant Candida isolates were recovered from 24% (6/25) of abdominal candidiasis patients exposed to echinocandin. Candida glabrata (29%) and Candida albicans (14%) mutants were identified. Multidrug-resistant bacteria were recovered from 83% of FKS mutant infections. Mutations were associated with prolonged echinocandin exposure (P = 0.01), breakthrough infections (P = 0.03), and therapeutic failures despite source control interventions (100%). Abdominal candidiasis is a hidden reservoir for the emergence of echinocandin-resistant Candida. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Kakinuma, Yoshimi; Igarashi, Kazuei
1999-01-01
A K+/H+ antiporter regulates cytoplasmic pH in Enterococcus hirae growing at alkaline pH. Mutants defective in this antiport activity were alkaline pH sensitive. One mutant, Pop1, lacked both K+/methylamine exchange at pH 9.5 and concomitant acidification of cytoplasmic pH. Pop1 grew well at pHs below 8 but did not at pHs above 9, conditions under which cytoplasmic pH was not fully acidified. PMID:10383981
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Mutant E. coli strain with increased succinic acid production
Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy
1998-01-01
A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.
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Mutant E. coli strain with increased succinic acid production
Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy
2001-09-25
A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.
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Mutant E. coli strain with increased succinic acid production
Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy
2002-01-01
A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.
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D'Alessandro, Walmirton B; Humber, Richard A; Luz, Christian
2012-08-13
Two isolates of Beauveria bassiana and one of Purpureocillium lilacinum (=Paecilomyces lilacinus) were found infecting Amblyomma cajennense engorged females collected on horses (0.15% infection rate from a total of 1982 specimens) and another two isolates of P. lilacinum and one Metarhizium anisopliae detected in soils (2.1% from 144 samples) collected in typical pasture habitats of this tick in Central Brazil from October 2009 to March 2011. Fungi were isolated from soils with Rhipicephalus sanguineus as surrogate baits. No fungi were found in ticks or soils during the driest months (May to August). Testing pathogenicity of fungi all R. sanguineus females were killed regardless of the isolate and fungi sporulated abundantly on the cadavers. A. cajennense was less susceptible to infection with P. lilacinum within 20 days than R. sanguineus. All three fungal species probably act as natural antagonists of A. cajennense particularly in the rainy season and have interest for integrate control of vectors of Rocky Mountain spotted fever. Copyright © 2012 Elsevier B.V. All rights reserved.
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Cruz-Ramírez, Alfredo; López-Bucio, José; Ramírez-Pimentel, Gabriel; Zurita-Silva, Andrés; Sánchez-Calderon, Lenin; Ramírez-Chávez, Enrique; González-Ortega, Emmanuel; Herrera-Estrella, Luis
2004-01-01
Phosphocholine (PCho) is an essential metabolite for plant development because it is the precursor for the biosynthesis of phosphatidylcholine, which is the major lipid component in plant cell membranes. The main step in PCho biosynthesis in Arabidopsis thaliana is the triple, sequential N-methylation of phosphoethanolamine, catalyzed by S-adenosyl-l-methionine:phosphoethanolamine N-methyltransferase (PEAMT). In screenings performed to isolate Arabidopsis mutants with altered root system architecture, a T-DNA mutagenized line showing remarkable alterations in root development was isolated. At the seedling stage, the mutant phenotype is characterized by a short primary root, a high number of lateral roots, and short epidermal cells with aberrant morphology. Genetic and biochemical characterization of this mutant showed that the T-DNA was inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT (XIPOTL1). Further analyses revealed that inhibition of PCho biosynthesis in xpl1 mutants not only alters several root developmental traits but also induces cell death in root epidermal cells. Epidermal cell death could be reversed by phosphatidic acid treatment. Taken together, our results suggest that molecules produced downstream of the PCho biosynthesis pathway play key roles in root development and act as signals for cell integrity. PMID:15295103
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Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri.
Jacob, C; Nouzières, F; Duret, S; Bové, J M; Renaudin, J
1997-01-01
The helical mollicute Spiroplasma citri, when growing on low-agar medium, forms fuzzy colonies with occasional surrounding satellite colonies due to the ability of the spiroplasmal cells to move through the agar matrix. In liquid medium, these helical organisms flex, twist, and rotate rapidly. By using Tn4001 insertion mutagenesis, a motility mutant was isolated on the basis of its nondiffuse, sharp-edged colonies. Dark-field microscopy observations revealed that the organism flexed at a low frequency and had lost the ability to rotate about the helix axis. In this mutant, the transposon was shown to be inserted into an open reading frame encoding a putative polypeptide of 409 amino acids for which no significant homology with known proteins was found. The corresponding gene, named scm1, was recovered from the wild-type strain and introduced into the motility mutant by using the S. citri oriC plasmid pBOT1 as the vector. The appearance of fuzzy colonies and the observation that spiroplasma cells displayed rotatory and flexional movements showed the motile phenotype to be restored in the spiroplasmal transformants. The functional complementation of the motility mutant proves the scm1 gene product to be involved in the motility mechanism of S. citri. PMID:9244268
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Bowen, Timothy L.; Whitman, William B.
1987-01-01
Methanococcus voltae incorporated exogenous adenine, guanine, hypoxanthine, and uracil, but not thymine. Growth of M. voltae was also sensitive to purine and pyrimidine analogs. Of the 20 analogs tested, 12 were inhibitory at 1 mg/ml. The most effective inhibitors were purine analogs with endocyclic substitutions. Nucleoside analogs and analogs with exocyclic substitutions or additions were less effective. Four purine analogs, 8-aza-2,6-diaminopurine, 8-azaguanine, 8-azahypoxanthine, and 6-mercaptopurine and one pyrimidine analog, 6-azauracil, were especially toxic. The MICs were 20, 0.5, 2.0, 80, and 10 μg/ml, respectively. Spontaneous resistance mutants were isolated for these five analogs. The MICs for these mutants were 20.5, 8.2, >65, >41, and 20.5 mg/ml, respectively. These concentrations far exceeded the solubilities of the analogs and represented an increase in resistance of at least three orders of magnitude. In addition to demonstrating cross resistance to several of the analogs, four of these mutants lost the ability to incorporate exogenous bases. These appeared to be mutations in the salvage pathways for purines and pyrimidines. In contrast, the mutant resistant to 6-mercaptopurine was not defective in purine uptake. Instead, it degraded 6-mercaptopurine. In the presence or absence of high concentrations of the analogs, the growth rates of the resistant mutants were no less than one-half of the growth rate of the wild type in the absence of the analog. The high level of resistance and rapid growth are very desirable properties for the application of the mutants in genetic experiments. PMID:16347408
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Dahl, Marlis; Müller, Susanne; Voll, Lars M.; Koch, Christian
2015-01-01
We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H+-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi. PMID:25992547
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Chen, M X; Bouquin, N; Norris, V; Casarégola, S; Séror, S J; Holland, I B
1991-01-01
We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein. Images PMID:1915285
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Wu, Chang-Jing; Yi, Le; Cui, Cheng-Bin; Li, Chang-Wei; Wang, Nan; Han, Xiao
2015-01-01
Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1–5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1–5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways. PMID:25913704
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Brockmeier, Susan L.; Loving, Crystal L.; Register, Karen B.; Kehrli, Marcus E.; Stibitz, Scott E.; Shore, Sarah M.
2012-01-01
The majority of virulence gene expression in Bordetella is regulated by a two-component sensory transduction system encoded by the bvg locus. In response to environmental cues, the BvgAS regulatory system controls expression of a spectrum of phenotypic phases, transitioning between a virulent (Bvg+) phase and a nonvirulent (Bvg−) phase, a process referred to as phenotypic modulation. We hypothesized that the ability of Bordetella bronchiseptica to undergo phenotypic modulation is required at one or more points during the infectious cycle in swine. To investigate the Bvg phase-dependent contribution to pathogenesis of B. bronchiseptica in swine, we constructed a series of isogenic mutants in a virulent B. bronchiseptica swine isolate and compared each mutant to the wild-type isolate for its ability to colonize and cause disease. We additionally tested whether a BvgAS system capable of modulation is required for direct or indirect transmission. The Bvg− phase-locked mutant was never recovered from any respiratory tract site at any time point examined. An intermediate phase-locked mutant (Bvgi) was found in numbers lower than the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, colonization of the respiratory tract and disease caused by the Bvg+ phase-locked mutant and the wild-type strain were indistinguishable. The Bvg+ phase-locked mutant transmitted to naïve pigs by both direct and indirect contact with efficiency equal to that of the wild-type isolate. These results indicate that while full activation of the BvgAS regulatory system is required for colonization and severe disease, it is not deleterious to direct and indirect transmission. Overall, our results demonstrate that the Bvg+ phase is sufficient for respiratory infection and host-to-host transmission of B. bronchiseptica in swine. PMID:22158743
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Lister, Philip D
2008-09-01
In a previous study, levofloxacin 750 mg eradicated 4 ciprofloxacin-resistant isolates of Streptococcus pneumoniae from an in vitro pharmacodynamic model (IVPM). However, quinolone resistance-determining region (QRDR) mutations were not detected among those isolates. This study further evaluates levofloxacin 500 mg and 750 mg against S pneumoniae strains with characterized QRDR mutations. Six isolates with levofloxacin minimum inhibitory concentrations (MICs) of 2 to 4 microg/mL were selected for this study. Strains 5401, 5409, and 5437 contained only parC mutations. Three additional strains contained 2 mutations each: strain 5429 (parC and parE ), strain 5442 (parC and gyrA), and strain 5445 (parC and gyrB). Logarithmic-phase cultures (approximately 1 x 10(7) CFU/mL) were inoculated into the peripheral compartment of the IVPM and exposed to peak free-drug concentrations achieved with levofloxacin 500 mg and 750 mg (PO) and ciprofloxacin 750 mg (PO). Elimination pharmacokinetics were simulated and changes in viable counts were measured over 30 h. Ciprofloxacin exhibited very little antibacterial activity against the 6 strains, while levofloxacin 750 mg rapidly killed and eradicated the 3 parC mutant strains and the dual parC/parE mutant strains. Although levofloxacin 500 mg initially decreased viable counts by 4.5 to 6 logs, inoculum regrowth was observed between 12 and 24 h for the 6 strains. Regrowth was not due to the selection of mutant subpopulations. The pharmacodynamics of both levofloxacin doses were substantially diminished against the 2 strains with dual mutations in both parC and gyrA/B. The rapid eradication of single parC and dual parC/parE mutants with levofloxacin 750 mg demonstrates that this dose may slow the emergence of resistance due to these mutations. The decreased efficacy of both levofloxacin doses against the double parC and gyrA/B mutants highlights the importance of preventing the development and spread of double mutants.
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Wu, Chang-Jing; Yi, Le; Cui, Cheng-Bin; Li, Chang-Wei; Wang, Nan; Han, Xiao
2015-04-22
Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α, 6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1-5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1-5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.
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Serratia marcescens arn, a PhoP-regulated locus necessary for polymyxin B resistance.
Lin, Quei Yen; Tsai, Yi-Lin; Liu, Ming-Che; Lin, Wei-Cheng; Hsueh, Po-Ren; Liaw, Shwu-Jen
2014-09-01
Polymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly against Serratia marcescens. To investigate the underlying mechanisms, Tn5 mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5 inserted into the arnB and arnC genes. In other bacteria, arnB and arnC belong to the seven-gene arn operon, which is involved in lipopolysaccharide (LPS) modification. LPSs of arn mutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility in S. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression of phoP and arn in the wild-type strain but not in the phoP mutant. Complementation of the phoP mutant with the full-length phoP gene restored the PB MIC and induction by PB and low Mg2+ levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to the arn promoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+ levels protected S. marcescens from a PB challenge in the wild-type strain but not in the phoP mutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression of ugd, a gene required for LPS modification, in S. marcescens through a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression of arnA upon exposure to PB than did susceptible isolates. This is the first report to describe the role of S. marcescens arn in PB resistance and its modulation by PB and Mg2+ through the PhoP protein. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Serratia marcescens arn, a PhoP-Regulated Locus Necessary for Polymyxin B Resistance
Lin, Quei Yen; Tsai, Yi-Lin; Liu, Ming-Che; Lin, Wei-Cheng; Hsueh, Po-Ren
2014-01-01
Polymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly against Serratia marcescens. To investigate the underlying mechanisms, Tn5 mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5 inserted into the arnB and arnC genes. In other bacteria, arnB and arnC belong to the seven-gene arn operon, which is involved in lipopolysaccharide (LPS) modification. LPSs of arn mutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility in S. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression of phoP and arn in the wild-type strain but not in the phoP mutant. Complementation of the phoP mutant with the full-length phoP gene restored the PB MIC and induction by PB and low Mg2+ levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to the arn promoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+ levels protected S. marcescens from a PB challenge in the wild-type strain but not in the phoP mutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression of ugd, a gene required for LPS modification, in S. marcescens through a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression of arnA upon exposure to PB than did susceptible isolates. This is the first report to describe the role of S. marcescens arn in PB resistance and its modulation by PB and Mg2+ through the PhoP protein. PMID:24957827
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James M. Slavicek; Melissa J. Mercer; Mary Ellen Kelly; Nancy Hayes-Plazolles
1996-01-01
The formation of few polyhedra mutants during serial propagation of baculoviruses in cell culture encumbers commercial scale production in this system. A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) variant (isolate A21-MPV) has been isolated and the traits of budded virus (BV) production, synthesis of polyhedra, the...
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Investigating the link between imipenem resistance and biofilm formation by Pseudomonas aeruginosa.
Musafer, Hadeel K; Kuchma, Sherry L; Naimie, Amanda A; Schwartzman, Joseph D; Al-Mathkhury, Harith J Fahad; O'Toole, George A
2014-07-01
Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC ≤ 2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC ≥ 8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.
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Yassien, M A M; Elfaky, M A
2015-11-01
A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.
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Marzec, M; Muszynska, A; Melzer, M; Sas-Nowosielska, H; Kurczynska, E U; Wick, S
2014-01-01
It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. ‘Karat’ with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants. PMID:23927737
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Parkinson, Elizabeth I; Bair, Joseph S; Nakamura, Bradley A; Lee, Hyang Y; Kuttab, Hani I; Southgate, Emma H; Lezmi, Stéphane; Lau, Gee W; Hergenrother, Paul J
2015-04-24
Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections.
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Fukuda, K; Watanabe, M; Asano, K; Ouchi, K; Takasawa, S
1991-12-01
p-Fluoro-DL-phenylalanine (PFP)-resistant mutants which produce a large amount of beta-phenethyl-alcohol, a rose-like flavor component, were isolated from the isogenic strains X2180-1A and X2180-1B of Saccharomyces cerevisiae. Cells of these mutants accumulated phenylalanine and tryptophan more than 3-fold times that of wild-type cells, while they accumulated less than half the tyrosine. The activity of prephenate dehydrogenase (PDG) (EC 1.3.1.12) was markedly decreased while that of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) was increased. Genetic analysis revealed that the mutation occurred at the TYR1 locus, encoding PDG, and that the mutated TYR1 gene, try1-pfp, caused both PFP resistance and beta-phenethyl-alcohol overproduction. This was supported by molecular genetic studies with cloned tyr1-pfp DNA.
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Thompson, L A; Gates, D M; Ingledew, W M; Jones, G A
1976-01-01
The Hungate anaerobic technique was used with a standard procedure for bacterial mutagenesis employing N-methyl-N-nitro-N'-nitrosoguanidine to obtain mutants of an obligate anaerobe. Three mutant strains were derived from a Coprococcus sp., strain Pe15, a rumen anaerobe capable of growing on phloroglucinol. The mutants did not grow on phloroglucinol but did degrade the compound in anaerobic washed-cell suspensions, producing the same end products in approximately the same proportions as the wild type. It was concluded that the mutants were blocked in a unique step or steps necessary for carbon skeleton or energy synthesis from phloroglucinol and not in formation of an enzyme involved in the pathway of phloroglucinol degradation. PMID:782358
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Thompson, L A; Gates, D M; Ingledew, W M; Jones, G A
1976-01-01
The Hungate anaerobic technique was used with a standard procedure for bacterial mutagenesis employing N-methyl-N-nitro-N'-nitrosoguanidine to obtain mutants of an obligate anaerobe. Three mutant strains were derived from a Coprococcus sp., strain Pe15, a rumen anaerobe capable of growing on phloroglucinol. The mutants did not grow on phloroglucinol but did degrade the compound in anaerobic washed-cell suspensions, producing the same end products in approximately the same proportions as the wild type. It was concluded that the mutants were blocked in a unique step or steps necessary for carbon skeleton or energy synthesis from phloroglucinol and not in formation of an enzyme involved in the pathway of phloroglucinol degradation.
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Sevastos, A; Markoglou, A; Labrou, N E; Flouri, F; Malandrakis, A
2016-03-01
Six benzimidazole (BMZ)-resistant Fusarium graminearum strains were obtained after UV mutagenesis and selection on carbendazim (MBC)-amended medium. In vitro bioassays resulted in the identification of two resistant phenotypes that were highly HR (Rf: 40-170, based on EC50) and moderately MR (Rf: 10-20) resistant to carbendazim. Cross resistance studies with other fungicides showed that all mutant strains tested were also resistant to other BMZs, such as benomyl and thiabendazole, but retained their parental sensitivity to fungicides belonging to other chemical groups. A point mutation at codon 6 (His6Asn) was found in the β2-tubulin gene of MR isolates while another mutation at codon 200 (Phe200Tyr) was present in one MR and one HR isolates. Interestingly, low temperatures suppressed MBC-resistance in all isolates bearing the H6N mutation. The three-dimensional homology model of the wild-type and mutants of β-tubulins were constructed, and the possible carbendazim binding site was analyzed. Studies on fitness parameters showed that the mutation(s) for resistance to BMZs did not affect the mycelial growth rate whereas adverse effects were found in sporulation and conidial germination in most of the resistant mutants. Pathogenicity tests on corn cobs revealed that mutants were less or equally aggressive to the wild-type strain but expressed their BMZ-resistance after inoculation on maize cobs treated with MBC. Analysis of mycotoxin production by high performance liquid chromatography revealed that only two HR strains produced zearalenone (ZEA) at concentrations similar to that of the wild-type strain, while no ZEA levels were detected in the rest of the mutants. Copyright © 2015 Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hoffman, R.M.; Wood, T.M.
1985-01-01
Clearing of agar plates containing ball-milled, delignified straw has been used for screening mutants of Penicillium funiculosum IMI 87160 III. The effects of glycerol and a number of sugars on the clearing were investigated for selecting derepressed mutants. The ..beta..-glucosidase synthesis by one such mutant, C22c, in shake flasks containing straw was not repressed by 5% glycerol, whereas activities on filter paper, CM-cellulose, and p-nitrophenyl-..beta..-xylosidase were only partially derepressed; xylanase was extensively derepressed. The evidence for separate control of the enzymes involved in the solubilization of straw is discussed. 23 references.
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FOOD MICROORGANISMS INFLUENCING THE GROWTH OF STAPHYLOCOCCUS AUREUS.
GRAVES, R R; FRAZIER, W C
1963-11-01
Some 870 cultures of predominating micro-organisms were isolated from market samples of hamburger, fresh pork sausage, fresh fish fillets, stewing beef, frozen chicken pot pie, frozen corn, frozen peas, and pasteurized and raw milk, before and after storage at different temperatures. The isolates were screened for their ability to influence the growth of Staphylococcus aureus strain 196E by means of spot-plate tests on APT and nutrient agars at 25 C. The 438 cultures that influenced the growth of S. aureus were retested on spot plates at 15, 30, and 42 C. After elimination of replicates, the 143 remaining cultures were classified into species, genera, or groups, and 14 different cultures were tested for their influence on the growth of S. aureus in APT broth at 25 C. Over half of the effective cultures inhibited S. aureus and less than half were stimulatory. Pork sausage had the highest proportion of inhibitory cultures, and stewing beef had the lowest. APT agar was better than nutrient agar for screening, and incubation at 15 C gave more effector organisms than at 30 and 42 C. Most of the lactic acid bacteria were inhibitory, but other groups of bacteria contained more stimulatory cultures than inhibitory ones. The three Escherichia coli cultures were stimulatory, but most other Escherichia cultures were inhibitory. Aerobacter and Paracolobactrum isolates were mostly stimulatory. Cultures of other kinds of bacteria were more or less evenly distributed between inhibitory ones and stimulatory ones. Genera containing mostly inhibitory bacteria were Streptococcus, Leuconostoc, and Lactobacillus. Inhibitory species were E. freundii and E. intermedia. Tests with S. aureus in broth indicated that all cultures inhibitory according to spot plates were inhibitory in broth, but stimulation on spot plates did not always indicate the same phenomenon in broth.
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Analysis of Binding Site Hot Spots on the Surface of Ras GTPase
DOE Office of Scientific and Technical Information (OSTI.GOV)
Buhrman, Greg; O; #8242
2012-09-17
We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond themore » active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.« less
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Demography of Northern Spotted Owls in southwestern Oregon
Zabel, Cynthia J.; Salmons, Susan E.; Forsman, Eric D.; DeStefano, Stephen; Raphael, Martin G.; Gutierrez, R.J.
1996-01-01
Northern Spotted Owls (Strix occidentalis caurina) are associated with lower elevation, commercially valuable, late-successional coniferous forests in the Pacific Northwest. Meta-analyses of demographic parameters indicate that Northern Spotted Owl populations are declining throughout their range (Anderson and Burnham 1992, Burnham et al. this volume). Recent research has attempted to determine whether management activities have affected the viability of Spotted Owl populations, and results have led to development of conservation plans for the species (Dawson et al. 1987, Thomas et al. 1990, Murphy and Noon 1992, USDI 1992, Thomas et al. 1993b).In the Recovery Plan for the Northern Spotted Owl (USDI 1992b) threats to the species were identified as small population sizes, declining populations, limited amounts of habitat, continued loss and fragmentation of habitat, geographically isolated populations, and predation and competition from other avian species. Weather and fire are natural processes that also may affect reproductive success of Spotted Owls. Weather may be a factor in the high annual variability in fecundity of Spotted Owls, as has been suggested for other predatory bird species (Newton, 1979, 1986). However, these factors have not been addressed in previous studies of Spotted Owls.Our objectives were to estimate survival, fecundity, and annual rates of population change (l) for resident, territorial female Spotted Owls at two study areas in the coastal mountains of southwestern Oregon. We tested if the amount of rainfall was correlated with reproduction of Spotted Owls. While surveying for Spotted Owls, we documented the increased presence of Barred Owls (Strix varia), a potential competitor of Spotted Owls.
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Diago-Navarro, Elizabeth; Mora, Liliana; Buckingham, Richard H; Díaz-Orejas, Ramón; Lemonnier, Marc
2009-01-01
Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.
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Bissett, Donald L.; Anderson, Richard L.
1974-01-01
Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain. PMID:4277494
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Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji
2013-12-01
The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.
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Hermsen, Elizabeth D.; Hovde, Laurie B.; Konstantinides, George N.; Rotschafer, John C.
2005-01-01
The purpose of this study was to compare the mutant prevention concentration (MPC) of ABT-492 to those of levofloxacin, moxifloxacin, and gatifloxacin against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The fluoroquinolones had comparable mutation selection windows, which is the ratio of MPC/MIC, for all isolates. PMID:15793158
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Rensch, Ulrike; Klein, Guenter; Kehrenberg, Corinna
2013-01-01
The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPCTRC values were 8 - 64-fold higher than MIC values and ranged between 1 - 16 µg/ml. The frequencies at which mutants were selected varied between 1.3 x 10-10 - 9.9 x 10-11. Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly93→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 µg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly93→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased susceptibility to triclosan. PMID:24205194
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Jahn, B; Koch, A; Schmidt, A; Wanner, G; Gehringer, H; Bhakdi, S; Brakhage, A A
1997-01-01
Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. The factors contributing to the predominance of A. fumigatus as an opportunistic pathogen are largely unknown. Since the survival of conidia in the host is a prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, important for infection. Therefore, an A. fumigatus mutant strain (white [W]) lacking conidial pigmentation was isolated. Scanning electron microscopy revealed that conidia of the W mutant also differed in their surface morphology from those of the wild type (WT). Mutant (W) and WT conidia were compared with respect to their capacities to stimulate an oxidative response in human phagocytes, their intracellular survival in human monocytes, and virulence in a murine animal model. Luminol-dependent chemiluminescence was 10-fold higher when human neutrophils or monocytes were challenged with W conidia compared with WT conidia. Furthermore, mutant conidia were more susceptible to killing by oxidants in vitro and were more efficiently damaged by human monocytes in vitro than WT conidia. In a murine animal model, the W mutant strain showed reduced virulence compared with the WT. A reversion analysis of the W mutant demonstrated that all phenotypes associated with the W mutant, i.e., altered conidial surface, amount of reactive oxygen species release, susceptibility to hydrogen peroxide, and reduced virulence in an murine animal model, coreverted in revertants which had regained the ability to produce green spores. This finding strongly suggests that the A. fumigatus mutant described here carries a single mutation which caused all of the observed phenotypes. Our results suggest that the conidium pigment or a structural feature related to it contributes to fungal resistance against host defense mechanisms in A. fumigatus infections. PMID:9393803
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Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gibson, Susan I.
Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affectmore » sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation. Characterization of mutant and wild-type plants has revealed that sugars inhibit breakdown of seed storage lipids. In addition, high concentrations of exogenous sugars largely eliminate the development of mature chloroplasts by developing seedlings. Affymetrix GeneChip experiments have revealed that expression of many plant genes is partially regulated by sugar levels, with approximately two percent of genes exhibiting alterations in steady-state mRNA levels in response to changing sugar concentrations. Ultimately, a better understanding of plant sugar responses may allow improvements in rates of carbon fixation and manipulation of carbon partitioning. These improvements will be needed to help make production of energy from biomass more economically attractive.« less
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Martin, F; Reinbolt, J; Dirheimer, G; Gangloff, J; Eriani, G
1996-01-01
Elements that confer identity to a tRNA in the cellular environment, where all aminoacyl-tRNA synthetases are competing for substrates, may be delineated by in vivo experiments using suppressor tRNAs. Here we describe the selection of active Escherichia coli tRNAAsp amber mutants and analyze their identity. Starting from a library containing randomly mutated tRNA(CUA)Asp genes, we isolated four amber suppressors presenting either lysine, alanine, or glutamine activity. Two of them, presenting mainly alanine or lysine activity, were further submitted to a second round of mutagenesis selection in order to improve their efficiency of suppression. Eleven suppressors were isolated, each containing two or three mutations. Ten presented identities of the two parental mutants, whereas one had switched from lysine to arginine identity. Analysis of the different mutants revealed (or confirmed for some nucleotides) their role as positive and/or negative determinants in AlaRS, LysRS, and ArgRS recognition. More generally, it appears that tRNAAsp presents identity characteristics closely related to those of tRNALys, as well as a structural basis for acquiring alanine or arginine identity upon moderate mutational changes; these consist of addition or suppression of the corresponding positive or negative determinants, as well as tertiary interactions. Failure to isolate aspartic acid-inserting suppressors is probably due to elimination of the important G34 identity element and its replacement by an antideterminant when changing the anticodon of the tRNAAsp to the CUA triplet. PMID:8809018
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Abouelfetouh, Alaa; Kassem, Mervat; Naguib, Marwa; El-Nakeeb, Moustafa
2017-01-01
Methicillin resistance among staphylococci isolated from patients in northern Egypt has escalated alarmingly in the past decade. Data about the prevalence of fusidic acid (FA) resistance in Egyptian clinical isolates are limited. This work investigates the prevalence and mechanism of FA resistance among 81 methicillin-resistant staphylococcal isolates from major hospitals of Alexandria, Egypt. Some combinations for treating infections due to resistant isolates were studied. Twenty-six isolates (32.1%) were FA resistant (minimum inhibitory concentrations [MICs] = 2-1,024 μg/ml), and fusB and fusC genes coding for FA resistance were detected in 30.77% and 34.62% of the FA-resistant strains, respectively. One highly resistant isolate, S502 (MIC = 1,024 μg/ml), possessed both genes. Plasmid curing resulted in fusB loss and MIC decrease by 16-64 folds. Conjugation caused acquisition of FA resistance among susceptible isolates. Serial passages in subinhibitory FA concentrations produced mutants with increased MIC by 4-32 folds. The combination of FA with rifampin, gentamicin, or ampicillin/sulbactam, in a subinhibitory concentration, was synergistic against the isolates, including serial passage mutants, decreasing number of survivors by an average of 2-4 logs. A relatively moderate rate of FA resistance was detected in Alexandria hospitals. Combination therapy with gentamicin, rifampin, or ampicillin/sulbactam is crucial to preserve the effectiveness of FA.
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Estimation of repetitive interval of periodic bands in laser electrophotographic printer output
NASA Astrophysics Data System (ADS)
Zhang, Jia; Allebach, Jan P.
2015-01-01
In the printing industry, electrophotography (EP) is a commonly used technology in laser printers and copiers. In the EP printing process, there are many rotating components involved in the six major steps: charging, exposure, development, transfer, fusing, and cleaning. If there is any irregularity in one of the rotating components, repetitive defects, such as isolated bands or spots, will occur on the output of the printer or copier. To troubleshoot these types of repetitive defect issues, the repeating interval of these isolated bands or spots is an important clue to locate the irregular rotating component. In our previous work, we have effectively identified the presence of isolated large pitch bands in the output from EP printers. In this paper, we describe an algorithm to estimate the repetitive interval of periodic bands, when the data is corrupted by the presence of aperiodic bands, missing periodic bands, and noise. We will also illustrate the effectiveness and robustness of our method with example results.
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Pomini, Armando M; Paccola-Meirelles, Luzia D; Marsaioli, Anita J
2007-02-21
The "maize white spot" foliar disease is a problem of increasing importance to Brazilian maize crops. A bacterium isolated from water-soaked lesions from infected maize leaves was pathogenic in biological assays in vivo. It was identified as a Gram-negative, nonsporulating, facultative anaerobic bacterium, belonging to the genus Pantoea. Chemical study of the extracts from bacterial cultivation media allowed the identification of (S)-(-)-N-butanoyl-homoserine lactone and trace amounts of N-hexanoyl-homoserine lactone, widely recognized quorum-sensing signaling substances employed in cell-to-cell communication systems. The absolute configuration of natural (S)-(-)-N-butanoyl-homoserine lactone was determined by gas chromatography-flame ionization detection with a chiral stationary phase and by comparison of circular dichroism spectroscopic data with enantiopure synthetic substances. Biological evaluations with reporter Agrobacterium tumefaciens NTL4(pZLR4) were carried out with synthetic and natural products and also with extracts from maize leaves contaminated with the isolated bacterium, as well as from healthy leaves.
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Pham, Cau D; Iqbal, Naureen; Bolden, Carol B; Kuykendall, Randall J; Harrison, Lee H; Farley, Monica M; Schaffner, William; Beldavs, Zintars G; Chiller, Tom M; Park, Benjamin J; Cleveland, Angela A; Lockhart, Shawn R
2014-08-01
Candida glabrata is the second leading cause of candidemia in U.S. hospitals. Current guidelines suggest that an echinocandin be used as the primary therapy for the treatment of C. glabrata disease due to the high rate of resistance to fluconazole. Recent case reports indicate that C. glabrata resistance to echinocandins may be increasing. We performed susceptibility testing on 1,380 isolates of C. glabrata collected between 2008 and 2013 from four U.S. cities, Atlanta, Baltimore, Knoxville, and Portland. Our analysis showed that 3.1%, 3.3%, and 3.6% of the isolates were resistant to anidulafungin, caspofungin, and micafungin, respectively. We screened 1,032 of these isolates, including all 77 that had either a resistant or intermediate MIC value with respect to at least one echinocandin, for mutations in the hot spot regions of FKS1 and FKS2, the major mechanism of echinocandin resistance. Fifty-one isolates were identified with hot spot mutations, 16 in FKS1 and 35 in FKS2. All of the isolates with an FKS mutation except one were resistant to at least one echinocandin by susceptibility testing. Of the isolates resistant to at least one echinocandin, 36% were also resistant to fluconazole. Echinocandin resistance among U.S. C. glabrata isolates is a concern, especially in light of the fact that one-third of those isolates may be multidrug resistant. Further monitoring of U.S. C. glabrata isolates for echinocandin resistance is warranted. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Li, Han-Guang; Ofosu, Fred Kwame; Li, Kun-Tai; Gu, Qiu-Ya; Wang, Qiang; Yu, Xiao-Bin
2014-11-01
To obtain native strains resistant to butanol toxicity, a new isolating method and serial enrichment was used in this study. With this effort, mutant strain SE36 was obtained, which could withstand 35g/L (compared to 20g/L of the wild-type strain) butanol challenge. Based on 16s rDNA comparison, the mutant strain was identified as Clostridium acetobutylicum. Under the optimized condition, the phase shift was smoothly triggered and fermentation performances were consequently enhanced. The maximum total solvent and butanol concentration were 23.6% and 24.3%, respectively higher than that of the wild-type strain. Furthermore, the correlation between butanol produced and the butanol tolerance was investigated, suggesting that enhancing butanol tolerance could improve butanol production. These results indicate that the simple but effective isolation method and acclimatization process are a promising technique for isolation and improvement of butanol tolerance and production. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Kim, Yonghwan; Koo, Bong-Seong; Lee, Hyeon-Cheol; Yoon, Youngdae
2015-03-01
Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.
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Mo, Hongmei; Parkin, Neil; Stewart, Kent D.; Lu, Liangjun; Dekhtyar, Tatyana; Kempf, Dale J.; Molla, Akhteruzzaman
2007-01-01
Two novel human immunodeficiency virus protease mutations, I84C and I84A, were identified in patient isolates. The mutants with I84C displayed high-level resistance (median, at least 56-fold) to nelfinavir and saquinavir, but the majority remained susceptible to lopinavir. In contrast, isolates with the I84A mutation exhibited ≥33-fold median increased levels of resistance to nelfinavir, indinavir, amprenavir, ritonavir, lopinavir, saquinavir, and atazanavir. Isolates with the I84A or I84C mutation tended to be more resistant than the isolates with the I84V mutation. Modeling of the structure of the mutant proteases indicated that the I84V, I84C, and I84A mutations all create unoccupied volume in the active site, with I84A introducing the greatest change in the accessible surface area from that of the wild-type structure. PMID:17101675
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Ribosomal protein L24 defect in Belly spot and tail (Bst), a mouse Minute
Oliver, Edward R.; Saunders, Thomas L.; Tarlé, Susan A.; Glaser, Tom
2008-01-01
Summary Ribosomal protein mutations, termed Minutes, have been instrumental in studying the coordination of cell and tissue growth in Drosophila. Although abundant in flies, equivalent defects in mammals are relatively unknown. Belly spot and tail (Bst) is a semidominant mouse mutation that disrupts pigmentation, somitogenesis and retinal cell fate determination. Here, we identify Bst as a deletion within the Rpl24 riboprotein gene. Bst significantly impairs Rpl24 splicing and ribosome biogenesis. Bst/+ cells have decreased rates of protein synthesis and proliferation, and are outcompeted by wild-type cells in C57BLKS↔ROSA26 chimeras. Bacterial artificial chromosome (BAC) and cDNA transgenes correct the mutant phenotypes. Our findings establish Bst as a mouse Minute and provide the first detailed characterization of a mammalian ribosomal protein mutation. PMID:15289434
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Burke, John J.; Steinback, Katherine E.; Arntzen, Charles J.
1979-01-01
we have compared chloroplast lamellae isolated from a chlorophyll-b-less mutant and wild type barley (Hordeum vulgare). The results demonstrate that: (a) one of the two major polypeptides comprising the lightharvesting complex (LHC) is present in the chlorophyll-b-less mutant; (b) higher cation concentrations are required to maintain grana stacks in the mutant; and (c) cation effects on excitation energy distribution are present in the chlorophyll-b-less mutant but are reduced in amount and are dependent on higher concentrations of cations. We interpret these data to support the concept that the LHC mediates cation-induced grana stacking and cation regulation of excitation energy distribution between photosystems I and Ii in chloroplast lamellae. A partial LHC complement in the mutant alters the quantitative cation requirement for both phenomena, but not the over-all qualitative response. Images PMID:16660704
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Das, Manuj K; Chetry, Sumi; Kalita, Mohan C; Dutta, Prafulla
2016-12-01
North-east region of India has consistent role in the spread of multi drug resistant Plasmodium (P.) falciparum to other parts of Southeast Asia. After rapid clinical treatment failure of Artemisinin based combination therapy-Sulphadoxine/Pyrimethamine (ACT-SP) chemoprophylaxis, Artemether-Lumefantrine (ACT-AL) combination therapy was introduced in the year 2012 in this region for the treatment of uncomplicated P. falciparum malaria. In a DNA sequencing based polymorphism analysis, seven codons of P. falciparum dihydropteroate synthetase ( Pf dhps) gene were screened in a total of 127 P. falciparum isolates collected from Assam, Arunachal Pradesh and Tripura of North-east India during the year 2014 and 2015 to document current sulfadoxine resistant haplotypes. Sequences were analyzed to rearrange both nucleotide and protein haplotypes. Molecular diversity indices were analyzed in DNA Sequence Polymorphism software (DnaSP) on the basis of Pf dhps gene sequences. Disappearance from selective neutrality was assessed based on the ratio of non-synonomous to synonomous nucleotide substitutions [dN/dS ratio]. Moreover, two-tailed Z test was performed in search of the significance for probability of rejecting null hypothesis of strict neutrality [dN = dS]. Presence of mutant P. falciparum multidrug resistance protein1 ( Pf mdr1) was also checked in those isolates that were present with new Pf dhps haplotypes. Phylogenetic relationship based on Pf dhps gene was reconstructed in Molecular Evolutionary Genetics Analysis (MEGA). Among eight different sulfadoxine resistant haplotypes found, IS GNG A haplotype was documented in a total of five isolates from Tripura with association of a new mutant M538 R allele. Sequence analysis of Pf mdr1 gene in these five isolates came to notice that not all but only one isolate was mutant at codon 86 (N86 Y ; Y YSND) in the multidrug resistance protein. Molecular diversity based on Pf dhps haplotypes revealed that P. falciparum populations in Assam and Tripura were under balancing selection for sulfadoxine resistant haplotypes but population from Arunachal Pradesh was under positive selection with comparatively high haplotype diversity ( h = 0.870). In reconstructed phylogenetic analysis, isolates having IS GNG A haplotype were grouped into two separate sub-clusters from the other isolates based on their genetic distances and diversities. This study suggests that sulfadoxine resistant isolates are still migrating from its epicenter to the other parts of Southeast Asia and hence control and elimination of the drug resistant isolates have become impedimental. Moreover, P. falciparum populations in different areas may undergo selection of particular sulfadoxine resistant haplotypes either in the presence of drug or after its removal to maintain their plasticity.
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Silva-Oliveira, R G; Orsolin, P C; Nepomuceno, J C
2016-09-01
Losartan potassium is an antihypertensive drug in the angiotensin II receptor antagonist (ARA) class. Some studies claim that, in addition to regulating blood pressure, this class of drug has anticancer properties. The objective of this study was to evaluate the genotoxic and antigenotoxic potential of losartan potassium using the SMART (Somatic Mutation and Recombination Test) assay on the somatic cells of Drosophila melanogaster, as well as the possible modulating effects of this drug, when associated with doxorubicin (DXR). Third instar larvae, descendents of standard and high bioactivation (ST and HB) crosses, were chronically treated with different concentrations of losartan potassium (0.25; 0.5; 1; 2; and 4 mM) alone or in association (co-treatment) with doxorubicin (DXR 0.125 mg/mL). The results showed an absence of a mutagenic effect of losartan potassium. In the co-treatment of losartan with DXR, the results showed that losartan is capable of reducing the number of mutant spots induced by DXR without altering the recombinogenic effect of the chemotherapeutic agent. Antiproliferative action appears to be the main mechanism involved in reducing the frequency of mutant spots and consequent modulation of alterations induced by DXR, although this parameter has not been directly assessed in this study. Copyright © 2016. Published by Elsevier Ltd.
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Mutant E. coli strain with increased succinic acid production
Donnelly, M.; Millard, C.S.; Stols, L.
1998-06-23
A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.
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Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni.
Gencay, Yilmaz Emre; Birk, Tina; Sørensen, Martine Camilla Holst; Brøndsted, Lone
2017-01-01
Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.
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Morphological and Genetic Analysis of Four Color Morphs of Bean Leaf Beetle.
Tiroesele, Bamphitlhi; Skoda, Steven R; Hunt, Thomas E; Lee, Donald J; Ullah, Muhammad Irfan; Molina-Ochoa, Jaime; Foster, John E
2018-03-01
Bean leaf beetle (BLB), Cerotoma trifurcata (Forster; Coleoptera: Chrysomelidae), exhibits considerable color variation but little is known about the underlying genetic structure and gene flow among color phenotypes. Genetic and morphological variation among four color phenotypes-green with spots (G+S), green without spots (G-S), red with spots (R+S) and red without spots (R-S)-were analyzed using amplified fragment length polymorphisms (AFLP) and morphometrics, respectively. AFLP generated 175 markers that showed ≥80% polymorphism. Analysis of molecular variance (AMOVA) indicated that genetic variation was greatest within phenotypes (82.6-84.0%); gene flow among the four phenotypes was relatively high (Nm = 3.82). The dendrogram and STRUCTURE analysis indicated some population divergence of G-S from the other phenotypes. Morphological parameters were similar among phenotypes except that R+S showed significant differences in weight and body-length. Canonical variables 1 and 2, based on average morphometric characters, accounted for 98% of the total variation; some divergence was indicated between G+S and R+S from each other and from the G-S/R-S BLB color morphs. The pattern of genetic variation indicated potential divergence of G-S and G+S from each other and from R-S and R+S. Although these results indicate that the four different color morphs are not genetically or reproductively isolated, there is some genetic differentiation/structure and morphological dissimilarity suggesting weak/incomplete isolation.
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Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene†
Wysong, Deborah R.; Christin, Laurent; Sugar, Alan M.; Robbins, Phillips W.; Diamond, Richard D.
1998-01-01
Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans. PMID:9573075
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Margis-Pinheiro, Marcia; Zhou, Xue-Rong; Zhu, Qian-Hao; Dennis, Elizabeth S; Upadhyaya, Narayana M
2005-03-01
We have isolated a severe dwarf transposon (Ds) insertion mutant in rice (Oryza sativa L.), which could be differentiated early in the seedling stage by reduced shoot growth and dark green leaves, and later by severe dwarfism and failure to initiate flowering. These mutants, however, showed normal seed germination and root growth. One of the sequences flanking Ds, rescued from the mutant, was of a chromosome 4-located putative ent-kaurene synthase (KS) gene, encoding the enzyme catalyzing the second step of the gibberellin (GA) biosynthesis pathway. Dwarf mutants were always homozygous for this Ds insertion and no normal plants homozygous for this mutation were recovered in the segregating progeny, indicating that the Ds insertion mutation is recessive. As mutations in three recently reported rice GA-responsive dwarf mutant alleles and the dwarf mutation identified in this study mapped to the same locus, we designate the corresponding gene OsKS1. The osks1 mutant seedlings were responsive to exogenous gibberellin (GA3). OsKS1 transcripts of about 2.3 kb were detected in leaves and stem of wild-type plants, but not in germinating seeds or roots, suggesting that OsKS1 is not involved in germination or root growth. There are at least five OsKS1-like genes in the rice genome, four of which are also represented in rice expressed sequence tag (EST) databases. All OsKS1-like genes are transcribed with different expression patterns. ESTs corresponding to all six OsKS genes are represented in other cereal databases including barley, wheat and maize, suggesting that they are biologically active.
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NASA Astrophysics Data System (ADS)
McAllister, P. E.; Newman, M. W.; Sauber, J. H.; Owens, W. J.
1984-03-01
Two significant fish kills occurred in the Pamlico River estuary (North Carolina, USA), one in December 1981 and January 1982, and the other in June 1982. The first involved only the southern flounder (Paralichthys lethostigma). Histopathologic examination of morbid and moribund flounder revealed extensive sloughing and necrosis of the mucosa of the pyloric caeca and intestine, and inflammation of the submucosa of the pyloric caeca. Brain and internal organ homogenates from morbid and moribund flounder were assayed on CHSE-214 cells, and a virus was isolated. Virus titers ranged from≤8.4 · 102 to 6.3 · 107 TCID50 per gram of tissue. Cross-plaque neutralization assays indicated that the southern flounder virus was infectious pancreatic necrosis virus serotype Ab. Immersion challenge showed the isolate is only slightly virulent for fry of brook trout (Salvelinus fontinalis). The second fish kill involved the southern flounder and six other species: hogchoker (Trinectes maculatus), Atlantic silverside (Menidia menidia), spot (Leiostomus xanthurus), Atlantic croaker (Micropogon undulatus), silver perch (Bairdiella chrysura), and striped mullet (Mugil cephalus). Virus was isolated from southern founder, hogchoker, Atlantic silverside, and spot. Neutralization assays indicated that the four isolates were nearly identical; however, the diversity of species affected suggests that the virus might not have been the specific cause of mortality.
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Almási, Asztéria; Csilléry, Gábor; Csömör, Zsófia; Nemes, Katalin; Palkovics, László; Salánki, Katalin; Tóbiás, István
2015-02-01
Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistance-breaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010-2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.
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El-Bebany, Ahmed F; Rampitsch, Christof; Daayf, Fouad
2010-01-01
Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.
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Tamada, Hiromi; Kiyama, Hiroshi
2015-01-01
Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice. PMID:26727725
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Tamada, Hiromi; Kiyama, Hiroshi
2015-01-01
Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.
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Tamada, Hiromi; Kiyama, Hiroshi
2015-01-01
Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.
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Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti.
Platero, Raúl A; Jaureguy, Melina; Battistoni, Federico J; Fabiano, Elena R
2003-01-21
Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.
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Ip, H; D'Aoust, F; Begum, A A; Zhang, H; Smith, D L; Driscoll, B T; Charles, T C
2001-12-01
Bradyrhizobium japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. Plasmid pZB32, carrying a nodY::lacZ transcriptional gene fusion, was introduced into B. japonicum cells that had been subjected to UV mutagenesis. Ten independent transformants producing a blue color on plates containing 5bromo-4chloro-3indolyl-beta-D-galactopyranoside but lacking genistein, indicative of constitutive expression of the nodY::lacZ reporter gene, were isolated. Beta-galactosidase activity assays revealed that while all of the 10 strains were sensitive to low concentrations of genistein, none exhibited truly constitutive nodY::lacZ expression in liquid culture. Soybean plants inoculated with three of the mutants were chlorotic and stunted, with shoot dry weights close to those of the uninoculated plants, indicating the absence of nitrogen fixation. Differences in the kinetics of nodY::lacZ expression and lipochitin oligosaccharide Nod signal production suggested that the strains carried different mutations. Some of these strains may be useful in mitigating the low root zone temperature-associated delay in soybean nodulation at the northern extent of soybean cultivation.
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Marin, E; Nussaume, L; Quesada, A; Gonneau, M; Sotta, B; Hugueney, P; Frey, A; Marion-Poll, A
1996-01-01
Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location. Images PMID:8665840
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Marin, E; Nussaume, L; Quesada, A; Gonneau, M; Sotta, B; Hugueney, P; Frey, A; Marion-Poll, A
1996-05-15
Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location.
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Singh, Susheel Kumar; Yadav, Deepti; Lal, Raj Kishori; Gupta, Madan M; Dhawan, Sunita Singh
2017-04-01
To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.
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Hindersin, Laura; Traulsen, Arne
2015-11-01
We analyze evolutionary dynamics on graphs, where the nodes represent individuals of a population. The links of a node describe which other individuals can be displaced by the offspring of the individual on that node. Amplifiers of selection are graphs for which the fixation probability is increased for advantageous mutants and decreased for disadvantageous mutants. A few examples of such amplifiers have been developed, but so far it is unclear how many such structures exist and how to construct them. Here, we show that almost any undirected random graph is an amplifier of selection for Birth-death updating, where an individual is selected to reproduce with probability proportional to its fitness and one of its neighbors is replaced by that offspring at random. If we instead focus on death-Birth updating, in which a random individual is removed and its neighbors compete for the empty spot, then the same ensemble of graphs consists of almost only suppressors of selection for which the fixation probability is decreased for advantageous mutants and increased for disadvantageous mutants. Thus, the impact of population structure on evolutionary dynamics is a subtle issue that will depend on seemingly minor details of the underlying evolutionary process.
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Zhang, Guo-Chang; Kong, In Iok; Kim, Heejin; Liu, Jing-Jing; Cate, Jamie H D; Jin, Yong-Su
2014-12-01
Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Van Hofwegen, Dustin J.; Hovde, Carolyn J.
2016-01-01
ABSTRACT The isolation of aerobic citrate-utilizing Escherichia coli (Cit+) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of E. coli Cit+ mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type E. coli strain B and with K-12 and three K-12 derivatives: an E. coli ΔrpoS::kan mutant (impaired for stationary-phase survival), an E. coli ΔcitT::kan mutant (deleted for the anaerobic citrate/succinate antiporter), and an E. coli ΔdctA::kan mutant (deleted for the aerobic succinate transporter). E. coli underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent E. coli Cit+ mutants were isolated from all E. coli derivatives except the E. coli ΔcitT::kan mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry. E. coli Cit+ mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four E. coli Cit+ mutants revealed the required sequence of mutational events leading to a refined Cit+ mutant. These events showed amplified citT and dctA loci followed by DNA rearrangements consistent with promoter capture events for citT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE. IMPORTANCE E. coli cannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and R. E. Lenski, Nature 489:513–518, 2012, http://dx.doi.org/10.1038/nature11514 ) found a single aerobic, citrate-utilizing E. coli strain after 33,000 generations (15 years). This was interpreted as a speciation event. Here we show why it probably was not a speciation event. Using similar media, 46 independent citrate-utilizing mutants were isolated in as few as 12 to 100 generations. Genomic DNA sequencing revealed an amplification of the citT and dctA loci and DNA rearrangements to capture a promoter to express CitT, aerobically. These are members of the same class of mutations identified by the LTEE. We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved. PMID:26833416
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Intracistronic complementation in the simian virus 40 A gene.
Tornow, J; Cole, C N
1983-01-01
A set of eight simian virus 40 mutants was constructed with lesions in the A gene, which encodes the large tumor (T) antigen. These mutants have small deletions (3-20 base pairs) at either 0.497, 0.288, or 0.243 map units. Mutants having both in-phase and frameshift mutations at each site were isolated. Neither plaque formation nor replication of the mutant DNAs could be detected after transfection of monkey kidney cells. Another nonviable mutant, dlA2459, had a 14-base-pair deletion at 0.193 map unit and was positive for viral DNA replication. Each of the eight mutants were tested for ability to form plaques after cotransfection with dlA2459 DNA. The four mutants that had in-phase deletions were able to complement dlA2459. The other four, which had frameshift deletions, did not. No plaques were formed after cotransfection of cells with any other pair of group A mutants. This suggests that the defect in dlA2459 defines a distinct functional domain of simian virus 40 T antigen. Images PMID:6312452
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Kasugamycin-dependent mutants of Escherichia coli.
Dabbs, E R
1978-01-01
Kasugamycin-dependent mutants have been isolated from Escherichia coli B. They were obtained through mutagenesis with ethyl methane sulfonate or nitrosoguanidine in conjunction with an antibiotic underlay technique. In the case of nitrosoguanidine, dependent mutants were obtained at a frequency of about 3% of survivors growing up in the selection. In the case of ethyl methane sulfonate, the corresponding value was 1%. Nineteen mutants showing a kasugamycin-dependent phenotype were studied. In terms of response to various temperatures and antibiotic concentrations, they were very heterogeneous, although most fell into two general classes. Genetic analysis indicated that in at least some cases, the kasugamycin-dependent phenotype was the product of two mutations. Two-dimensional gel electropherograms revealed alterations in the ribosomal proteins of seven mutants. One mutant had an alteration in protein S13, and one had an alteration in protein L14. Three showed changes in protein S9. Each of two mutants had changes in two proteins, S18 and L11. Three of these mutants additionally had protein S18 occurring in a partly altered, partly unaltered form. Images PMID:363701
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USDA-ARS?s Scientific Manuscript database
Two isolates of Beauveria bassiana and one of Purpureocillium lilacinum (=Paecilomyces lilacinus) were found affecting A. cajennense engorged females collected on horses (0.15 percent infection rate from a total of 1982 specimens) and another two isolates of P. lilacinum and one M. anisopliae detect...
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Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus
Hodgkin, Jonathan; Kaiser, Dale
1977-01-01
A large number of nonmotile mutants of the gliding bacterium Myxococcus xanthus have been isolated and partly characterized. About [unk] of these mutants are conditional mutants of a novel kind: mutant cells become transiently motile after contact with nonmutant cells or with cells of a different mutant type. These “stimulatable” mutants fall into five phenotypic classes (types B, C, D, E, and F). Most mutants are nonstimulatable (type A) and never become motile, but type A cells (and wild-type cells) can stimulate cells of any of the other five types. Stimulatable mutants of different types are capable of stimulating each other. For example, in a mixture of B and C cells, both become motile. Linkage analysis using a generalized transducing phage has shown that each of types B, C, D, E, and F corresponds to a single distinct genetic locus. Type A mutants, by contrast, belong to at least 17 different loci. Stimulation depends on close apposition of interacting cells, because stimulation does not occur when contact between cells is prevented. It is possible that the stimulatable mutants are defective in components of the gliding mechanism that can be exchanged between cells. Alternatively, they may be defective in a system of cell communication controlling the coordinated cell movements observed in Myxococcus. Images PMID:16592422
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Characterization of a Weak Allele of Zebrafish cloche Mutant
Ma, Ning; Huang, Zhibin; Chen, Xiaohui; He, Fei; Wang, Kun; Liu, Wei; Zhao, Linfeng; Xu, Xiangmin; Liao, Wangjun; Ruan, Hua; Luo, Shenqiu; Zhang, Wenqing
2011-01-01
Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche 172 (clo 172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo 172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo 172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo s5 mutant. In contrast, primitive myeloid cells were totally lost in clo 172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo 172 mutant, confirmed by the dramatic decrease of lyc in clo 172 runx1w84x double mutant. Collectively, the clo 172 mutant is a weak allele compared to the clo s5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene. PMID:22132109
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Radiation effects in nematodes: Results from IML-1 experiments
NASA Technical Reports Server (NTRS)
Nelson, G. A.; Schubert, W. W.; Kazarians, G. A.; Richards, G. F.; Benton, E. V.; Benton, E. R.; Henke, R.
1994-01-01
The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Flight sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Not Available
The plasmids present in M. ethanolicum have been characterized as to size, relatedness, and curing rate. Auxotrophs have been isolated and are being tested for the ability of several plasmids to promote mobilization of these markers. A cloning vector has been identified which can be used not only to clone the genes of interest but to isolate mutants in these genes and place a selectable marker on each of the plasmids. Isolation of a series of methane and methanol mutants, determination of which of the plasmids carries the mtn gene(s), and identification of methane-specific proteins on two-dimensonal O'Farrell gels shouldmore » all be completed shortly. In addition, the cloning of the methane genes and development of a genetic system should be well underway. A more detailed appraisal of future experiments is presented in the accompanying renewal proposal. (ERB)« less
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Diago-Navarro, Elizabeth; Mora, Liliana; Buckingham, Richard H; Díaz-Orejas, Ramón; Lemonnier, Marc
2008-01-01
Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin–antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a ∼10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1. PMID:19019162
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Cole, Ashley E.; Hani, Fatmah M.; Altman, Ronni; Meservy, Megan; Roth, John R.; Altman, Elliot
2017-01-01
While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants. PMID:27974497
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Duarte, M.; Sousa, R.; Videira, A.
1995-01-01
We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly. PMID:7768434
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Morrison, Sandra G; Giebel, Amanda M; Toh, Evelyn C; Spencer, Horace J; Nelson, David E; Morrison, Richard P
2018-07-01
Some members of the genus Chlamydia , including the human pathogen Chlamydia trachomatis , infect multiple tissues, including the genital and gastrointestinal (GI) tracts. However, it is unknown if bacterial targeting to these sites is mediated by multifunctional or distinct chlamydial factors. We previously showed that disruption of individual large clostridial toxin homologs encoded within the Chlamydia muridarum plasticity zone were not critical for murine genital tract infection. Here, we assessed whether cytotoxin genes contribute to C. muridarum GI tropism. Infectivity and shedding of wild-type (WT) C. muridarum and three mutants containing nonsense mutations in different cytotoxin genes, tc0437 , tc0438 , and tc0439 , were compared in mouse genital and GI infection models. One mutant, which had a nonsense mutation in tc0439 , was highly attenuated for GI infection and had a GI 50% infectious dose (ID 50 ) that was 1,000 times greater than that of the WT. GI inoculation with this mutant failed to elicit anti-chlamydial antibodies or to protect against subsequent genital tract infection. Genome sequencing of the tc0439 mutant revealed additional chromosomal mutations, and phenotyping of additional mutants suggested that the GI attenuation might be linked to a nonsense mutation in tc0600 The molecular mechanism underlying this dramatic difference in tissue-tropic virulence is not fully understood. However, isolation of these mutants demonstrates that distinct chlamydial chromosomal factors mediate chlamydial tissue tropism and provides a basis for vaccine initiatives to isolate chlamydia strains that are attenuated for genital infection but retain the ability to colonize the GI tract and elicit protective immune responses. Copyright © 2018 Morrison et al.
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Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI.
Chater, K F; Wilde, L C
1976-01-01
The bacteriophage Pa16, isolated from soil on Streptomyces albus G, was restricted when transferred from an alternative host back to S. albus G. Extracted unmodified Pa16 deoxyribonucleic acid was cleaved at a single site by a cell-free extract of S. albus G. Fractions cleaving Pal6 deoxyribonucleic acid contained the endonuclease SalI first described by J. Arrand, P. Myers, and R. J. Roberts (unpublished data). A mutant of S. albus G was isolated which was defective in both restriction and modification of Pal6. This mutant lacked SalI activity. It is concluded that SalI is the agent of restriction of Pal6 by S. albus G. Images PMID:977549
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Yelina, Nataliya E; Lambing, Christophe; Hardcastle, Thomas J; Zhao, Xiaohui; Santos, Bruno; Henderson, Ian R
2015-10-15
During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes. © 2015 Yelina et al.; Published by Cold Spring Harbor Laboratory Press.
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Coffman, Valerie C.; Nile, Aaron H.; Lee, I-Ju; Liu, Huayang
2009-01-01
Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis. PMID:19864459
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Zhao, Guozhong; Hou, Lihua; Yao, Yunping; Wang, Chunling; Cao, Xiaohong
2012-07-16
Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.
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Tricarboxylic acid cycle without malate dehydrogenase in Streptomyces coelicolor M-145.
Takahashi-Íñiguez, Tóshiko; Barrios-Hernández, Joana; Rodríguez-Maldonado, Marion; Flores, María Elena
2018-06-23
The oxidation of malate to oxaloacetate is catalysed only by a nicotinamide adenine dinucleotide-dependent malate dehydrogenase encoded by SCO4827 in Streptomyces coelicolor. A mutant lacking the malate dehydrogenase gene was isolated and no enzymatic activity was detected. As expected, the ∆mdh mutant was unable to grow on malate as the sole carbon source. However, the mutant grew less in minimal medium with glucose and there was a delay of 36 h. The same behaviour was observed when the mutant was grown on minimal medium with casamino acids or glycerol. For unknown reasons, the mutant was not able to grow in YEME medium with glucose. The deficiency of malate dehydrogenase affected the expression of the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase genes, decreasing the expression of both genes by approximately two- to threefold.
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Li, Xianggan; Volrath, Sandy L.; Nicholl, David B.G.; Chilcott, Charles E.; Johnson, Marie A.; Ward, Eric R.; Law, Marcus D.
2003-01-01
In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil. PMID:12972658
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Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana
2015-12-08
Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.
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Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana
2015-01-01
Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701
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Hirose, K; Kawasaki, Y; Kotani, K; Abiko, K; Sato, H
2004-05-01
Quinolone-resistant (QR) mutants of Mycoplasma bovirhinis strain PG43 (type strain) were generated by stepwise selection in increasing concentrations of enrofloxacin (ENR). An alteration was found in the quinolone resistance-determining region (QRDR) of the parC gene coding for the ParC subunit of topoisomerase IV from these mutants, but not in the gyrA, gyrB, and parE gene coding for the GyrA and GyrB subunits of DNA gyrase and the ParE subunit of topoisomerase IV. Similarly, such an alteration in QRDR of parC was found in the field isolates of M. bovirhinis, which possessed various levels of QR. The substitution of leucine (Leu) by serine (Ser) at position 80 of QRDR of ParC was observed in both QR-mutants and QR-isolates. This is the first report of QR based on a point mutation of the parC gene in M. bovirhinis.
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Parkinson, Elizabeth I.; Bair, Joseph S.; Nakamura, Bradley A.; Lee, Hyang Y.; Kuttab, Hani I.; Southgate, Emma H.; Lezmi, Stéphane; Lau, Gee W.; Hergenrother, Paul J.
2015-01-01
Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections. PMID:25907309
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Controlling plant architecture by manipulation of gibberellic acid signalling in petunia
Liang, Yin-Chih; Reid, Michael S; Jiang, Cai-Zhong
2014-01-01
Since stem elongation is a gibberellic acid (GA) response, GA inhibitors are commonly used to control plant height in the production of potted ornamentals and bedding plants. In this study, we investigated interfering with GA signaling by using molecular techniques as an alternative approach. We isolated three putative GID1 genes (PhGID1A, PhGID1B and PhGID1C) encoding GA receptors from petunia. Virus-induced gene silencing (VIGS) of these genes results in stunted growth, dark-green leaves and late-flowering. We also isolated the gai mutant gene (gai-1) from Arabidopsis. We have generated transgenic petunia plants in which the gai mutant protein is over-expressed under the control of a dexamethasone-inducible promoter. This system permits induction of the dominant Arabidopsis gai mutant gene at a desired stage of plant development in petunia plants by the application of dexamethasone (Dex). The induction of gai in Dex-treated T1 petunia seedlings caused dramatic growth retardation with short internodes. PMID:26504556
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Li, M; Zhang, H Y; Liang, B
2013-01-01
Twelve-low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe-the molecular lesion likely to be responsible-for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at-amino acid 168, having Phe (TTC) instead of Ser (TCC)', was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5'-flanking regions in 12 LR mutants of T. harzianum.
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Dictyostelium discoideum mutants with conditional defects in phagocytosis
1994-01-01
We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s). PMID:7519624
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A Proteomic Study of Brassinosteroid Response in Arabidopsis
Deng, Zhiping; Zhang, Xin; Tang, Wenqiang; Oses-Prieto, Juan A; Suzuki, Nagi; Gendron, Joshua M; Chen, Huanjing; Guan, Shenheng; Chalkley, Robert J.; Peterman, T. Kaye; Burlingame, Alma L.; Wang, Zhi-Yong
2010-01-01
Summary The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we have performed a proteomic study of BR-regulated proteins in Arabidopsis using two-dimensional difference gel electrophoresis (2-D DIGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR insensitive mutant bri1-116 and BR hypersensitive mutant bzr1-1D identified 5 proteins (PATL1, PATL2, THI1, AtMDAR3 and NADP-ME2) affected by both BR-treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knockout mutants or immunoblotting. Interestingly, about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein- and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore, BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in 2-D DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses. PMID:17848588
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Yao, Jun; Qian, Xuli; Bao, Jingxiao; Wei, Qinjun; Lu, Yajie; Zheng, Heng; Cao, Xin; Xing, Guangqian
2015-06-02
A Chinese family was identified with clinical features of enlarged vestibular aqueduct syndrome (EVAS). The mutational analysis showed that the proband (III-2) had EVAS with bilateral sensorineural hearing loss and carried a rare compound heterozygous mutation of SLC26A4 (IVS7-2A>G, c.2167C>G), which was inherited from the same mutant alleles of IVS7-2A>G heterozygous father and c.2167C>G heterozygous mother. Compared with another confirmed pathogenic biallelic mutation in SLC26A4 (IVS7-2A>G, c.2168A>G), these two biallelic mutations shared one common mutant allele and the same codon of the other mutant allele, but led to different changes of amino acid (p.H723D, p.H723R) and both resulted in the deafness phenotype. Structure-modeling indicated that these two mutant alleles changed the shape of pendrin protein encoded by SLC26A4 with increasing randomness in conformation, and might impair pendrin's ability as an anion transporter. The molecular dynamics simulations also revealed that the stability of mutant pendrins was reduced with increased flexibility of backbone atoms, which was consistent with the structure-modeling results. These evidences indicated that codon 723 was a hot-spot region in SLC26A4 with a significant impact on the structure and function of pendrin, and acted as one of the genetic factors responsible for the development of hearing loss.
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Engineered Human Antibody Constant Domains with Increased Stability*S⃞
Gong, Rui; Vu, Bang K.; Feng, Yang; Prieto, DaRue A.; Dyba, Marzena A.; Walsh, Joseph D.; Prabakaran, Ponraj; Veenstra, Timothy D.; Tarasov, Sergey G.; Ishima, Rieko; Dimitrov, Dimiter S.
2009-01-01
The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (Tm = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (Tm = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases. PMID:19307178
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Garcia, J P; Adams, V; Beingesser, J; Hughes, M L; Poon, R; Lyras, D; Hill, A; McClane, B A; Rood, J I; Uzal, F A
2013-07-01
Clostridium perfringens type D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX to C. perfringens type D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulent C. perfringens type D wild-type strain (WT), an isogenic ETX null mutant (etx mutant), and a strain where the etx mutation has been reversed (etx complemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenic etx mutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation of etx knockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis.
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USDA-ARS?s Scientific Manuscript database
Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr 1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr 1-1 gene was over-expressed under the control of a c...
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Wiebe, M G; Robson, G D; Oliver, S G; Trinci, A P
1994-11-01
Variants (designated A23-S and A24-S) of the Quorn myco-protein fungus, Fusarium graminearum A3/5 were isolated from a series of glucose-limited cultures grown at a dilution rate of 0.18 h-1 for a combined total of 109 d. These variants had unchanged mycelial morphologies but, when grown in mixed culture with the parental strain (A3/5) in glucose-limited chemostat culture at 0.18 h-1, A23-S and A24-S had selection coefficients of 0.013 and 0.017 h-1, respectively, and supplanted A3/5. When a monoculture of A23-S was grown in a glucose-limited culture at a dilution rate of 0.18 h-1, the appearance of highly branched (so-called colonial) mutants was delayed compared with their appearance in chemostat cultures of the parental strain. Furthermore, when a monoculture of A24-S was grown in glucose-limited culture at 0.18 h-1, the appearance of colonial mutants was delayed even further. Thus, it is possible to isolate advantageous (relative to A3/5) variants of F. graminearum A3/5 which have unchanged mycelial morphologies, but in which the appearance of colonial mutants is delayed.
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Genetic diversity of Plasmodium falciparum and distribution of drug resistance haplotypes in Yemen
2013-01-01
Background Despite evident success of malaria control in many sites in the Arabian Peninsula, malaria remains endemic in a few spots, in Yemen and south-west of Saudi Arabia. In addition to local transmission, imported malaria sustains an extra source of parasites that can challenge the strengths of local control strategies. This study examined the genetic diversity of Plasmodium falciparum in Yemen and mutations of drug resistant genes, to elucidate parasite structure and distribution of drug resistance genotypes in the region. Methods Five polymorphic loci (MSP-2, Pfg377 and three microsatellites on chromosome 8) not involved in anti-malarial drug resistance, and four drug resistant genes (pfcrt, pfmdr1, dhfr and dhps) were genotyped in 108 P. falciparum isolates collected in three sites in Yemen: Dhamar, Hodeidah and Taiz. Results High diversity was seen in non-drug genes, pfg377 (He = 0.66), msp-2 (He = 0.80) and three microsatellites on chr 8, 7.7 kb (He = 0.88), 4.3 kb (He = 0.77) and 0.8 kb (He = 0.71). There was a high level of mixed-genotype infections (57%), with an average 1.8 genotypes per patient. No linkage disequilibrium was seen between drug resistant genes and the non-drug markers (p < 0.05). Genetic differentiation between populations was low (most pair-wise FST values <0.03), indicating extensive gene flow between the parasites in the three sites. There was a high prevalence of mutations in pfmdr1, pfcrt and dhfr; with four mutant pfmdr1 genotypes (NFCDD[57%], NFSND[21%], YFCDD[13%] and YFSND[8% ]), two mutant pfcrt genotypes (CVIET[89%] and SVMNT[4%]) and one mutant dhfr genotype (ICNI[53.7%]). However, no dhps mutations were detected. Conclusion The high diversity of P. falciparum in Yemen is indicative of a large parasite reservoir, which represents a challenge to control efforts. The presence of two distinct pfcrt genotype, CVIET and SVMNT, suggests that chloroquine resistance can possibly be related to a migratory path from Africa and Asia. The absence of the triple mutant dhfr genotype (IRN) and dhps mutations supports the use of artesunate + sulphadoxine-pyrimethamine as first-line therapy. However, the prevalent pfmdr1 genotype NFSND [21%] has previously been associated with tolerance/resistance response to artemisinin combination therapy (ACT). Regular surveys are, therefore, important to monitor spread of pfmdr1 and dhfr mutations and response to ACT. PMID:23855834
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A new genetic factor for root gravitropism in rice (Oryza sativa L.).
Shi, Jiang-hua; Hao, Xi; Wu, Zhong-chang; Wu, Ping
2009-10-01
Root gravitropism is one of the important factors to determine root architecture. To understand the mechanism underlying root gravitropism, we isolated a rice (Xiushui63) mutant defective in root gravitropism, designated as gls1. Vertical sections of root caps revealed that gls1 mutant displayed normal distribution of amyloplast in the columella cells compared with the wild type. The gls1 mutant was less sensitive to 2,4-dichlorophenoxyacetic acid (2,4-D) and alpha-naphthaleneacetic acid (NAA) than the wild type. Genetic analysis indicated that the phenotype of gls1 mutant was caused by a single recessive mutation, which is mapped in a 255-kb region between RM16253 and CAPS1 on the short arm of chromosome 4.
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Strain improvement of Lactobacillus lactis for D-lactic acid production.
Joshi, D S; Singhvi, M S; Khire, J M; Gokhale, D V
2010-04-01
Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased D: -lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest D: -lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into D-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain D-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.
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Mutant prevention concentrations of four carbapenems against gram-negative rods.
Credito, Kim; Kosowska-Shick, Klaudia; Appelbaum, Peter C
2010-06-01
We tested the propensities of four carbapenems to select for resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii mutants by determining the mutant prevention concentrations (MPCs) for 100 clinical strains with various ss-lactam phenotypes. Among the members of the Enterobacteriaceae family and A. baumannii strains, the MPC/MIC ratios were mostly 2 to 4. In contrast, for P. aeruginosa the MPC/MIC ratios were 4 to > or =16. The MPC/MIC ratios for beta-lactamase-positive K. pneumoniae and E. coli isolates were much higher (range, 4 to >16 microg/ml) than those for ss-lactamase-negative strains.
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Yin, L B; Zhang, C F; Xia, Q L; Yang, Y; Xiao, K; Zhao, L Z
2016-09-23
Fusarium oxysporum strain BM-201 was treated with ultraviolet (UV) radiation to obtain a high pectinase-producing strain. Mutant UV-10-41 was obtained and then treated by diethyl sulfate. Next, the mutant UV-diethyl sulfate-43 derived from UV-10-41 was selected as high pectinase-producing strain. Mutant UV-diethyl sulfate-43 was incubated on slant for 10 generations, demonstrating that the pectinase-producing genes were stable. Pectinase activity reached 391.2 U/mL, which is 73.6% higher than that of the original strain.
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Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.
2003-01-01
Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments. PMID:14660416
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Van Dien, Stephen J; Marx, Christopher J; O'Brien, Brooke N; Lidstrom, Mary E
2003-12-01
Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.
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Agrawal, Poonam; Laddha, Kirti
2017-04-01
This study was undertaken to isolate and quantify aristolochic acid in Aristolochia indica stem and Apama siliquosa root. Aristolochic acid is an important biomarker component present in the Aristolochiaceae family. The isolation method involved simple solvent extraction, precipitation and further purification, using recrystallization. The structure of the compound was confirmed using infrared spectroscopy, mass spectrometry and nuclear magnetic resonance. A specific and rapid high-performance thin layer chromatography (HPTLC) method was developed for analysis of aristolochic acid. The method involved separation on the silica gel 60 F 254 plates using the single solvent system of n-hexane: chloroform: methanol. The method showed good linear relationship in the range 0.4-2.0 μg/spot with r 2 = 0.998. The limit of detection and limit of quantification were 62.841 ng/spot and 209.47 ng/spot, respectively. The proposed validated HPTLC method was found to be an easy to use, accurate and convenient method that could be successfully used for standardization and quality assessment of herbal material as well as formulations containing different species of the Aristolochiaceae family. Copyright © 2016. Published by Elsevier B.V.
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Proton acceleration by irradiation of isolated spheres with an intense laser pulse
Ostermayr, Tobias M.; Haffa, D.; Hilz, P.; ...
2016-09-26
We report on experiments irradiating isolated plastic spheres with a peak laser intensity of 2–3 × 10 20 W cm –2. With a laser focal spot size of 10 μm full width half maximum (FWHM) the sphere diameter was varied between 520 nm and 19.3 μm. Maximum proton energies of ~ 25 MeV are achieved for targets matching the focal spot size of 10 μm in diameter or being slightly smaller. For smaller spheres the kinetic energy distributions of protons become nonmonotonic, indicating a change in the accelerating mechanism from ambipolar expansion towards a regime dominated by effects caused bymore » Coulomb repulsion of ions. The energy conversion efficiency from laser energy to proton kinetic energy is optimized when the target diameter matches the laser focal spot size with efficiencies reaching the percent level. The change of proton acceleration efficiency with target size can be attributed to the reduced cross-sectional overlap of subfocus targets with the laser. Reported experimental observations are in line with 3D3V particle in cell simulations. In conclusion, they make use of well-defined targets and point out pathways for future applications and experiments.« less
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Proton acceleration by irradiation of isolated spheres with an intense laser pulse
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ostermayr, Tobias M.; Haffa, D.; Hilz, P.
We report on experiments irradiating isolated plastic spheres with a peak laser intensity of 2–3 × 10 20 W cm –2. With a laser focal spot size of 10 μm full width half maximum (FWHM) the sphere diameter was varied between 520 nm and 19.3 μm. Maximum proton energies of ~ 25 MeV are achieved for targets matching the focal spot size of 10 μm in diameter or being slightly smaller. For smaller spheres the kinetic energy distributions of protons become nonmonotonic, indicating a change in the accelerating mechanism from ambipolar expansion towards a regime dominated by effects caused bymore » Coulomb repulsion of ions. The energy conversion efficiency from laser energy to proton kinetic energy is optimized when the target diameter matches the laser focal spot size with efficiencies reaching the percent level. The change of proton acceleration efficiency with target size can be attributed to the reduced cross-sectional overlap of subfocus targets with the laser. Reported experimental observations are in line with 3D3V particle in cell simulations. In conclusion, they make use of well-defined targets and point out pathways for future applications and experiments.« less
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Identifying geographic hot spots of reassortment in a multipartite plant virus
Savory, Fiona R; Varma, Varun; Ramakrishnan, Uma
2014-01-01
Reassortment between different species or strains plays a key role in the evolution of multipartite plant viruses and can have important epidemiological implications. Identifying geographic locations where reassortant lineages are most likely to emerge could be a valuable strategy for informing disease management and surveillance efforts. We developed a predictive framework to identify potential geographic hot spots of reassortment based upon spatially explicit analyses of genome constellation diversity. To demonstrate the utility of this approach, we examined spatial variation in the potential for reassortment among Cardamom bushy dwarf virus (CBDV; Nanoviridae, Babuvirus) isolates in Northeast India. Using sequence data corresponding to six discrete genome components for 163 CBDV isolates, a quantitative measure of genome constellation diversity was obtained for locations across the sampling region. Two key areas were identified where viruses with highly distinct genome constellations cocirculate, and these locations were designated as possible geographic hot spots of reassortment, where novel reassortant lineages could emerge. Our study demonstrates that the potential for reassortment can be spatially dependent in multipartite plant viruses and highlights the use of evolutionary analyses to identify locations which could be actively managed to facilitate the prevention of outbreaks involving novel reassortant strains. PMID:24944570
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Characterization of two brassinosteroid C-6 oxidase genes in pea.
Jager, Corinne E; Symons, Gregory M; Nomura, Takahito; Yamada, Yumiko; Smith, Jennifer J; Yamaguchi, Shinjiro; Kamiya, Yuji; Weller, James L; Yokota, Takao; Reid, James B
2007-04-01
C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.
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Nascimento, Heloisa H; Silva, Lucas E P; Souza, Renata T; Silva, Neusa P; Scaletsky, Isabel C A
2014-07-10
Biofilm formation by enteropathogenic Escherichia coli (EPEC) have been recently described in the prototype typical EPEC E2348/69 strain and in an atypical EPEC O55:H7 strain. In this study, we sought to evaluate biofilm formation in a collection of 126 atypical EPEC strains isolated from 92 diarrheic and 34 nondiarrheic children, belonging to different serotypes. The association of biofilm formation and adhesin-related genes were also investigated. Biofilm formation occurred in 37 (29%) strains of different serotypes, when the assays were performed at 26°C and 37°C for 24 h. Among these, four strains (A79, A87, A88, and A111) formed a stronger biofilm than did the others. The frequency of biofilm producers was higher among isolates from patients compared with isolates from controls (34.8% vs 14.7%; P = 0.029). An association was found between biofilm formation and expression of type 1 fimbriae and curli (P < 0.05). Unlike the previously described aEPEC O55:H7, one aEPEC O119:HND strain (A111) formed a strong biofilm and pellicle at the air-liquid interface, but did not express curli. Transposon mutagenesis was used to identify biofilm-deficient mutants. Transposon insertion sequences of six mutants revealed similarity with type 1 fimbriae (fimC, fimD, and fimH), diguanylate cyclase, ATP synthase F1, beta subunit (atpD), and the uncharacterized YjiC protein. All these mutants were deficient in biofilm formation ability. This study showed that the ability to adhere to abiotic surfaces and form biofilm is present in an array of aEPEC strains. Moreover, it seems that the ability to form biofilms is associated with the presence of type 1 fimbriae and diguanylate cyclase. Characterization of additional biofilm formation mutants may reveal other mechanisms involved in biofilm formation and bring new insights into aEPEC adhesion and pathogenesis.
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Sow, Fatimata; Bonnot, Guillaume; Ahmed, Bilal Rabah; Diagana, Sidi Mohamed; Kebe, Hachim; Koita, Mohamedou; Samba, Ba Malado; Al-Mukhaini, Said K; Al-Zadjali, Majed; Al-Abri, Seif S; Ali, Osama A M; Samy, Abdallah M; Hamid, Muzamil Mahdi Abdel; Ali Albsheer, Musab M; Simon, Bruno; Bienvenu, Anne-Lise; Petersen, Eskild; Picot, Stéphane
2017-02-02
Plasmodium vivax is the second most important human malaria parasite, widely spread across the world. This parasite is associated with important issues in the process toward malaria elimination, including potential for relapse and increased resistance to chloroquine. Plasmodium vivax multi-drug resistant (pvmdr1) is suspected to be a marker of resistance although definitive evidence is lacking. Progress has been made in knowledge of biological factors affecting parasite growth, including mechanisms of regulated cell death and the suspected role of metacaspase. Plasmodium vivax metacaspase1 (PvMCA1-cd) has been described with a catalytic domain composed of histidine (H372) and cysteine (C428) residues. The aim of this study was to test for a link between the conserved histidine and cysteine residues in PvMCA1-cd, and the polymorphism of the P. vivax multi-drug resistant gene (pvmdr1). Thirty P. vivax isolates were collected from Mauritania, Sudan, and Oman. Among the 28 P. vivax isolates successfully sequenced, only 4 samples showed the conserved His (372)-Cys (428) residues in PvMCA1-cd. Single nucleotide polymorphisms observed were H372T (46.4%), H372D (39.3%), and C428R (85.7%). A new polymorphic catalytic domain was observed at His (282)-Cys (305) residues. Sequences alignment analysis of pvmdr1 showed SNP in the three codons 958, 976 and 1076. A single SNP was identified at the codon M958Y (60%), 2 SNPs were found at the position 976: Y976F (13%) and Y976V (57%), and 3 SNPs were identified at the position 1076: F1076L (40%), F1076T (53%) and F1076I (3%). Only one isolate was wildtype in all three codons (MYF), 27% were single MYL mutants, and 10% were double MFL mutants. Three new haplotypes were also identified: the triple mutant YVT was most prevalent (53.3%) distributed in the three countries, while triple YFL and YVI mutants (3%), were only found in samples from Sudan and Mauritania. Triple or quadruple mutants for metacaspase genes and double or triple mutants for Pvmdr1 were observed in 24/28 and 19/28 samples. There was no difference in the frequency of mutations between PvMCA1-cd and Pvmdr1 (P > 0.2). Histidine and cysteine residues in PvMCA1-cd are highly polymorphic and linkage disequilibrium with SNPs of Pvmdr1 gene may be expected from these three areas with different patterns of P. vivax transmission.
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The circling mutant Pcdh15roda is a new mouse model for hearing loss.
Torres, Adriana Amorim; Rzadzinska, Agnieszka K; Ribeiro, Andrea Frozino; Silva, Daniel Almeida da Silva E; Guénet, Jean-Louis; Massironi, Sílvia Maria Gomes; Godard, Ana Lúcia Brunialti
2013-01-01
Mouse mutagenesis is a key tool for studying gene function and several mutant alleles have been described and constitute mouse models for human hereditary diseases. Genetic hearing loss represents over 50% of all hearing loss cases in children and, due to the heterogeneity of the disorder, there is still a demand for the isolation and characterization of new genes and alleles. Here we report phenotypic and molecular characterization of a new mouse model for hereditary hearing loss. The mutant rodador, isolated by Massironi and colleagues in 2006, presents an autosomal recessive disorder characterized by deafness and balance dysfunction associated with abnormal stereocilia in the inner ear. The mutation was mapped to mouse chromosome 10, and characterization of the gene Pcdh15 revealed an AT-to-GC transition in intron 23 of mutant animals. The alteration led to the switch of a dinucleotide ApA for ApG, creating a novel intronic acceptor splice site, which leads to incorporation of eight intronic bases into the processed mRNA and alteration of the downstream reading frame. In silico analysis indicated that the mutated protein is truncated and lacks two cadherin domains, and the transmembrane and cytoplasmic domains. Real Time PCR analyses revealed a significantly reduced Pcdh15 mRNA level in the brain of mutant mice, which might be due to the mechanism of non-sense mediated decay. In man, mutations in the orthologue PCDH15 cause non-syndromic deafness and Usher Syndrome Type 1F, a genetic disorder characterized by hearing loss and retinitis pigmentosa. Rodador mouse constitutes a new model for studying deafness in these conditions and may help in the comprehension of the pathogeneses of the disease, as well as of the mechanisms involved in the morphogenesis and function of inner ear stereocilia. This is a new ENU-induced allele and the first isolated in a BALB/c background. Copyright © 2013 Elsevier B.V. All rights reserved.
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Soge, Olusegun O; Salipante, Stephen J; No, David; Duffy, Erin; Roberts, Marilyn C
2016-05-01
We evaluated the in vitro activity of delafloxacin against a panel of 117 Neisseria gonorrhoeae strains, including 110 clinical isolates collected from 2012 to 2015 and seven reference strains, compared with the activities of seven antimicrobials currently or previously recommended for treatment of gonorrhea. We examined the potential for delafloxacin to select for resistant mutants in ciprofloxacin-susceptible and ciprofloxacin-resistant N. gonorrhoeae We characterized mutations in the gyrA, gyrB, parC, and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) by PCR and sequencing and by whole-genome sequencing. The MIC50, MIC90, and MIC ranges of delafloxacin were 0.06 μg/ml, 0.125 μg/ml, and ≤0.001 to 0.25 μg/ml, respectively. The frequency of spontaneous mutation ranged from 10(-7) to <10(-9) The multistep delafloxacin resistance selection of 30 daily passages resulted in stable resistant mutants. There was no obvious cross-resistance to nonfluoroquinolone comparator antimicrobials. A mutant with reduced susceptibility to ciprofloxacin (MIC, 0.25 μg/ml) obtained from the ciprofloxacin-susceptible parental strain had a novel Ser91Tyr alteration in the gyrA gene. We also identified new mutations in the gyrA and/or parC and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) of two mutant strains with elevated delafloxacin MICs of 1 μg/ml. Although delafloxacin exhibited potent in vitro activity against N. gonorrhoeae isolates and reference strains with diverse antimicrobial resistance profiles and demonstrated a low tendency to select for spontaneous mutants, it is important to establish the correlation between these excellent in vitro data and treatment outcomes through appropriate randomized controlled clinical trials. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Salipante, Stephen J.; No, David; Duffy, Erin; Roberts, Marilyn C.
2016-01-01
We evaluated the in vitro activity of delafloxacin against a panel of 117 Neisseria gonorrhoeae strains, including 110 clinical isolates collected from 2012 to 2015 and seven reference strains, compared with the activities of seven antimicrobials currently or previously recommended for treatment of gonorrhea. We examined the potential for delafloxacin to select for resistant mutants in ciprofloxacin-susceptible and ciprofloxacin-resistant N. gonorrhoeae. We characterized mutations in the gyrA, gyrB, parC, and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) by PCR and sequencing and by whole-genome sequencing. The MIC50, MIC90, and MIC ranges of delafloxacin were 0.06 μg/ml, 0.125 μg/ml, and ≤0.001 to 0.25 μg/ml, respectively. The frequency of spontaneous mutation ranged from 10−7 to <10−9. The multistep delafloxacin resistance selection of 30 daily passages resulted in stable resistant mutants. There was no obvious cross-resistance to nonfluoroquinolone comparator antimicrobials. A mutant with reduced susceptibility to ciprofloxacin (MIC, 0.25 μg/ml) obtained from the ciprofloxacin-susceptible parental strain had a novel Ser91Tyr alteration in the gyrA gene. We also identified new mutations in the gyrA and/or parC and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) of two mutant strains with elevated delafloxacin MICs of 1 μg/ml. Although delafloxacin exhibited potent in vitro activity against N. gonorrhoeae isolates and reference strains with diverse antimicrobial resistance profiles and demonstrated a low tendency to select for spontaneous mutants, it is important to establish the correlation between these excellent in vitro data and treatment outcomes through appropriate randomized controlled clinical trials. PMID:26976873
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Analysis of the Fluoroquinolone Antibiotic Resistance Mechanism of Salmonella enterica Isolates.
Kim, Soo-Young; Lee, Si-Kyung; Park, Myeong-Soo; Na, Hun-Taek
2016-09-28
Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.
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Li, Jihong; Sayeed, Sameera; Robertson, Susan; Chen, Jianming; McClane, Bruce A
2011-12-01
Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.
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Huergo, Luciano F; Noindorf, Lilian; Gimenes, Camila; Lemgruber, Renato S P; Cordellini, Daniela F; Falarz, Lucas J; Cruz, Leonardo M; Monteiro, Rose A; Pedrosa, Fábio O; Chubatsu, Leda S; Souza, Emanuel M; Steffens, Maria B R
2010-07-01
This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.
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Genetic and molecular dosimetry of HZE radiation (US-1 RADIAT)
NASA Technical Reports Server (NTRS)
Nelson, Gregory A.; Schubert, W. W.; Kazarians, G. A.; Richards, G. F.; Benton, E. V.; Benton, E. R.; Henke, R. P.
1995-01-01
In order to estimate radiation exposure in space, experiments were conducted during the 1st International Microgravity Laboratory (IML-1) mission in order to isolate genetic changes in animal cells caused by cosmic rays. The space measurements were evaluated against results from synthetic cosmic rays produced by particle accelerators on the ground. The biological material used was the tiny soil nematode, Caenorhabditis elegans. The measurements were made by thermoluminescent detectors and plastic nuclear track detectors. The development and the chromosome mechanics in microgravity were studied, and the mutagenesis induced by radiation exposure was analyzed. The results showed that there are no obvious differences in the development, behavior and chromosome mechanics, as a function of gravity unloading (reproduction, self-fertilization and mating of males with hermaphrodites, gross anatomy, symmetry and gametogenesis, pairing, disjoining and recombination of chromosomes). A variety of mutants were isolated, and it was noted that mutants isolated from regions of identified high particles were more severely affected than those isolated by random screening. Linear energy transfer particles seem to favor large scale genetic lesions.
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The characterization of a zebrafish mid-hindbrain mutant, mid-hindbrain gone (mgo).
Shima, Takaki; Znosko, Wade; Tsang, Michael
2009-04-01
The vertebrate mid-hindbrain boundary (MHB) is a crucial morphological structure required for patterning and neural differentiation of the midbrain and anterior hindbrain. We isolated a novel zebrafish mutant, MHB gone (mgo), that exhibited a defective MHB. Expression of engrailed3 in the prospective MHB was absent at the 1-somite stage, suggesting that initiation of the isthmic organizer was disrupted in mgo mutants. Complementation test with mgo and noi, in which the pax2a gene is mutated, infer that the mgo mutant may represent a novel noi allele. However, pronephric, otic vesicle, and commissural axonal defects described in noi mutants were not associated with mgo mutants. Genetic mapping revealed that the mgo mutation is linked to the Pax2a locus, but no mutation was detected in pax2a exons or within intron-exon boundaries. Based on these findings, we propose that the mgo mutation genetically interacts with pax2a required for the initiation of MHB formation. Copyright 2009 Wiley-Liss, Inc.
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Enhancing the Production of D-Mannitol by an Artificial Mutant of Penicillium sp. T2-M10.
Duan, Rongting; Li, Hongtao; Li, Hongyu; Tang, Linhuan; Zhou, Hao; Yang, Xueqiong; Yang, Yabin; Ding, Zhongtao
2018-05-26
D-Mannitol belongs to a linear polyol with six-carbon and has indispensable usage in medicine and industry. In order to obtain more efficient D-mannitol producer, this study has screened out a stable mutant Penicillium sp. T2-M10 that was isolated from the initial D-mannitol-produced strain Penicillium sp.T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. The mutant had a considerable enhancement in yield of D-mannitol based on optimizing fermentation. The production condition was optimized as the PDB medium with 24 g/L glucose for 9 days. The results showed that the production of D-mannitol from the mutant strain T2-M10 increased 125% in contrast with the parental strain. Meanwhile, the fact that D-mannitol is the main product in the mutant simplified the process of purification. Our finding revealed the potential value of the mutant strain Penicillium sp. T2-M10 to be a D-mannitol-producing strain.
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Mutation-Screening of Pleurotus Ferulae with High Temperature Tolerance by Nitrogen Ion Implantation
NASA Astrophysics Data System (ADS)
Chen, Henglei; Wan, Honggui; Zhang, Jun; Zeng, Xianxian
2008-08-01
In order to obtain Pleurotus ferulae with high temperature tolerance, conidiophores of wild type strain ACK were implanted with nitrogen ions in energy of 5 ~15 keV and dose of 1.5 × 1015 ~ 1.5 × 1016 cm-2, and a mutant CGMCC1763 was isolated subsequently through thermotolerant screening method. It was found that during riper period the surface layer mycelium of the mutant in mushroom bag wasn't aging neither grew tegument even above 30° C. The mycelium endurable temperature of the mutant was increased by 5°C compared to that of the wild type strain. The fruiting bodies growth temperature of the mutant was 18 ~22°C in daytime and 8~14°C at night. The highest growth temperature of fruiting bodies of the mutant was increased about 7°C w.r.t. that of original strain. Through three generations investigations, it was found that the mutant CGMCC1763 was stable with high temperature tolerance.
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Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants.
Nicolson, T; Rüsch, A; Friedrich, R W; Granato, M; Ruppersberg, J P; Nüsslein-Volhard, C
1998-02-01
The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.
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Chakraborty, Somnath; Ghosh, Upasana; Balasubramanian, Thangavel; Das, Punyabrata
2014-01-01
Objective To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host–pathogen interaction model. Methods Thirty species of marine plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug. Results Nine plant isolates exhibited significant survivability in host. The drug MP07X thus formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1 000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection. Conclusions The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug. PMID:25183065
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Vermaas, Wim F. J.; Williams, John G. K.; Rutherford, A. William; Mathis, Paul; Arntzen, Charles J.
1986-01-01
CP-47 is absent in a genetically engineered mutant of cyanobacterium Synechocystis 6803, in which the psbB gene [encoding the chlorophyll-binding photosystem II (PSII) protein CP-47] was interrupted. Another chlorophyll-binding PSII protein, CP-43, is present in the mutant, and functionally inactive PSII-enriched particles can be isolated from mutant thylakoids. We interpret these data as indicating that the PSII core complex of the mutant still assembles in the absence of CP-47. The mutant lacks a 77 K fluorescence emission maximum at 695 nm, suggesting that the PSII reaction center is not functional. The absence of primary photochemistry was indicated by EPR and optical measurements: no chlorophyll triplet originating from charge recombination between P680+ and Pheo- was observed in the mutant, and there were no flash-induced absorption changes at 820 nm attributable to chlorophyll P680 oxidation. These observations lead us to conclude that CP-47 plays an essential role in the activity of the PSII reaction center. Images PMID:16593788
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Improved Properties of Baker's Yeast Mutants Resistant to 2-Deoxy-d-Glucose
Rincón, Ana M.; Codón, Antonio C.; Castrejón, Francisco; Benítez, Tahía
2001-01-01
We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-d-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-d-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties. PMID:11526034
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Tiwari, Sameeksha; Awasthi, Manika; Singh, Swati; Pandey, Veda P; Dwivedi, Upendra N
2017-10-23
Protein-protein interactions (PPI) are a new emerging class of novel therapeutic targets. In order to probe these interactions, computational tools provide a convenient and quick method towards the development of therapeutics. Keeping this in view the present study was initiated to analyse interaction of tumour suppressor protein p53 (TP53) and breast cancer associated protein (BRCA1) as promising target against breast cancer. Using computational approaches such as protein-protein docking, hot spot analyses, molecular docking and molecular dynamics simulation (MDS), stepwise analyses of the interactions of the wild type and mutant TP53 with that of wild type BRCA1 and their modulation by alkaloids were done. Protein-protein docking method was used to generate both wild type and mutant complexes of TP53-BRCA1. Subsequently, the complexes were docked using sixteen different alkaloids, fulfilling ADMET and Lipinski's rule of five criteria, and were compared with that of a well-known inhibitor of PPI, namely nutlin. The alkaloid dicentrine was found to be the best docked alkaloid among all the docked alklaloids as well as that of nutlin. Furthermore, MDS analyses of both wild type and mutant complexes with the best docked alkaloid i.e. dicentrine, revealed higher stability of mutant complex than that of the wild one, in terms of average RMSD, RMSF and binding free energy, corroborating the results of docking. Results suggested more pronounced interaction of BRCA1 with mutant TP53 leading to increased expression of mutated TP53 thus showing a dominant negative gain of function and hampering wild type TP53 function leading to tumour progression.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Bible, Amber; Fletcher, Sarah J; Pelletier, Dale A
The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically-important plants. Pantoea sp. YR 343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid. Pantoea sp. YR 343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plantmore » hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR 343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of indole-3-acetic acid. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR 343.« less
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Bible, Amber N.; Fletcher, Sarah J.; Pelletier, Dale A.; Schadt, Christopher W.; Jawdy, Sara S.; Weston, David J.; Engle, Nancy L.; Tschaplinski, Timothy; Masyuko, Rachel; Polisetti, Sneha; Bohn, Paul W.; Coutinho, Teresa A.; Doktycz, Mitchel J.; Morrell-Falvey, Jennifer L.
2016-01-01
The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343. PMID:27148182
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Bible, Amber; Fletcher, Sarah J; Pelletier, Dale A; ...
2016-04-18
The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically-important plants. Pantoea sp. YR 343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid. Pantoea sp. YR 343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plantmore » hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR 343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of indole-3-acetic acid. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR 343.« less
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Brossier, Florence; Boudinet, Marlène; Jarlier, Vincent; Petrella, Stéphanie; Sougakoff, Wladimir
2016-09-01
Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG protein. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Identification of gram-negative bacilli using the Autobac IDX.
Burdash, N M; Welborn, A L; Teti, G; Bannister, E R; Manos, J P
1985-01-01
The Autobac IDX is a new system for the rapid identification of clinically significant members of the Enterobacteriaceae and Aeromonas, Acinetobacter, Alcaligenes, Flavobacterium, Moraxella, and Pseudomonas species. The use of 18 differentially inhibitory compounds such as dyes and antibiotics along with a computerized algorithm based on a multivariate analysis provides the basis for the identification of 30 different groups of gram-negative bacilli. Required preliminary tests include observations on the presence or absence of swarming on a sheep blood agar plate and noting the following: growth, lactose fermentation, and bile precipitation from a MacConkey plate. Spot indole and spot oxidase tests must be performed as well. Identification by the Autobac IDX System takes 3-6 hr after completion of the preliminary tests. From a total of 403 isolates tested, the Autobac system agreed with the MicroID AND N/F systems on 382 identifications (94.8%). Four isolates, two Acinetobacter anitratus, one Serratia marcescens and one Moraxella osloensis could not be identified by IDX. Additional testing was required on 35 (8.7%) of the isolates.
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Involvement of Two Plasmids in the Degradation of Carbaryl by Arthrobacter sp. Strain RC100
Hayatsu, Masahito; Hirano, Motoko; Nagata, Tadahiro
1999-01-01
A bacterium capable of utilizing carbaryl (1-naphthyl N-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as Arthrobacter and was designated strain RC100. RC100 hydrolyzes the N-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah−) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat−) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah− mutants or Nat− mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes. PMID:10049857
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A Direct Screening Procedure for Gravitropism Mutants in Arabidopsis thaliana (L.) Heynh. 1
Bullen, Bertha L.; Best, Thérèse R.; Gregg, Mary M.; Barsel, Sara-Ellen; Poff, Kenneth L.
1990-01-01
In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable. PMID:11537704
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A direct screening procedure for gravitropism mutants in Arabidopsis thaliana (L.) Heynh
NASA Technical Reports Server (NTRS)
Bullen, B. L.; Best, T. R.; Gregg, M. M.; Poff, K. L.; Barsel, S-E (Principal Investigator)
1990-01-01
In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable.
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Bowman, Shaun M; Piwowar, Amy; Ciocca, Maria; Free, Stephen J
2005-01-01
Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.
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A detailed study of gerJ mutants of Bacillus subtilis.
Warburg, R J; Buchanan, C E; Parent, K; Halvorson, H O
1986-08-01
A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Stenger, Drake C., E-mail: drake.stenger@ars.usda.
Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of themore » corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection. -- Highlights: •Sampling protocol minimized differential selection/history among isolates. •Polymorphism among consensus sequences dominated by negative/purifying selection. •Within-isolate N:S ratio corrected for RT-PCR error by bootstrapping. •Within-isolate mutant spectrum dominated by new mutations yet to undergo selection.« less
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Brown, Steven; Guss, Adam; Yang, Shihui; Karpinets, Tatiana; Lynd, Lee; Shao, Xiongjun
2014-01-14
The present invention provides isolated nucleic acid molecules which encode a mutant acetaldehyde-CoA/alcohol dehydrogenase or mutant alcohol dehydrogenase and confer enhanced tolerance to ethanol. The invention also provides related expression vectors, genetically engineered microorganisms having enhanced tolerance to ethanol, as well as methods of making and using such genetically modified microorganisms for production of biofuels based on fermentation of biomass materials.
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Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing.
Garcia, Virginie; Bres, Cécile; Just, Daniel; Fernandez, Lucie; Tai, Fabienne Wong Jun; Mauxion, Jean-Philippe; Le Paslier, Marie-Christine; Bérard, Aurélie; Brunel, Dominique; Aoki, Koh; Alseekh, Saleh; Fernie, Alisdair R; Fraser, Paul D; Rothan, Christophe
2016-12-01
The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F 2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F 2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.
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Islam, Md Tarikul; Sarkar, Suprovath Kumar; Sultana, Nusrat; Begum, Mst Noorjahan; Bhuyan, Golam Sarower; Talukder, Shezote; Muraduzzaman, A K M; Alauddin, Md; Islam, Mohammad Sazzadul; Biswas, Pritha Promita; Biswas, Aparna; Qadri, Syeda Kashfi; Shirin, Tahmina; Banu, Bilquis; Sadya, Salma; Hussain, Manzoor; Sarwardi, Golam; Khan, Waqar Ahmed; Mannan, Mohammad Abdul; Shekhar, Hossain Uddin; Chowdhury, Emran Kabir; Sajib, Abu Ashfaqur; Akhteruzzaman, Sharif; Qadri, Syed Saleheen; Qadri, Firdausi; Mannoor, Kaiissar
2018-01-02
Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.
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Kim, W; Whitman, W B
1999-01-01
To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A plasmid, pWDK104, was recovered from this mutant by electroporation of a plasmid preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1). PMID:10430573
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Min, Sharon; Ingraham, Karen; Huang, Jianzhong; McCloskey, Lynn; Rilling, Sarah; Windau, Anne; Pizzollo, Jason; Butler, Deborah; Aubart, Kelly; Miller, Linda A; Zalacain, Magdalena; Holmes, David J; O'Dwyer, Karen
2015-08-01
The continuous emergence of multidrug-resistant pathogenic bacteria is compromising the successful treatment of serious microbial infections. GSK1322322, a novel peptide deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated safety and efficacy in human proof-of-concept clinical studies. In vitro studies were performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major pathogens that cause respiratory tract and skin infections. Resistance to GSK1322322 occurred at high frequency through loss-of-function mutations in the formyl-methionyl transferase (FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4 strains) and via missense mutations in Streptococcus pneumoniae (6/21 strains), but the mutations were associated with severe in vitro and/or in vivo fitness costs. The overall FoR to GSK1322322 was very low in Haemophilus influenzae, with only one PDF mutant being identified in one of four strains. No target-based mutants were identified from S. pyogenes, and only one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S. pneumoniae, PDF mutants were isolated from only six of 21 strains tested; an additional 10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants characterized from those three organisms (35/37 mutants) carried mutations in residues at or in close proximity to one of three highly conserved motifs that are part of the active site of the PDF protein, with 30 of the 35 mutations occurring at position V71 (using the S. pneumoniae numbering system). Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Identification of Two Loci in Tomato Reveals Distinct Mechanisms for Salt Tolerance
Borsani, Omar; Cuartero, Jesus; Fernández, José A.; Valpuesta, Victoriano; Botella, Miguel A.
2001-01-01
Salt stress is one of the most serious environmental factors limiting the productivity of crop plants. To understand the molecular basis for salt responses, we used mutagenesis to identify plant genes required for salt tolerance in tomato. As a result, three tomato salt-hypersensitive (tss) mutants were isolated. These mutants defined two loci and were caused by single recessive nuclear mutations. The tss1 mutant is specifically hypersensitive to growth inhibition by Na+ or Li+ and is not hypersensitive to general osmotic stress. The tss2 mutant is hypersensitive to growth inhibition by Na+ or Li+ but, in contrast to tss1, is also hypersensitive to general osmotic stress. The TSS1 locus is necessary for K+ nutrition because tss1 mutants are unable to grow on a culture medium containing low concentrations of K+. Increased Ca2+ in the culture medium suppresses the growth defect of tss1 on low K+. Measurements of membrane potential in apical root cells were made with an intracellular microelectrode to assess the permeability of the membrane to K+ and Na+. K+-dependent membrane potential measurements indicate impaired K+ uptake in tss1 but not tss2, whereas no differences in Na+ uptake were found. The TSS2 locus may be a negative regulator of abscisic acid signaling, because tss2 is hypersensitive to growth inhibition by abscisic acid. Our results demonstrate that the TSS1 locus is essential for K+ nutrition and NaCl tolerance in tomato. Significantly, the isolation of the tss2 mutant demonstrates that abscisic acid signaling is also important for salt and osmotic tolerance in glycophytic plants. PMID:11283342
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kuge, O.; Nishijima, M.; Akamatsu, Y.
1986-05-05
Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with /sup 32/Pi and L-(U-/sup 14/C)serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% ofmore » that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells.« less
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Ingraham, Karen; Huang, Jianzhong; McCloskey, Lynn; Rilling, Sarah; Windau, Anne; Pizzollo, Jason; Butler, Deborah; Aubart, Kelly; Miller, Linda A.; Zalacain, Magdalena; Holmes, David J.; O'Dwyer, Karen
2015-01-01
The continuous emergence of multidrug-resistant pathogenic bacteria is compromising the successful treatment of serious microbial infections. GSK1322322, a novel peptide deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated safety and efficacy in human proof-of-concept clinical studies. In vitro studies were performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major pathogens that cause respiratory tract and skin infections. Resistance to GSK1322322 occurred at high frequency through loss-of-function mutations in the formyl-methionyl transferase (FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4 strains) and via missense mutations in Streptococcus pneumoniae (6/21 strains), but the mutations were associated with severe in vitro and/or in vivo fitness costs. The overall FoR to GSK1322322 was very low in Haemophilus influenzae, with only one PDF mutant being identified in one of four strains. No target-based mutants were identified from S. pyogenes, and only one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S. pneumoniae, PDF mutants were isolated from only six of 21 strains tested; an additional 10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants characterized from those three organisms (35/37 mutants) carried mutations in residues at or in close proximity to one of three highly conserved motifs that are part of the active site of the PDF protein, with 30 of the 35 mutations occurring at position V71 (using the S. pneumoniae numbering system). PMID:26014938
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NASA Astrophysics Data System (ADS)
Pommerol, Antoine; Thomas, Nicolas; Antonella Barucci, M.; Bertaux, Jean-Loup; Davidsson, Björn; Ramy El-Maarry, Mohamed; La Forgia, Fiorengela; Fornasier, Sonia; Gracia, Antonio; Groussin, Olivier; Jost, Bernhard; Keller, Horst Uwe; Kuehrt, Ekkehard; Marschall, Raphael; Massironi, Matteo; Motolla, Stefano; Naletto, Giampiero; Oklay, Nilda; Pajola, Maurizio; Poch, Olivier
2015-04-01
Since the beginning of Rosetta's orbital observations, over a hundred small bright spots have been identified in images returned by its OSIRIS NAC camera, in all types of morphological regions on the nucleus. Bright spots are found as clusters of several tens of individuals in the vicinity of cliffs, or isolated without clear structural relation to the surrounding terrain. They are however mostly observed in the areas of the nucleus currently receiving the lowest amount of insolation and some of the best examples appear completely surrounded by shadows. Their typical sizes are of the order of a few metres and they are often observed at the surfaces of boulders of larger dimension. The brightness of these spots is up to ten times the average brightness of the surrounding terrain and multi-spectral analyses show a significantly bluer spectrum over the 0.3-1µm range. Comparisons of images taken in September and November 2014 under similar illumination conditions do not show any significant change of these features. Analysis of the results of past and present laboratory experiments with H2O-ice/dust mixtures provide interesting insights about the nature and origin of the bright spots. In particular, recent sublimation experiments conducted at the University of Bern reproduce the spectro-photometric variability observed at the surface of the nucleus by sequences of formation and ejection of a mantle of refractory organic-rich dust at the surface of the icy material. The formation of hardened layers of ice by sintering/re-condensation below the uppermost dust layer can also have strong implications for both the photometric and mechanical properties of the subsurface layer. Based on the comparison between OSIRIS observations and laboratory results, our favoured interpretation of the observed features is that the bright spots are exposures of water ice, resulting from the removal of the uppermost layer of refractory dust that covers the rest of the nucleus. Some of the observations of clusters of bright spots are very indicative of a formation process, which involves the breakage and collapse of brittle layers of ice to form fields of large boulders, some of them showing bright spots on part of their surface. Some of the isolated spots observed elsewhere on the nucleus might as well have been formed by similar processes and then have been transported over large distances by multiple bounces. These surface exposures of water ice must be more recent than the last passage at perihelion, as they would rapidly sublimate at short heliocentric distance. The hypothesis formulated here will thus easily be tested as the comet approaches the Sun, by checking if and how fast the bright spots vanish and disappear.
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Wang, Y J; Liu, T; Hou, J M; Zuo, Y H
2013-09-01
In this report, 156 hygromycin-resistant mutants were generated via restriction enzyme-mediated insertional (REMI) mutagenesis. All mutants were subjected to a bioassay on detached leaves. Five mutants (T4, T39, T71, T91, and T135) showed reduced symptom development, whereas one mutant (T120) did not exhibit any symptoms on the leaves compared with the wild type. The pathogenicity of these mutants was further assayed through the spray inoculation of whole seedlings. The results demonstrated that the pathogenicity of the T4, T39, T71, T91, and T135 mutants was reduced, whereas the T120 mutant lost its pathogenicity. Southern blot analysis revealed that the plasmids were inserted at different sites in the genome with different copy numbers. Flanking sequences approximately 550, 860, and 150 bp were obtained from T7, T91, and T120, respectively through plasmids rescue. Sequence analysis of the flanking sequences from T7 and T91 showed no homology to any known sequences in GenBank. The flanking sequence from the T120 mutant was highly homologous to MAPKK kinases, which regulates sexual/asexual development, melanization, pathogenicity from Cochliobolus heterostrophus. These results indicate that REMI and plasmids rescue have great potential for finding pathogenicity genes.
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Andrographolide induces degradation of mutant p53 via activation of Hsp70.
Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu
2018-05-22
The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.
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Pruvost, O; Roumagnac, P; Gaube, C; Chiroleu, F; Gagnevin, L
2005-01-01
Mango bacterial black spot, caused by Xanthomonas campestris pv. mangiferaeindicae, is a potentially severe disease in several tropical and subtropical areas. Data describing the life cycle of the pathogen are needed for improving integrated pest management strategies. Because of the important bacterial microflora associated with mango leaves, isolation of the pathogen is often difficult using nonselective agar media. A previously developed medium, BVGA, failed to inhibit several Gram-negative saprophytic bacteria, especially those belonging to Enterobacteriaceae. Two new semiselective media were developed. The selectivity of KC and NCTM3 media was achieved using cephalexin 40 mg l(-1), kasugamycin 20 mg l(-1) and neomycin 1 mg l(-1), cephalexin 100 mg l(-1), trimethoprime 5 mg l(-1), pivmecillinam 100 mg l(-1) respectively. Plating efficiencies ranged from 76 to 104% and from 78 to 132% for KC and NCTM3 respectively. The new media allowed the growth of X. campestris pv. mangiferaeindicae whatever its country of isolation. The pathogen was repeatedly isolated with these media from asymptomatic leaves sampled in growth chamber experiments. This work provides a description of new semiselective media, which should be valuable tools to study the ecology and epidemiology of X. campestris pv. mangiferaeindicae.
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The role of proline substitutions within flexible regions on thermostability of luciferase.
Yu, Haoran; Zhao, Yang; Guo, Chao; Gan, Yiru; Huang, He
2015-01-01
Improving the stability of firefly luciferase has been a critical issue for its wider industrial applications. Studies about hyperthermophile proteins show that flexibility could be an effective indicator to find out weak spots to engineering thermostability of proteins. However, the relationship among flexibility, activity and stability in most of proteins is unclear. Proline is the most rigid residue and can be introduced to rigidify flexible regions to enhance thermostability of proteins. We firstly apply three different methods, molecular dynamics (MD) simulation, B-FITTER and framework rigidity optimized dynamics algorithm (FRODA) to determine the flexible regions of Photinus pyralis luciferase: Fragment 197-207; Fragment 471-481 and Fragment 487-495. Then, introduction of proline is used to rigidify these flexible regions. Two mutants D476P and H489P within most flexible regions are finally designed. In the results, H489P mutant shows improved thermostability while maintaining its catalytic efficiency compared to that of wild type luciferase. Flexibility analysis confirms that the overall rigidity and local rigidity of H489P mutant are greatly strengthened. D476P mutant shows decreased thermosatbility and the reason for this is elucidated at the molecular level. S307P mutation is randomly chosen outside the flexible regions as a control. Thermostability analysis shows that S307P mutation has decreased kinetic stability and enhanced thermodynamic stability. Copyright © 2014 Elsevier B.V. All rights reserved.
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Characterization of some isolates of newly recovered avian sarcoma virus. [X Radiation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Halpern, C.C.; Hayward, W.S.; Hanafusa, H.
1979-01-01
We previously reported the isolation of a newly recovered avian sarcoma virus (rASV) from tumors of chickens injected with transformation-defective (td) mutants of the Schmidt--Ruppin strain of Rous sarcoma virus (SR--RSV). In this paper, we present further biological and biochemical characterization of the recovered sarcoma viruses. High titers of rASV's were generally obtained by cocultivation of tumor cells with normal chicken embryo fibroblasts or by homogenization of tumor tissues. Most rASV isolates were similar to SR--RSV, subgroup A (SR--RSV--A), in their growth characteristics and were nondefective in replication. The subgroup specificity of rASV's and the electrophoretic mobilities of their structuralmore » proteins were the same as those of parental td viruses. The nondefectiveness of rASV's was further substantiated by the size of their genomic RNA, which was indistinguishable from that of SR--RSV--AA and substantially larger than that of parental td RNA. Molecular hybridization using complementary DNA specific to the src gene of SR--RSV (cDNA/sub src/) showed that the RNAs of td mutants used in this study contained extensive deletions within the src gene (7 to 30% hybridization with cDNA/sub src/); the same probe hybridized up to 90% with RNA from two isolates of rASV. These data indicate that rASV has regained genetic information which had been deleted in the td mutants and strongly suggest that the generation of rASV involves a genetic interaction between td virus and host cell genetic information.« less
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Kumar, Ayush; Worobec, Elizabeth A
2002-10-01
To determine the presence of a proton gradient-dependent efflux of fluoroquinolone drugs in Serratia marcescens. Thirteen clinical isolates of S. marcescens were screened for resistance to four fluoroquinolones: ofloxacin, ciprofloxacin, norfloxacin and nalidixic acid by determining MICs. The presence of a proton gradient-dependent efflux mechanism was assessed using ethidium bromide accumulation assays. Drug accumulation studies for norfloxacin, ciprofloxacin and ofloxacin were performed to determine the drug specificity of efflux. Western transfer of cellular proteins, followed by immunodetection using anti-AcrA (Escherichia coli) antibodies were used to demonstrate the presence of a resistance-nodulation-cell division (RND) pump protein. PCR was used to identify a RND pump-encoding gene using primers for two conserved motifs within inner membrane components of RND proteins. A mutant strain of S. marcescens, UOC-67WL, was isolated by culturing the wild-type strain in the presence of ciprofloxacin in T-soy media and was subjected to the same studies as described above for the clinical isolates. Ethidium bromide accumulation assays confirmed the presence of a proton gradient-dependent efflux mechanism in S. marcescens. One clinical isolate, T-861, and the mutant strain, UOC-67WL, were found to efflux ciprofloxacin and ofloxacin. Western immunoblot results confirmed overexpression of an AcrA-like protein in T-861 and UOC-67WL. Sequencing of the PCR product showed the presence of a mexF-like gene, which is overexpressed in nfxC mutants of Pseudomonas aeruginosa. This study reports the presence of a proton gradient-dependent efflux mechanism in S. marcescens.
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Sng, L H; Cornish, N; Knapp, C C; Ludwig, M D; Hall, G S; Washington, J A
1998-04-01
Bacteremia due to a vancomycin-dependent enterococcus (VDE) occurred during long-term vancomycin therapy in a renal transplant recipient with underlying pancreatitis and a vancomycin-resistant enterococcal (VRE) wound infection and bacteremia. The VDE was isolated from blood during vancomycin therapy and grew only in the presence of vancomycin and D-alanine-D-alanine (DADA), a substance required for cell-wall synthesis. Colonies beyond the periphery of growth of the VDE around a vancomycin disk contained vancomycin-independent revertant mutants after 48 hours of incubation. Pulsed-field gel electrophoresis of the VDE, revertant mutant, the initial blood culture isolate of VRE, and an autopsy isolate showed that the four strains were identical. Antimicrobial susceptibility testing was performed using standard macrobroth and microbroth dilution methods. DADA was used as a growth supplement for macrobroth dilution susceptibility testing of the VDE isolate. Minimum inhibitory concentrations (MICs) were similar for the VRE isolate and the VDE revertant, which were both resistant to ampicillin, high-level gentamicin, ciprofloxacin, imipenem, vancomycin, and daptomycin, and were susceptible to fusidic acid, high-level streptomycin, rifampin, and a quinupristin-dalfopristin combination. The MICs of teicoplanin were 2 microg/mL or less and 16 microg/mL for the clinical VRE isolate and the VDE revertant, respectively. The autopsy isolate was resistant to all antimicrobials tested and showed a fourfold increase in MICs for quinupristin-dalfopristin compared with that of the original blood isolate. The VDE was susceptible to all drugs tested except vancomycin.
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Vödisch, Martin; Albrecht, Daniela; Lessing, Franziska; Schmidt, André D; Winkler, Robert; Guthke, Reinhard; Brakhage, Axel A; Kniemeyer, Olaf
2009-03-01
The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. We established a 2-D reference map for A. fumigatus. Using MALDI-TOF-MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.
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NASA Technical Reports Server (NTRS)
Stevens, G. H.; Anzic, G.
1979-01-01
Issues and results in a NASA study of the potential concepts and markets for a multibeam 30/20 GHz domestic satellite system in the 1990s are presented. Issues considered include the reduction of signal attenuation due to rain, beam-beam interference isolation in the multibeam system, the method of access/modulation (FDMA, TDMA or hybrid) and the market for reduced reliability and wideband services. A hypothetical demonstration payload configuration which would attempt to resolve these issues is illustrated. The communications payload would employ a system of seven contiguous coverage spots in order to demonstrate a typical cell in a contiguous beam system having extensive frequency re-use, as in a direct-to-user system, and a single spot, typical of a trunking system, to determine signal isolation. The payload could be carried on several existing buses and is illustrated on an MMS bus.
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Detection and Identification of the First Viruses in Chia (Salvia hispanica)
Celli, Marcos G.; Perotto, Maria C.; Martino, Julia A.; Flores, Ceferino R.; Conci, Vilma C.; Pardina, Patricia Rodriguez
2014-01-01
Chia (Salvia hispanica), an herbaceous plant native to Latin America, has become important in the last 20 years due to its beneficial effects on health. Here, we present the first record and identification of two viruses in chia plants. The comparison of the complete nucleotide sequences showed the presence of two viral species with the typical genome organization of bipartite New World begomovirus, identified as Sida mosaic Bolivia virus 2 and Tomato yellow spot virus, according to the ICTV taxonomic criteria for begomovirus classification. DNA-A from Sida mosaic Bolivia virus 2 exhibited 96.1% nucleotide identity with a Bolivian isolate of Sida micrantha, and Tomato yellow spot virus showed 95.3% nucleotide identity with an Argentine bean isolate. This is the first report of begomoviruses infecting chia as well as of the occurrence of Sida mosaic Bolivia virus 2 in Argentina. PMID:25243369
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Mutagenizing brewing yeast strain for improving fermentation property of beer.
Liu, Zengran; Zhang, Guangyi; Sun, Yunping
2008-07-01
A brewing yeast mutant with perfect sugar fermentation capacity was isolated by mutagenizing the Saccharomyces pastorianus transformant, which carries an integrated glucoamylase gene and has one copy of non-functional alpha-acetolactate synthase gene. The mutant was able to utilize maltotriose efficiently, and the maltotriose fermentability in YNB-2% maltotriose medium increased from 32.4% to 72.0% after 5 d in shaking culture. The wort fermentation test confirmed that the sugar fermentation property of the mutant was greatly improved, while its brewing performances were analogous to that of the wild-type strain and the characteristic trait of shortened beer maturation period was retained. Therefore, we believe that the brewing yeast mutant would benefit the beer industry and would be useful for low caloric beer production.
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Purkan; Ihsanawati; Natalia, D; Syah, Y M; Retnoningrum, D S; Kusuma, H S
2016-01-01
Mutations in katG gene are often associated with isoniazid (INH) resistance in Mycobacterium tuberculosis strain. This research was perfomed to identify the katG mutation in clinical isolate (L8) that is resistant to INH at 1 μg/ml. In addition to characterize the catalase-peroxidase of KatG L8 and perform the ab initio structural study of the protein to get a more complete understanding in drug activation and the resistance mechanism. The katG gene was cloned and expressed in Escherichia coli, then followed by characterization of catalase-peroxidase of KatG. The structure modelling was performed to know a basis of alterations in enzyme activity. A substitution of A713G that correspond to Asn238Ser replacement was found in the L8 katG. The Asn238Ser modification leads to a decline in the activity of catalase-peroxidase and INH oxidation of the L8 KatG protein. The catalytic efficiency (Kcat/KM) of mutant KatGAsn238Ser respectively decreases to 41 and 52% for catalase and peroxidase. The mutant KatGAsn238Ser also shows a decrease of 62% in INH oxidation if compared to a wild type KatG (KatGwt). The mutant Asn238Ser might cause instability in the substrate binding site of KatG, because of removal of a salt bridge connecting the amine group of Asn238 to the carboxyl group of Glu233, which presents in KatGwt. The lost of the salt bridge in the substrate binding site in mutant KatGAsn238Ser created changes unfavorable for enzyme activities, which in turn emerge as INH resistance in the L8 isolate of M. tuberculosis.
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Grigg, Matthew J; Barber, Bridget E; Marfurt, Jutta; Imwong, Mallika; William, Timothy; Bird, Elspeth; Piera, Kim A; Aziz, Ammar; Boonyuen, Usa; Drakeley, Christopher J; Cox, Jonathan; White, Nicholas J; Cheng, Qin; Yeo, Tsin W; Auburn, Sarah; Anstey, Nicholas M
2016-01-01
Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.
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Wang, Won-Bo; Yuan, Yu-Han; Hsueh, Po-Ren; Liaw, Shwu-Jen
2012-01-01
In this study, we demonstrated that 10′(Z), 13′(E)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof Proteus mirabilis which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and rppA mutant but not in the rcsB mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the rcsB mutant. Moreover, the finding that HQ17-2 inhibits the expression of flhDC gene in the wild-type and rcsB-complemented strain but not in the rcsB mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and rcsB mutant but not in the rppA mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of rppA and pmrI, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the rppA mutant. The inhibition of rppA and pmrI expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications. PMID:23029100
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Faille, Christine; Sadovskaya, Irina; Charbit, Alain; Benezech, Thierry; Shen, Yang; Loessner, Martin J.; Bautista, Jean Romain; Midelet-Bourdin, Graziella
2018-01-01
The wall teichoic acid (WTA) is the major carbohydrate found within the extracellular matrix of the Listeria monocytogenes biofilm. We first addressed the frequency of spontaneous mutations in two genes (lmo2549 and lmo2550) responsible for the GlcNAcylation in 93 serotype 1/2a strains that were mainly isolated from seafood industries. We studied the impact of mutations in lmo2549 or lmo2550 genes on biofilm formation by using one mutant carrying a natural mutation inactivating the lmo2550 gene (DSS 1130 BFA2 strain) and two EGD-e mutants that lack respective genes by in-frame deletion of lmo2549 or lmo2550 using splicing-by-overlap-extension PCR, followed by allelic exchange mutagenesis. The lmo2550 gene mutation, occurring in around 50% isolates, caused a decrease in bacterial adhesion to stainless steel compared to wild-type EGD-e strain during the adhesion step. On the other hand, bacterial population weren’t significantly different after 24h-biofilm formation. The biofilm architecture was different between the wild-type strain and the two mutants inactivated for lmo2549 or lmo2550 genes respectively with the presence of bacterial micro-colonies for mutants which were not observed in the wild-type EGD-e strain biofilm. These differences might account for the stronger hydrophilic surface exhibited by the mutant cells. Upon a water flow or to a cleaning procedure at a shear stress of 0.16 Pa, the mutant biofilms showed the higher detachment rate compared to wild-type strain. Meanwhile, an increase in the amount of residual viable but non-culturable population on stainless steel was recorded in two mutants. Our data suggests that the GlcNAc residue of WTA played a role in adhesion and biofilm formation of Listeria monocyctogenes. PMID:29320565