Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines

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Robert Silas Allen

2013-09-01

Full Text Available Forward genetic screens have identified numerous genes involved in development and metabolism, and remain a cornerstone of biological research. However to locate a causal mutation, the practice of crossing to a polymorphic background to generate a mapping population can be problematic if the mutant phenotype is difficult to recognise in the hybrid F2 progeny, or dependent on parental specific traits. Here in a screen for leaf hyponasty mutants, we have performed a single backcross of an Ethane Methyl Sulphonate (EMS generated hyponastic mutant to its parent. Whole genome deep sequencing of a bulked homozygous F2 population and analysis via the Next Generation EMS mutation mapping pipeline (NGM unambiguously determined the causal mutation to be a single nucleotide polymorphisim (SNP residing in HASTY, a previously characterised gene involved in microRNA biogenesis. We have evaluated the feasibility of this backcross approach using three additional SNP mapping pipelines; SHOREmap, the GATK pipeline, and the samtools pipeline. Although there was variance in the identification of EMS SNPs, all returned the same outcome in clearly identifying the causal mutation in HASTY. The simplicity of performing a single parental backcross and genome sequencing a small pool of segregating mutants has great promise for identifying mutations that may be difficult to map using conventional approaches.

Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

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Diana J M van den Wollenberg

Full Text Available Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people

International Nuclear Information System (INIS)

Kyoizumi, Seishi; Umeki, Shigeko; Akiyama, Mitoshi

1991-06-01

The frequency of mutant T lymphocytes defective in T-cell receptor gene (α or β) expression was measured using the two-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of ≥ 1.5 Gy) and 125 of whom were distally exposed (DS86 doses of 228 Th formerly used for radiodiagnosis. In addition, thyroid disease patients treated with 131 I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for the measurement of recent exposures to genotoxic agents. (author)

Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

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Yano, Koichi; Masuda, Kenta; Akanuma, Genki; Wada, Tetsuya; Matsumoto, Takashi; Shiwa, Yuh; Ishige, Taichiro; Yoshikawa, Hirofumi; Niki, Hironori; Inaoka, Takashi; Kawamura, Fujio

2016-01-01

The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

Contribution of single-strand breaks and alkali-labile bonds to the loss of infectivity of γ-irradiated phiX174 RF-DNA in E. coli cells mutant in various repair functions

International Nuclear Information System (INIS)

McKee, R.H.

1975-01-01

Twenty-one radiation sensitive mutants have been examined for their capacity to support gamma-irradiated phiX174 RF-DNA. The survival of phiX174 RF-DNA was reduced in essentially all of the sensitive mutants. The irradiated phiX174 RF-DNA was then separated into populations containing either single-strand breaks or alkali-labile bonds to examine the capacity of the mutants to repair each of the classes of lesions. It was found that all E. coli strains are unable to repair 22 percent of the single-strand breaks and all sensitive mutants are unable to repair an additional 10 percent of the breaks. All the repair functions examined are involved in single-strand break repair and none are more or less necessary than any of the others. PhiX174 RF-DNA is also inactivated by alkali-labile bonds. In the normal strains the inactivation efficiency is 0.16 lethal events per lesion with a threshold dose of 15 to 20 krads. The mutants are divided into two classes by their sensitivity to alkali-labile bonds. Both classes of mutants are also inactivated by alkali-labile bonds with efficiencies of about 0.17 and 0.29 lethal events per lesion, respectively. It is proposed that the differences seen in survival curves of phiX174 measured in the sensitive mutants is due to this difference. Although in normal cells the efficiency of inactivation of phiX174 by single-strand breaks is 50 percent greater than by alkali-labile bonds, alkali-labile bonds are produced at approximately twice the rate of single-strand breaks so alkali-labile bonds account for about 61 percent of the overall inactivation. In the mutants of least sensitivity alkali-labile bonds account for about 54 percent of the inactivating events and in the most sensitive about 67 percent

Non-oncogenic T-region mutants of Agrobacterium tumefaciens do transfer T-DNA into plant cells

NARCIS (Netherlands)

Hille, Jacques; Wullems, George; Schilperoort, Rob

1983-01-01

A new procedure for site-directed mutagenesis has been applied to the shooting and rooting loci of T-DNA of an octopine Ti-plasmid of Agrobacterium tumefaciens. Mutants have been obtained which induced tumours that either developed shoots or produced more roots than normally observed. Double

Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10).

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Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

2011-08-26

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)*

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Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

2011-01-01

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1–332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant. PMID:21719701

Construction and functional analysis of Trichoderma harzianum mutants that modulate maize resistance to the pathogen Curvularia lunata.

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Fan, Lili; Fu, Kehe; Yu, Chuanjin; Ma, Jia; Li, Yaqian; Chen, Jie

2014-01-01

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate an insertional mutant library of the mycelial fungus Trichoderma harzianum. From a total of 450 mutants, six mutants that showed significant influence on maize resistance to C. lunata were analyzed in detail. Maize coated with these mutants was more susceptible to C. lunata compared with those coated with a wild-type (WT) strain. Similar to other fungal ATMT libraries, all six mutants were single copy integrations, which occurred preferentially in noncoding regions (except two mutants) and were frequently accompanied by the loss of border sequences. Two mutants (T66 and T312) that were linked to resistance were characterized further. Maize seeds coated with T66 and T312 were more susceptible to C. lunata than those treated with WT. Moreover, the mutants affected the resistance of maize to C. lunata by enhancing jasmonate-responsive gene expression. T66 and T312 induced maize resistance to C. lunata infection through a jasmonic acid-dependent pathway.

Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

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Qi Jia

2012-01-01

Full Text Available In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

Rapid selection of escape mutants by the first CD8 T cell responses in acute HIV-1 infection

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Korber, Bette Tina Marie [Los Alamos National Laboratory

2008-01-01

The recent failure of a vaccine that primes T cell responses to control primary HIV-1 infection has raised doubts about the role of CD8+ T cells in early HIV-1 infection. We studied four patients who were identified shortly after HIV-1 infection and before seroconversion. In each patient there was very rapid selection of multiple HIV-1 escape mutants in the transmitted virus by CD8 T cells, including examples of complete fixation of non-synonymous substitutions within 2 weeks. Sequencing by single genome amplification suggested that the high rate of virus replication in acute infection gave a selective advantage to virus molecules that contained simultaneous and gained sequential T cell escape mutations. These observations show that whilst early HIV-1 specific CD8 T cells can act against virus, rapid escape means that these T cell responses are unlikely to benefit the patient and may in part explain why current HIV-1 T cell vaccines may not be protective.

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs.

Science.gov (United States)

Mandal, Debabrata; Köhrer, Caroline; Su, Dan; Babu, I Ramesh; Chan, Clement T Y; Liu, Yuchen; Söll, Dieter; Blum, Paul; Kuwahara, Masayasu; Dedon, Peter C; Rajbhandary, Uttam L

2014-02-01

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2(Ile)) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2(Ile) binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants.

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Shis, David L; Bennett, Matthew R

2013-03-26

The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host's native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits.

Transcriptional and proteomic analysis of the Aspergillus fumigatus ΔprtT protease-deficient mutant.

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Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W P; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

2012-01-01

Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus.

Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

Science.gov (United States)

Tedesco, D; Fischer-Fantuzzi, L; Vesco, C

1993-03-01

Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes

International Nuclear Information System (INIS)

Hakoda, Masayuki; Akiyama, Mitoshi; Kyoizumi, Seishi; Kobuke, Kyoko; Awa, A.A.

1987-07-01

Approximately 80 % of human peripheral blood T-lymphocytes could be cloned in the presence of crude Interleukin-2, phytohemagglutinin, and X-irradiated autologous lymphocytes and Raji B-cells. This modified cloning method was used to measure the in vivo frequency of HGPRT-deficient mutant T-lymphocytes. Repeated experiments using blood from the same individuals revealed that the frequency of mutant cells was almost constant for each individual even though the cloning efficiency of lymphocytes varied somewhat from experiment to experiment. Approximately 80 % of both wild-type unselected and 6-thioguanine-resistant colonies had helper/inducer and about 20 % had suppressor/cytotoxic T-lymphocyte markers. No difference was observed in the distribution of lymphocyte subsets between wild and mutant lymphocyte colonies. (author)

Detailed conformation dynamics and activation process of wild type c-Abl and T315I mutant

Science.gov (United States)

Yang, Li-Jun; Zhao, Wen-Hua; Liu, Qian

2014-10-01

Bcr-Abl is an important target for therapy against chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The synergistic effect between myristyl pocket and the ATP pocket has been found. But its detailed information based on molecular level still has not been achieved. In this study, conventional molecular dynamics (CMD) and target molecular dynamics (TMD) simulations were performed to explore the effect of T315I mutation on dynamics and activation process of Abl containing the N-terminal cap (Ncap). The CMD simulation results reveal the increasing flexibility of ATP pocket in kinase domain (KD) after T315I mutation which confirms the disability of ATP-pocket inhibitors to the Abl-T315I mutant. On the contrary, the T315I mutation decreased the flexibility of remote helix αI which suggests the synergistic effect between them. The mobility of farther regions containing Ncap, SH3, SH2 and SH2-KD linker were not affected by T315I mutation. The TMD simulation results show that the activation process of wild type Abl and Abl-T315I mutant experienced global conformation change. Their differences were elucidated by the activation motion of subsegments including A-loop, P-loop and Ncap. Besides, the T315I mutation caused decreasing energy barrier and increasing intermediate number in activation process, which results easier activation process. The TMD and CMD results indicate that a drug targeting only the ATP pocket is not enough to inhibit the Abl-T315I mutant. An effective way to inhibit the abnormal activity of Abl-T315I mutant is to combine the ATP-pocket inhibitors with inhibitors binding at non-ATP pockets mainly related to Ncap, SH2-KD linker and myristyl pocket.

Molecular analysis of human argininosuccinate lyase: Mutant characterization and alternative splicing of the coding region

International Nuclear Information System (INIS)

Walker, D.C.; McCloskey, D.A.; Simard, L.R.; McInnes, R.R.

1990-01-01

Argininosuccinic acid lyase (ASAL) deficiency is a clinically heterogeneous autosomal recessive urea cycle disorder. The authors previously established by complementation analysis that 29 ASAL-deficient patients have heterogeneous mutations in a single gene. To prove that the ASAL structural gene is the affected locus, they sequenced polymerase chain reaction-amplified ASAL cDNA of a representative mutant from the single complementation group. Fibroblast strain 944 from a late-onset patient who was the product of a consanguineous mating, had only a single base-pair change in the coding region, a C-283→ T transition at a CpG dinucleotide in exon 3. This substitution converts Arg-95 to Cys (R95C), occurs in a stretch of 13 residues that is identical in yeast and human ASAL, and was present in both of the patient's alleles but not in 14 other mutant or 10 normal alleles. They observed that amplified cDNA from mutant 944 and normal cells (liver, keratinocytes, lymphoblasts, and fibroblasts) contained, in addition to the expected 5' 513-base-pair band, a prominent 318-base-pair ASAL band formed by the splicing of exon 2 from the transcript. The short transcript maintains the ASAL reading frame but removes Lys-51, a residue that may be essential for catalysis, since it binds the argininosuccinate substrate. They conclude (i) that the identification of the R95C mutation in strain 944 demonstrates that virtually all ASAL deficiency results from defects in the ASAL structural gene and (ii) that minor alternative splicing of the coding region occurs at the ASAL locus

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

Science.gov (United States)

Shcherbakov, Victor P; Kudryashova, Elena

2014-09-01

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss

Using heavy-ion mutagenesis technology to select cellulose enzyme vitality of mutants of Aspergillium niger

International Nuclear Information System (INIS)

Tang Jiahui; Yang Fumin; Wang Shuyang

2012-01-01

In order to improve the cellulose ion beam at 20, 40, 60, 80, 100, 120Gy and 140 enzyme vitality of Aspergillus niger (=AS3.316), heavy Gy doses was used for inducing mutation. Higher cellulose enzyme vitality strains were screened through the primary screening and secondary screening. The result showed that 5 mutants T2-1, T3-1, T5-1, T6-3, T6-4 were selected, and T6-4 had the highest cellulose enzyme activity. The activity of filter paper cellulose enzyme, endo-glucanase, exo-glucanase and 13-glucosidase of T6-4 was 61.3, 116.2, 29.9 U/mL and 35.9 U/mL respectively. Compared with the original A. niger (=AS3.316), the cellulose enzyme activity was increased by 3.5, 3.78, 2.76 and 2.52 times in turn. The activity of cellulose enzyme of the rest mutants sorted from strong to the weak were T6-3T5-1T3-1T2-1. The dose at 120 Gy showed the best mutagenesis effect. Mutants had different degree of changes in the genetic stability, but overall, the performance showed relatively stable

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

Science.gov (United States)

Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

Directory of Open Access Journals (Sweden)

Peng Li

2017-09-01

Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

Energy Technology Data Exchange (ETDEWEB)

Jett, J. [Lawrence Livermore National Lab., CA (United States)

1994-12-01

While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

International Nuclear Information System (INIS)

Hasegawa, K.; Kuge, O.; Nishijima, M.; Akamatsu, Y.

1989-01-01

We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well

mPeriod2 Brdm1 and other single Period mutant mice have normal food anticipatory activity.

Science.gov (United States)

Pendergast, Julie S; Wendroth, Robert H; Stenner, Rio C; Keil, Charles D; Yamazaki, Shin

2017-11-14

Animals anticipate the timing of food availability via the food-entrainable oscillator (FEO). The anatomical location and timekeeping mechanism of the FEO are unknown. Several studies showed the circadian gene, Period 2, is critical for FEO timekeeping. However, other studies concluded that canonical circadian genes are not essential for FEO timekeeping. In this study, we re-examined the effects of the Per2 Brdm1 mutation on food entrainment using methods that have revealed robust food anticipatory activity in other mutant lines. We examined food anticipatory activity, which is the output of the FEO, in single Period mutant mice. Single Per1, Per2, and Per3 mutant mice had robust food anticipatory activity during restricted feeding. In addition, we found that two different lines of Per2 mutant mice (ldc and Brdm1) anticipated restricted food availability. To determine if FEO timekeeping persisted in the absence of the food cue, we assessed activity during fasting. Food anticipatory (wheel-running) activity in all Period mutant mice was also robust during food deprivation. Together, our studies demonstrate that the Period genes are not necessary for the expression of food anticipatory activity.

AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

Energy Technology Data Exchange (ETDEWEB)

O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)

2010-09-07

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

Energy Technology Data Exchange (ETDEWEB)

Henry, Rowan M; Caplan, David; Pomes, Regis [Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, M5G 1X8 (Canada); Fadda, Elisa, E-mail: pomes@sickkids.ca [Department of Chemistry, University of Galway (Ireland)

2011-06-15

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

International Nuclear Information System (INIS)

Henry, Rowan M; Caplan, David; Pomes, Regis; Fadda, Elisa

2011-01-01

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

Science.gov (United States)

Henry, Rowan M.; Caplan, David; Fadda, Elisa; Pomès, Régis

2011-06-01

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

High frequency of a single nucleotide substitution (c.-6-180T>G) of the canine MDR1/ABCB1 gene associated with phenobarbital-resistant idiopathic epilepsy in Border Collie dogs.

Science.gov (United States)

Mizukami, Keijiro; Yabuki, Akira; Chang, Hye-Sook; Uddin, Mohammad Mejbah; Rahman, Mohammad Mahbubur; Kushida, Kazuya; Kohyama, Moeko; Yamato, Osamu

2013-01-01

A single nucleotide substitution (c.-6-180T>G) associated with resistance to phenobarbital therapy has been found in the canine MDR1/ABCB1 gene in Border Collies with idiopathic epilepsy. In the present study, a PCR-restriction fragment length polymorphism assay was developed for genotyping this mutation, and a genotyping survey was carried out in a population of 472 Border Collies in Japan to determine the current allele frequency. The survey demonstrated the frequencies of the T/T wild type, T/G heterozygote, and G/G mutant homozygote to be 60.0%, 30.3%, and 9.8%, respectively, indicating that the frequency of the mutant G allele is extremely high (24.9%) in Border Collies. The results suggest that this high mutation frequency of the mutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies.

Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

Science.gov (United States)

Dahl, Joseph M; Wang, Hongyun; Lázaro, José M; Salas, Margarita; Lieberman, Kate R

2014-03-07

The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Science.gov (United States)

Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E.; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R.; Ross, Ian; Parton, Robert G.; Böcking, Till; Gambin, Yann

2016-01-01

Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils. PMID:27892477

A study of eukaryotic response mechanisms to atmospheric pressure cold plasma by using Saccharomyces cerevisiae single gene mutants

International Nuclear Information System (INIS)

Feng Hongqing; Wang Ruixue; Sun Peng; Wu Haiyan; Liu Qi; Li Fangting; Fang Jing; Zhang Jue; Zhu Weidong

2010-01-01

The mechanisms of eukaryotic cell response to cold plasma are studied. A series of single gene mutants of eukaryotic model organism Saccharomyces cerevisiae are used to compare their sensitivity to plasma treatment with the wild type. We examined 12 mutants in the oxidative stress pathway and the cell cycle pathway, in which 8 are found to be hypersensitive to plasma processing. The mutated genes' roles in the two pathways are analyzed to understand the biological response mechanisms of plasma treatment. The results demonstrate that genes from both pathways are needed for the eukaryotic cells to survive the complex plasma treatment.

Anomalous magnetic aftereffect in Nd3(Fe,T)29 (T = Ti or Re) intermetallic compounds

International Nuclear Information System (INIS)

Collocott, S.J.; Dunlop, J.B.; Gwan, P.B.

1999-01-01

Full text: The intermetallic compounds Nd 3 (Fe,Ti) 29 and Nd 3 (Fe,Re) 29 order ferromagnetically with Curie Temperatures, T c , of 430 and 370 K respectively. They have a monoclinic crystal structure, space group A2/m (Nd 3 (Fe,Ti) 29 type) with two rare earth sites and eleven Fe(T) sites, which is an intermediate structure between the rhombohedral Th 2 Zn 17 and tetragonal ThMn 12 structures, and is closely related to hexagonal CaCu 5 . Ferromagnetic materials, depending on their magnetic prehistory, may exhibit a time dependent magnetisation. The term 'magnetic aftereffect' is used to describe this behaviour, which may fall into three categories: 1. Reversible or diffusion aftereffect which is associated with the diffusion of impurity atoms or holes within the ferromagnetic lattice. 2. The irreversible or fluctuation aftereffect, which results in a logarithmic time dependence of magnetisation, J(t)=J(0)+Sln(t+t 0 ), where S is the magnetic viscosity and t 0 a parameter to establish the origin of the time scale measurements. 3. Quantum tunnelling of magnetisation which is observed at very low temperatures. A range of magnetic aftereffects have been observed in both Nd 3 (Fe,Ti) 29 and Nd 3 (Fe,Re) 29 . Of particular interest is the case where the material is fully saturated by application of a field in the positive direction, the applied field is then reversed to trace out part of the major demagnetisation curve into the third quadrant, and thence along a recoil curve, such that in zero applied field the magnetisation is zero (H=0, J=0). (This corresponds to dc field magnetisation.) This magnetic prehistory results in two interesting effects; spontaneous remagnetisation e.g. remagnetisation without application of an external field, and thermal remagnetisation e.g. an increase in magnetisation as the temperature is increased. Additionally, the behaviour of the magnetic viscosity has been explored on the major demagnetisation curve as a function of temperature

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains

International Nuclear Information System (INIS)

LingNah Su; Little, J.B.

1992-01-01

A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D o 192 ± 13 vs 127 ± 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. (author)

High Frequency of a Single Nucleotide Substitution (c.-6-180T>G of the Canine MDR1/ABCB1 Gene Associated with Phenobarbital-Resistant Idiopathic Epilepsy in Border Collie Dogs

Directory of Open Access Journals (Sweden)

Keijiro Mizukami

2013-01-01

Full Text Available A single nucleotide substitution (c.-6-180T>G associated with resistance to phenobarbital therapy has been found in the canine MDR1/ABCB1 gene in Border Collies with idiopathic epilepsy. In the present study, a PCR-restriction fragment length polymorphism assay was developed for genotyping this mutation, and a genotyping survey was carried out in a population of 472 Border Collies in Japan to determine the current allele frequency. The survey demonstrated the frequencies of the T/T wild type, T/G heterozygote, and G/G mutant homozygote to be 60.0%, 30.3%, and 9.8%, respectively, indicating that the frequency of the mutant G allele is extremely high (24.9% in Border Collies. The results suggest that this high mutation frequency of the mutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies.

Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

Science.gov (United States)

Regales, Lucia; Balak, Marissa N; Gong, Yixuan; Politi, Katerina; Sawai, Ayana; Le, Carl; Koutcher, Jason A; Solit, David B; Rosen, Neal; Zakowski, Maureen F; Pao, William

2007-08-29

The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M) alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M)-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M)-expressing animals develop tumors with longer latency than EGFR(L858R+T790M)-bearing mice and in the absence of additional kinase domain mutations. These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M) alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

Directory of Open Access Journals (Sweden)

Lucia Regales

2007-08-01

Full Text Available The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M-expressing animals develop tumors with longer latency than EGFR(L858R+T790M-bearing mice and in the absence of additional kinase domain mutations.These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

A mutant screening method by critical annealing temperature-PCR for site-directed mutagenesis.

Science.gov (United States)

Liu, Ying; Wu, Ting; Song, Jian; Chen, Xuelian; Zhang, Yu; Wan, Yu

2013-03-11

Distinguishing desired mutants from parental templates and undesired mutants is a problem not well solved in Quikchange™ mutagenesis. Although Dpn I digestion can eliminate methylated parental (WT) DNA, the efficiency is not satisfying due to the existence of hemi-methylated DNA in the PCR products, which is resistant to Dpn I. The present study designed a novel critical annealing temperature (T(c))-PCR to replace Dpn I digestion for more perfect mutant distinguishing, in which part-overlapping primers containing mutation(s) were used to reduce initial concentration of template DNA in mutagenic PCR. A T(c)-PCR with the same mutagenic primers was performed without Dpn I digestion. The T(c) for each pair of the primers was identified by gradient PCR. The relationship between PCR-identified T(c) and T(m) of the primers was analyzed and modeled with correlation and regression. Gradient PCR identified a T(c) for each of 14 tested mutagenic primers, which could discriminate mismatched parental molecules and undesired mutants from desired mutants. The PCR-identified T(c) was correlated to the primer's T(m) (r = 0.804, P<0.0001). Thus, in practical applications, the T(c) can be easily calculated with a regression equation, T(c)= 48.81 + 0.253*T(m). The new protocol introduced a novel T(c)-PCR method for mutant screening which can more efficiently and accurately select against parental molecules and undesired mutations in mutagenic sequence segments.

Identification of a Gravitropism-Deficient Mutant in Rice

Directory of Open Access Journals (Sweden)

He Yan

2017-03-01

Full Text Available A gravitropism-deficient mutant M96 was isolated from a mutant bank, generated by ethyl methane sulfonate (EMS mutagenesis of indica rice accession ZJ100. The mutant was characterized as prostrate growth at the beginning of germination, and the prostrate growth phenotype ran through the whole life duration. Tiller angle and tiller number of M96 increased significantly in comparison with the wild type. Tissue section observation analysis indicated that asymmetric stem growth around the second node occurred in M96. Genetic analysis and gene mapping showed that M96 was controlled by a single recessive nuclear gene, tentatively termed as gravitropism-deficient M96 (gdM96, which was mapped to a region of 506 kb flanked by markers RM5960 and InDel8 on the long arm of chromosome 11. Sequencing analysis of the open reading frames in this region revealed a nucleotide substitution from G to T in the third exon of LOC_Os11g29840. Additionally, real-time fluorescence quantitative PCR analysis showed that the expression level of LOC_Os11g29840 in the stems was much higher than in the roots and leaves in M96. Furthermore, the expression level was more than four times in M96 stem than in the wild type stem. Our results suggested that the mutant gene was likely a new allele to the reported gene LAZY1. Isolation of this new allele would facilitate the further characterization of LAZY1.

Monitoring hprt mutant frequency over time in T-lymphocytes of people accidentally exposed to high doses of ionizing radiation

International Nuclear Information System (INIS)

Cruz, A.D. da; Curry, J.; Glickman, B.W.

1996-01-01

Modern technologies have provided the opportunity to monitor mutations in people in vivo. The subjects of this study were accidentally exposed to 137 Cesium in a radiological accident that occurred in September 1987 in Goiania, Brazil, during which more than 150 people received doses greater than 0.1 Gy and as high as 7 Gy. The objective of this study was to determine how long the hprt mutant T-cells in the peripheral blood contribute to mutant T-cells in the peripheral blood contribute to mutant frequency by examining the timecourse of the T-lymphocyte response to ionizing radiation. This report describes the results obtained over a period of 2.3 to 4.5 years subsequent to the accident, from 11 subjects with doses ranging from 1 to 7 Gy, and from nine control subjects selected from the same population. The mean In MF (±SE) of the control group was 2.5 (±0.2) + In10 -6 . The exposed group had a significantly increased mutant frequency; the mean ln MF (±SE) were 3.3 (±0.3) + In10 -6 , 2.8 (±0.2) + In10 -6 , and 2.3 (±0.2) + In10 -6 , in the years 1990-1992 respectively and using Buckton's models, we demonstrated that mutant T-cells have a short-term memory with a half-life of 2.1 years. This relatively short half-half limits the effective use of the hprt assay as the method of choice to monitor past exposure. The data also demonstrate a positive correlation with age, and an inverse correlation with plating efficiency. 77 refs., 3 figs., 4 tabs

p21-ras effector domain mutants constructed by "cassette" mutagenesis

DEFF Research Database (Denmark)

Stone, J C; Vass, W C; Willumsen, B M

1988-01-01

A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...

Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.

Science.gov (United States)

Singh, Upinder; Brewer, Jeremy L; Boothroyd, John C

2002-05-01

Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

DEFF Research Database (Denmark)

Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.

2002-01-01

The fab1 mutant of Arabidopsis is partially deficient in activity of ß-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a c......DNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

Combined curative radiation therapy alone in (T1) T2-3 rectal adenocarcinoma: a pilot study of 29 patients

International Nuclear Information System (INIS)

Gerard, J.P.; Roy, P.; Coquard, R.; Barbet, N.; Romestaing, P.; Ayzac, L.; Ardiet, J.M.; Thalabard, J.C.

1996-01-01

Aim: Analysis of a pilot study including 29 consecutive patients with high surgical risk or refusal of colostomy treated with radiation therapy alone with curative intent. Patients: Between 1986 and 1992, 29 patients were treated for infiltrating adenocarcinoma of the rectum. Median age was 72 years. Transrectal ultrasound staging was used in 24 patients (T1, 2; T2, 14; T3, 13; N0, 23; N1, 6). In 20 patients the lower border of the tumor was at 5 cm or less from the anal verge and in 19 patients the diameter exceeded 3 cm. CEA was elevated in seven cases. Treatment: Contact X-ray (50 kV) was given first (70 Gy/3 fractions). External beam radiation therapy used a three-field technique in the prone position. Accelerated schedule (39 Gy/13 fractions/17 days) with a concomitant boost 'field within the field' (4 Gy/4 fractions). Six weeks later an iridium-192 implant was performed in 21 (20 Gy/22 h). Results: Median follow-up time was 46 months. Overall and specific survival at 5 years was 68% (SE = 0.09) and 76% (SE = 0.08). Local control was obtained in (21(29)) patients (72%). There was one grade 2 rectal bleeding and five grade 2 rectal necroses. The overall tolerance was good in these frail patients. Discussion: For T2. T3 or T1 > 3 cm diameter rectal adenocarcinoma, where contact X-ray alone is not recommended, a combined treatment with radiation therapy alone is able to give good local control with acceptable toxicity. This treatment should be restricted to inoperable patients

Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

International Nuclear Information System (INIS)

Chen Wenshu; Lee Yijang; Yu Yichu; Hsaio Chinghui

2010-01-01

Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of γ-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G 2 /M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

Calcium-Induced Activation of a Mutant G-Protein-Coupled Receptor Causes In Vitro Transformation of NIH/3T3 Cells

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Ana O. Hoff

1999-12-01

Full Text Available The calcium-sensing receptor (CaR is a G-proteincoupled receptor that is widely expressed, has tissuespecific functions, regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151 Met in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

Science.gov (United States)

Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki; Takai, Yuki; Okumura, Yoshiko; Baba, Miki; Datsenko, Kirill A; Tomita, Masaru; Wanner, Barry L; Mori, Hirotada

2006-01-01

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

Science.gov (United States)

Arai, Sayuri; Hayashihara, Kayoko; Kanamoto, Yuki; Shimizu, Kazunori; Hirokawa, Yasutaka; Hanai, Taizo; Murakami, Akio; Honda, Hiroyuki

2017-08-01

Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO 2 , low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72 h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

Science.gov (United States)

Yao, Jia-Long; Tomes, Sumathi; Gleave, Andrew P

2013-05-01

Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

On the structural affinity of macromolecules with different biological properties: Molecular dynamics simulations of a series of TEM-1 mutants

Energy Technology Data Exchange (ETDEWEB)

Giampaolo, Alessia Di [Dipartimento di Scienze Fisiche e Chimiche, Universita’ degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy); Mazza, Fernando [Department of Health Sciences, Univ. of L’Aquila, 67010 L’Aquila (Italy); Daidone, Isabella [Dipartimento di Scienze Fisiche e Chimiche, Universita’ degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy); Amicosante, Gianfranco; Perilli, Mariagrazia [Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy); Aschi, Massimiliano, E-mail: massimiliano.aschi@univaq.it [Dipartimento di Scienze Fisiche e Chimiche, Universita’ degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy)

2013-07-12

Highlights: •We have performed molecular dynamics simulations of TEM-1 mutants. •Mutations effects on the mechanical properties are considered. •Mutants do not significantly alter the average enzymes structure. •Mutants produce sharp alterations in enzyme conformational repertoire. •Mutants also produce changes in the active site volume. -- Abstract: Molecular Dynamics simulations have been carried out in order to provide a molecular rationalization of the biological and thermodynamic differences observed for a class of TEM β-lactamases. In particular we have considered the TEM-1(wt), the single point mutants TEM-40 and TEM-19 representative of IRT and ESBL classes respectively, and TEM-1 mutant M182T, TEM-32 and TEM-20 which differ from the first three for the additional of M182T mutation. Results indicate that most of the thermodynamic, and probably biological behaviour of these systems arise from subtle effects which, starting from the alterations of the local interactions, produce drastic modifications of the conformational space spanned by the enzymes. The present study suggests that systems showing essentially the same secondary and tertiary structure may differentiate their chemical–biological activity essentially (and probably exclusively) on the basis of the thermal fluctuations occurring in their physiological environment.

On the structural affinity of macromolecules with different biological properties: Molecular dynamics simulations of a series of TEM-1 mutants

International Nuclear Information System (INIS)

Giampaolo, Alessia Di; Mazza, Fernando; Daidone, Isabella; Amicosante, Gianfranco; Perilli, Mariagrazia; Aschi, Massimiliano

2013-01-01

Highlights: •We have performed molecular dynamics simulations of TEM-1 mutants. •Mutations effects on the mechanical properties are considered. •Mutants do not significantly alter the average enzymes structure. •Mutants produce sharp alterations in enzyme conformational repertoire. •Mutants also produce changes in the active site volume. -- Abstract: Molecular Dynamics simulations have been carried out in order to provide a molecular rationalization of the biological and thermodynamic differences observed for a class of TEM β-lactamases. In particular we have considered the TEM-1(wt), the single point mutants TEM-40 and TEM-19 representative of IRT and ESBL classes respectively, and TEM-1 mutant M182T, TEM-32 and TEM-20 which differ from the first three for the additional of M182T mutation. Results indicate that most of the thermodynamic, and probably biological behaviour of these systems arise from subtle effects which, starting from the alterations of the local interactions, produce drastic modifications of the conformational space spanned by the enzymes. The present study suggests that systems showing essentially the same secondary and tertiary structure may differentiate their chemical–biological activity essentially (and probably exclusively) on the basis of the thermal fluctuations occurring in their physiological environment

Single top t-channel

CERN Document Server

Faltermann, Nils

2017-01-01

The production of single top quarks allows to study the interplay of top quark physics and the electroweak sector of the standard model. Deviations from predictions can be a hint for physics beyond the standard model. The t-channel is the dominant production mode for single top quarks at the LHC. This talk presents the latest measurements from the ATLAS and CMS collaborations.

Canonical forms for single-qutrit Clifford+T operators

OpenAIRE

Glaudell, Andrew N.; Ross, Neil J.; Taylor, Jacob M.

2018-01-01

We introduce canonical forms for single qutrit Clifford+T circuits and prove that every single-qutrit Clifford+T operator admits a unique such canonical form. We show that our canonical forms are T-optimal in the sense that among all the single-qutrit Clifford+T circuits implementing a given operator our canonical form uses the least number of T gates. Finally, we provide an algorithm which inputs the description of an operator (as a matrix or a circuit) and constructs the canonical form for ...

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia.

Science.gov (United States)

Cleyrat, Cédric; Girard, Romain; Choi, Eun H; Jeziorski, Éric; Lavabre-Bertrand, Thierry; Hermouet, Sylvie; Carillo, Serge; Wilson, Bridget S

2017-09-26

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34 + cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

miR-155 Controls Lymphoproliferation in LAT Mutant Mice by Restraining T-Cell Apoptosis via SHIP-1/mTOR and PAK1/FOXO3/BIM Pathways.

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Alexandre K Rouquette-Jazdanian

Full Text Available Linker for Activation of T cells (LAT is an adapter protein that is essential for T cell function. Knock-in mice with a LAT mutation impairing calcium flux develop a fatal CD4+ lymphoproliferative disease. miR-155 is a microRNA that is correlated with hyperproliferation in a number of cancers including lymphomas and leukemias and is overexpressed in mutant LAT T cells. To test whether miR-155 was merely indicative of T cell activation or whether it contributes to lymphoproliferative disease in mutant LAT mice, we interbred LAT mutant and miR-155-deficient mice. miR-155 deficiency markedly inhibited lymphoproliferative disease by stimulating BIM-dependent CD4+ T cell apoptosis, even though ERK activation and T cell proliferation were increased in double mutant CD4+ T cells. Bim/Bcl2l11 expression is activated by the forkhead transcription factor FOXO3. Using miR-155-deficient, LAT mutant T cells as a discovery tool, we found two connected pathways that impact the nuclear translocation and activation of FOXO3 in T cells. One pathway is mediated by the inositide phosphatase SHIP-1 and the serine/threonine kinases AKT and PDK1. The other pathway involves PAK1 and JNK kinase activation. We define crosstalk between the two pathways via the kinase mTOR, which stabilizes PAK1. This study establishes a role for PAK1 in T cell apoptosis, which contrasts to its previously identified role in T cell proliferation. Furthermore, miR-155 regulates the delicate balance between PAK1-mediated proliferation and apoptosis in T cells impacting lymphoid organ size and function.

Single-step selection of drug resistant Acinetobacter baylyi ADP1 mutants reveals a functional redundancy in the recruitment of multidrug efflux systems.

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Anthony J Brzoska

Full Text Available Members of the genus Acinetobacter have been the focus recent attention due to both their clinical significance and application to molecular biology. The soil commensal bacterium Acinetobacter baylyi ADP1 has been proposed as a model system for molecular and genetic studies, whereas in a clinical environment, Acinetobacter spp. are of increasing importance due to their propensity to cause serious and intractable systemic infections. Clinically, a major factor in the success of Acinetobacter spp. as opportunistic pathogens can be attributed to their ability to rapidly evolve resistance to common antimicrobial compounds. Whole genome sequencing of clinical and environmental Acinetobacter spp. isolates has revealed the presence of numerous multidrug transporters within the core and accessory genomes, suggesting that efflux is an important host defense response in this genus. In this work, we used the drug-susceptible organism A. baylyi ADP1 as a model for studies into the evolution of efflux mediated resistance in genus Acinetobacter, due to the high level of conservation of efflux determinants across four diverse Acinetobacter strains, including clinical isolates. A single exposure of therapeutic concentrations of chloramphenicol to populations of A. baylyi ADP1 cells produced five individual colonies displaying multidrug resistance. The major facilitator superfamily pump craA was upregulated in one mutant strain, whereas the resistance nodulation division pump adeJ was upregulated in the remaining four. Within the adeJ upregulated population, two different levels of adeJ mRNA transcription were observed, suggesting at least three separate mutations were selected after single-step exposure to chloramphenicol. In the craA upregulated strain, a T to G substitution 12 nt upstream of the craA translation initiation codon was observed. Subsequent mRNA stability analyses using this strain revealed that the half-life of mutant craA mRNA was significantly

Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-yl carbamate.

Science.gov (United States)

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.

Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

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Norifumi Muraki

2016-05-01

Full Text Available Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

Isolation of UV-sensitive mutants of mouse L5178Y cells by a cell suspension spotting method

International Nuclear Information System (INIS)

Shiomi, T.; Hieda-Shiomi, N.; Sato, K.

1982-01-01

We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers. We divided the mutants into two groups, highly sensitive and moderately sensitive mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG

An Acquired HER2T798I Gatekeeper Mutation Induces Resistance to Neratinib in a Patient with HER2 Mutant-Driven Breast Cancer.

Science.gov (United States)

Hanker, Ariella B; Brewer, Monica Red; Sheehan, Jonathan H; Koch, James P; Sliwoski, Gregory R; Nagy, Rebecca; Lanman, Richard; Berger, Michael F; Hyman, David M; Solit, David B; He, Jie; Miller, Vincent; Cutler, Richard E; Lalani, Alshad S; Cross, Darren; Lovly, Christine M; Meiler, Jens; Arteaga, Carlos L

2017-06-01

We report a HER2 T798I gatekeeper mutation in a patient with HER2 L869R -mutant breast cancer with acquired resistance to neratinib. Laboratory studies suggested that HER2 L869R is a neratinib-sensitive, gain-of-function mutation that upon dimerization with mutant HER3 E928G , also present in the breast cancer, amplifies HER2 signaling. The patient was treated with neratinib and exhibited a sustained partial response. Upon clinical progression, HER2 T798I was detected in plasma tumor cell-free DNA. Structural modeling of this acquired mutation suggested that the increased bulk of isoleucine in HER2 T798I reduces neratinib binding. Neratinib blocked HER2-mediated signaling and growth in cells expressing HER2 L869R but not HER2 L869R/T798I In contrast, afatinib and the osimertinib metabolite AZ5104 strongly suppressed HER2 L869R/T798I -induced signaling and cell growth. Acquisition of HER2 T798I upon development of resistance to neratinib in a breast cancer with an initial activating HER2 mutation suggests HER2 L869R is a driver mutation. HER2 T798I -mediated neratinib resistance may be overcome by other irreversible HER2 inhibitors like afatinib. Significance: We found an acquired HER2 gatekeeper mutation in a patient with HER2 -mutant breast cancer upon clinical progression on neratinib. We speculate that HER2 T798I may arise as a secondary mutation following response to effective HER2 tyrosine kinase inhibitors (TKI) in other cancers with HER2 -activating mutations. This resistance may be overcome by other irreversible HER2 TKIs, such as afatinib. Cancer Discov; 7(6); 575-85. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 539 . ©2017 American Association for Cancer Research.

A detailed study of gerJ mutants of Bacillus subtilis.

Science.gov (United States)

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.

A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid.

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Man Hei Cheng

Full Text Available Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg was investigated by sequencing. A single base deletion (299delG in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

Science.gov (United States)

Martens, I; Nilsson, S A; Linder, S; Magnusson, G

1989-05-01

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.

Waiting times for the appearance of cytotoxic T-lymphocyte escape mutants in chronic HIV-1 infection

International Nuclear Information System (INIS)

Liu Yi; Mullins, James I.; Mittler, John E.

2006-01-01

The failure of HIV-1 to escape at some cytotoxic T-lymphocyte (CTL) epitopes has generally been explained in terms of viral fitness costs or ineffective or attenuated CTL responses. Relatively little attention has been paid to the evolutionary time required for escape mutants to be detected. This time is significantly affected by selection, mutation rates, the presence of other advantageous mutations, and the effective population size of HIV-1 in vivo (typically estimated to be ∼10 3 in chronically infected patients, though one study has estimated it to be ∼10 5 ). Here, we use a forward simulator with experimentally estimated HIV-1 parameters to show that these delays can be substantial. For an effective population size of 10 3 , even highly advantageous mutants (s = 0.5) may not be detected for a couple of years in chronically infected patients, while moderately advantageous escape mutants (s = 0.1) may not be detected for up to 10 years. Even with an effective population size of 10 5 , a moderately advantageous escape mutant (s = 0.1) may not be detected in the population within 2 years if it has to compete with other selectively advantageous mutants. Stochastic evolutionary forces, therefore, in addition to viral fitness costs and ineffective or attenuated CTL responses, must be taken into account when assessing the selection of CTL escape mutations

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

DEFF Research Database (Denmark)

Costa-Neto, Claudio M; Miyakawa, Ayumi A; Pesquero, João B

2002-01-01

and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I...... and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared...... with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain...

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid : Growth profiles and catalase activities in relation to microbody proliferation

NARCIS (Netherlands)

Klei, Ida J. van der; Rytka, Joanna; Kunau, Wolf H.; Veenhuis, Marten

The parental strain (A+T+) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A+T-), catalase A (A-T+) or both catalases (A-T-), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two

Isolation and charactarization of T-DNA-insertion Mutants of Arabidopsis thaliana that are Tolerant to Salt

International Nuclear Information System (INIS)

Njoroge, N.C.; Tremblay, L.; Lefebvre, D.D.

2006-01-01

In order to provide an insight into physiological mechanisms underlying salt tolerance in plants,T-DNA insertionally mutagenized seeds of Arabidopsis thaliana were screened on media containing 150-175 millimolar sodium chloride (mM Nacl) for an ability to germinate with formation of two green expanded cotyledons.Under these saline conditions the wild-type (WT) seeds of A.thaliana do not germinate. Two different mutants,NN3 and NN143 were isolated. Genetic analysis of the F1 and F2 generations indicates that the salt tolerance trait in mutant NN3 is recessive and dominant in mutant NN143. Allelism test indicates that mutants NN3 and NN143 are not allelic to each other, but they are alleic to aba and abi mutants respectively. When subjected to water stress imposed by 175mM Nacl for two weeks,kanamycin homozygous seeds of mutants NN3 and NN143 attained germination levels of 97% and 65% respectively. At this concentration of salt, the wild-type seeds are incapable of germination. On 300mM mannitol, a non-ionic osmoticum, mutants NN143 and NN3 and wild type attained a germination levels of 77%, 95% and 2% respectively. The biomass of mutant NN3 seedlings grown on a medium containing 150 mM NaCl was significanlly greater than that of mutant NN143.Between 104 and 145 hours after germination on media containing 175 mM NaCl and 300mM mannitol,germination levels of mutant NN3 were significantly higher than those of mutant NN143.However, both attain the same level of germination after 200 hours. Mutant NN43 is capable of germination on a medium containing 2-6 μM (micromolar) abscisic acid (ABA) with germination ranging from 11to100%. After two weeks on 2 μ ABA, it attained 100% germination and the wild type and mutant NN3 did not germinate. The biomass of NN143 seedlings grown on ABA-free medium and those grown on 2 μM ABA were not significantly different. In presence of both 1μABA and 250mM mannitol, mutant NN143 seedlings achieved 60% germination compared to 93

A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

Science.gov (United States)

Su, Shih-Heng; Krysan, Patrick J

2016-12-01

Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

A higher yielding mutant of black gram with improved nodule formation

International Nuclear Information System (INIS)

Singh, R.K.; Raghuvanshi, S.S.

1987-01-01

Dry seeds of black gram (Vigna mungo (L) Hopper) var. T 9 with 12.2% moisture content were irradiated at 10, 20 and 30 krad of gamma rays. This was followed by combined treatment of one set in each dose with freshly prepared 0. 25% EMS in phosphate buffer at 7.0 pH at 30± deg. C for 6 hours. In M 2 population of 20 krad two mutants with pentafoliate instead of trifoliate leaves were found. This character was true breeding in M 3 M 6 generation. Crosses revealed monogenic recessive inheritance of this character. The proposed gene symbol is p5. This mutant has normal maturity period and the plant height is the same as T 9 (ca. 50 cm). Preliminary yield trials indicate superiority of the mutant line over control. The mutant line also shows a significant improvement in number and weight of root nodules, potentially improving green manuring value. Improvement of root nodulation in mungbean mutants was reported before by others

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera[OPEN

Science.gov (United States)

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S.; Johnson, Marc T.J.; Wright, Stephen I.; Pellizzer, Tommaso; Sobanski, Johanna; Greiner, Stephan

2016-01-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. PMID:27053421

Further studies on a temperature-sensitive mutant of Escherichia coli with defective repair capacity

International Nuclear Information System (INIS)

Morfiadakis, I.; Geissler, E.; Akademie der Wissenschaften der DDR, Berlin. Zentralinstitut fuer Molekularbiologie)

1981-01-01

A temperature-sensitive mutant of E. coli, WG24, was studied with respect to its sensitivity to photodynamic action, its capacity to perform host controlled reactivation, and its sensitivity to transduction at elevated temperatures. Mutant cells are much more sensitive than wild type cells to photodynamic action by thiopyronine and visible light at elevated temperatures. As well defined rec mutants, WG24 cells are less able to reactivate UV irradiated lambdac phages at elevated temperatures, while their ability to repair T1 phages is less impaired. Mutant cells cannot be transduced to T6 resistance at a detectable rate at elevated temperature. It is concluded, therefore, that some rec gene carries a ts mutation in this mutant. (author)

Productive mutants of niger

International Nuclear Information System (INIS)

Misra, R.C.

2001-01-01

Seeds of six niger (Guizotia abyssinica Cass.) varieties ('GA-10', 'ONS-8', 'IGP-72', 'N-71', 'NB-9' and 'UN-4') were treated with 0.5, 0.75 and 1% ethyl methanesulphonate. After four generations of selection, 29 mutant lines were developed and those were evaluated from 1990-92 during Kharif (July to October) and Rabi (December to March) seasons. Average plant characteristics and yield data of four high yielding mutants along with 'IGP-76' (National Check), GA-10 (Zonal Check) and 'Semiliguda Local' (Local Check) are presented

Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

OpenAIRE

Gross, D J; Halban, P A; Kahn, C R; Weir, G C; Villa-Komaroff, L

1989-01-01

A patient with type II diabetes associated with hyperproinsulinemia has been shown to have a point mutation in one insulin gene allele, resulting in replacement of histidine with aspartic acid at position 10 of the B-chain. To investigate the basis of the proinsulin processing defect, we introduced an identical mutation in the rat insulin II gene and expressed both the normal and the mutant genes in the AtT-20 pituitary corticotroph cell line. Cells expressing the mutant gene showed increased...

Assemblies of amyloid-β30-36 hexamer and its G33V/L34T mutants by replica-exchange molecular dynamics simulation.

Directory of Open Access Journals (Sweden)

Zhenyu Qian

Full Text Available The aggregation of amyloid-β peptides is associated with the pathogenesis of Alzheimer's disease, in which the 30-36 fragments play an important part as a fiber-forming hydrophobic region. The fibrillar structure of Aβ30-36 has been detected by means of X-ray diffraction, but its oligomeric structural determination, biophysical characterization, and pathological mechanism remain elusive. In this study, we have investigated the structures of Aβ30-36 hexamer as well as its G33V and L34T mutants in explicit water environment using replica-exchange molecular dynamics (REMD simulations. Our results show that the wild-type (WT Aβ30-36 hexamer has a preference to form β-barrel and bilayer β-sheet conformations, while the G33V or L34T mutation disrupts the β-barrel structures: the G33V mutant is homogenized to adopt β-sheet-rich bilayers, and the structures of L34T mutant on the contrary get more diverse. The hydrophobic interaction plays a critical role in the formation and stability of oligomeric assemblies among all the three systems. In addition, the substitution of G33 by V reduces the β-sheet content in the most populated conformations of Aβ30-36 oligomers through a steric effect. The L34T mutation disturbs the interpeptide hydrogen bonding network, and results in the increased coil content and morphological diversity. Our REMD runs provide structural details of WT and G33V/L34T mutant Aβ30-36 oligomers, and molecular insight into the aggregation mechanism, which will be helpful for designing novel inhibitors or amyloid-based materials.

Inverse agonism of SQ 29,548 and Ramatroban on Thromboxane A2 receptor.

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Raja Chakraborty

Full Text Available G protein-coupled receptors (GPCRs show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera.

Science.gov (United States)

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S; Zupok, Arkadiusz; Johnson, Marc T J; Wright, Stephen I; Pellizzer, Tommaso; Sobanski, Johanna; Bock, Ralph; Greiner, Stephan

2016-04-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. © 2016 American Society of Plant Biologists. All rights reserved.

Synthesis of bacteriophage-coded gene products during infection of Escherichia coli with amber mutants of T3 and T7 defective in gene 1

DEFF Research Database (Denmark)

Issinger, O G; Hausmann, R

1973-01-01

During nonpermissive infection by a T7 amber mutant in gene 1 (phage RNA polymerase-deficient), synthesis of the products of the phage genes 3 (endonuclease), 3, 5 (lysozyme), 5 (DNA polymerase), and 17 (serum blocking power) was shown to occur at about half the rate as during wild-type infection...

A higher yielding mutant of black gram with improved nodule formation

Energy Technology Data Exchange (ETDEWEB)

Singh, R K; Raghuvanshi, S S [Plant Genetic Unit, Department of Botany, University of Lucknow (India)

1987-07-01

Dry seeds of black gram (Vigna mungo (L) Hopper) var. T{sub 9} with 12.2% moisture content were irradiated at 10, 20 and 30 krad of gamma rays. This was followed by combined treatment of one set in each dose with freshly prepared 0. 25% EMS in phosphate buffer at 7.0 pH at 30{+-} deg. C for 6 hours. In M{sub 2} population of 20 krad two mutants with pentafoliate instead of trifoliate leaves were found. This character was true breeding in M{sub 3} M{sub 6} generation. Crosses revealed monogenic recessive inheritance of this character. The proposed gene symbol is p5. This mutant has normal maturity period and the plant height is the same as T{sub 9} (ca. 50 cm). Preliminary yield trials indicate superiority of the mutant line over control. The mutant line also shows a significant improvement in number and weight of root nodules, potentially improving green manuring value. Improvement of root nodulation in mungbean mutants was reported before by others.

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

Directory of Open Access Journals (Sweden)

Shayla K Shorter

Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

Decreased uv mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, L.; Hinkle, D.; Prakash, S.

1978-01-01

A DNA replication mutant of yeast, cdc8, was found to decrease uv-induced reversion of lys2-1, arg4-17, tryl and ural. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following uv irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since uv-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme

Expression of spoT in Borrelia burgdorferi during Serum Starvation

OpenAIRE

Concepcion, Marc B.; Nelson, David R.

2003-01-01

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tick Ixodes scapularis. A 2.9-kb fragment containing a putative spoT gene was isolated from B. burgdorferi genomic DNA by PCR amplification and cloned into a pBAD24 vector. The cloned gene complemented Escherichia coli mutant strain CF1693, which contains deletions of both the relA and spoT genes. The spoT gene in E. coli encodes a bifunctional enzyme capable of synthesizing and degrading (p)ppGpp, which mediates...

Genetic analysis of the induced mutants of rice resistant to bacterial leaf blight

International Nuclear Information System (INIS)

Nakai, H.

1990-01-01

Full text: Seeds of the rice cultivar 'Harebare', which is susceptible to bacterial leaf blight (BLB), were treated with thermal neutrons, gamma-rays, ethyleneimine and ethylmethane-sulfonate. In the M2, plants with better resistance to BLB were identified through inoculation at the seedling and the flag leaf stages with an isolate (T7174) of the Japanese differential race I. Several mutant lines resistant to BLB were selected through tests of the M 3 or M 4 lines derived from selected resistant M 2 plants. The frequency of resistant mutants was significantly higher after the thermal neutron treatment than after treatments with other mutagens. Two mutants, which originated from the neutron treatment, showing a highly quantitative resistance to multiple BLB races were analysed for gene(s) for resistance. The resistance of one of them (M41) to the Japanese races I, II, III, IV, and V was found to be conditioned by a single recessive gene. Three other recessive genes for resistance are known, but their reaction to differential races is different. Therefore, this gene was thought to be new and was tentatively designated as xa-nm(t). The resistance of another mutant (M57) was found to be polygenically inherited. (author)

AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

Science.gov (United States)

O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim

2009-01-01

SUMMARY Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABLT315I mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and pre-clinical evaluation of AP24534, a potent, orally available multi-targeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABLT315I-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML. PMID:19878872

Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli

International Nuclear Information System (INIS)

Kumar, S.

1976-01-01

Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi 80 cya + and phi 80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants

Benomyl-resistant mutant strain of Trichoderma sp. with increased mycoparasitic activity.

Science.gov (United States)

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.

Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

DEFF Research Database (Denmark)

Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde

1996-01-01

Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants...... were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl....... Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA. Udgivelsesdato: 1996-Aug-23...

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid: Growth profiles and catalase activities in relation to microbody proliferation

OpenAIRE

Klei, Ida J. van der; Rytka, Joanna; Kunau, Wolf H.; Veenhuis, Marten

1990-01-01

The parental strain (A+T+) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A+T-), catalase A (A-T+) or both catalases (A-T-), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two oleic acid-grown A+-strains (A+T+ and A+T-) high catalase activities were found; catalase activity invariably remained low in the A-T+ strain and was never detected in the A-T- strain. The levels of β-...

denV gene of bacteriophage T4 restores DNA excision repair to mei-9 and mus201 mutants of Drosophila melanogaster

International Nuclear Information System (INIS)

Banga, S.S.; Boyd, J.B.; Valerie, K.; Harris, P.V.; Kurz, E.M.; de Riel, J.K.

1989-01-01

The denV gene of bacteriophage T4 was fused to a Drosophila hsp70 (70-kDa heat shock protein) promoter and introduced into the germ line of Drosophila by P-element-mediated transformation. The protein product of that gene (endonuclease V) was detected in extracts of heat-shocked transformants with both enzymological and immunoblotting procedures. That protein restores both excision repair and UV resistance to mei-9 and mus201 mutants of this organism. These results reveal that the denV gene can compensate for excision-repair defects in two very different eukayotic mutants, in that the mus201 mutants are typical of excision-deficient mutants in other organisms, whereas the mei-9 mutants exhibit a broad pleiotropism that includes a strong meiotic deficiency. This study permits an extension of the molecular analysis of DNA repair to the germ line of higher eukaryotes. It also provides a model system for future investigations of other well-characterized microbial repair genes on DNA damage in the germ line of this metazoan organism

A mutant of a mutant of a mutant of a ...: Irradiation of progressive radiation-induced mutants in a mutation-breeding programme with Chrysanthenum morifolium RAM

International Nuclear Information System (INIS)

Broertjes, C.; Koene, P.; Veen, J.W.H. van.

1980-01-01

Radiation-induced sports in Chrysanthemum morifolium RAM. have been reported for several years. It has become an everyday practice to produce flower-colour mutants from outstanding cross-breeding products, even before they are distributed for the commercial production of cut flowers. One of the most successful and recent examples is that of cv. Horim, of which hundreds of mutants were produced by successive use of radiation-induced mutants in the mutation-breeding programme. Over about 4 years a variety of flower-colour mutants was obtained, not only largely including the outstanding characteristics of the original cultivar but sometimes even with an appreciable improvement in quality and yield. It is expected that the latter types, the Miros group, will soon completely supersede the spontaneous or raditation-induced Horim sports and mutants and take over the leading position of the Horim group in the production of all-year-round (AYR) cut-flowers. (orig.)

Subthreshold membrane potential oscillations in inferior olive neurons are dynamically regulated by P/Q- and T-type calcium channels: a study in mutant mice.

Science.gov (United States)

Choi, Soonwook; Yu, Eunah; Kim, Daesoo; Urbano, Francisco J; Makarenko, Vladimir; Shin, Hee-Sup; Llinás, Rodolfo R

2010-08-15

The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming alpha1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming alpha1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1-/- mice. In the CaV3.1-/- mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1-/- mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1-/- mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1-/- and CaV3.1-/- mice. These results suggest that both alpha1A P/Q- and alpha1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.

Life cycle cost analysis of single slope hybrid (PV/T) active solar still

International Nuclear Information System (INIS)

Kumar, Shiv; Tiwari, G.N.

2009-01-01

This paper presents the life cycle cost analysis of the single slope passive and hybrid photovoltaic (PV/T) active solar stills, based on the annual performance at 0.05 m water depth. Effects of various parameters, namely interest rate, life of the system and the maintenance cost have been taken into account. The comparative cost of distilled water produced from passive solar still (Rs. 0.70/kg) is found to be less than hybrid (PV/T) active solar still (Rs. 1.93/kg) for 30 years life time of the systems. The payback periods of the passive and hybrid (PV/T) active solar still are estimated to be in the range of 1.1-6.2 years and 3.3-23.9 years, respectively, based on selling price of distilled water in the range of Rs. 10/kg to Rs. 2/kg. The energy payback time (EPBT) has been estimated as 2.9 and 4.7 years, respectively. (author)

Mutacin H-29B is identical to mutacin II (J-T8

Directory of Open Access Journals (Sweden)

Lavoie Marc C

2006-04-01

Full Text Available Abstract Background Streptococcus mutans produces bacteriocins named mutacins. Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. Some of them were found to be lantibiotics, defined as bacterial ribosomally synthesised lanthionine-containing peptides with antimicrobial activity. The goal of this study was to produce and characterize a new mutacin from S. mutans strain 29B, as it shows a promising activity spectrum against current human pathogens. Results Mutacin H-29B, produced by S. mutans strain 29B, was purified by successive hydrophobic chromatography from a liquid preparation consisting of cheese whey permeate (6% w/v supplemented with yeast extract (2% and CaCO3 (1%. Edman degradation revealed 24 amino acids identical to those of mutacin II (also known as J-T8. The molecular mass of the purified peptide was evaluated at 3246.08 ± 0.1 Da by MALDI-TOF MS. Conclusion A simple procedure for production and purification of mutacins along with its characterization is presented. Our results show that the amino acid sequence of mutacin H-29B is identical to the already known mutacin II (J-T8 over the first 24 residues. S. mutans strains of widely different origins may thus produce very similar bacteriocins.

Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

Science.gov (United States)

Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

2004-01-30

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

Mutants of alfalfa mosaic virus

International Nuclear Information System (INIS)

Roosien, J.

1983-01-01

In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

Characterization of a cold-adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

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Dong, Juan; Gasmalla, Mohammed A A; Zhao, Wei; Sun, Jingtao; Liu, Wenyu; Wang, Mingming; Han, Liang; Yang, Ruijin

2017-09-01

A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No. 1, Tianshan, People's Republic of China and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room temperature plasma method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30 ℃, pH 9.0 and 25 ℃, pH 8.5, respectively. EstTB11 was thermally more stable (50 ℃ for 1 H) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0 ℃ and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4 ℃. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS

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Estes, Patricia S.; Boehringer, Ashley; Zwick, Rebecca; Tang, Jonathan E.; Grigsby, Brianna; Zarnescu, Daniela C.

2011-01-01

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders. PMID:21441568

Primary study on lesion mimic mutants of rice (oryza sativa L.)

International Nuclear Information System (INIS)

Hao Zhongna; Zhang Hongzhi; Tao Rongixang

2007-01-01

Nineteen lesion mimic mutants (xsl1-19) of japonica rice Xiushui11 were obtained by γ-rays irradiation treatment. All mutants belonged to whole life lesion mimic. Lesion mimic of mutants didn't largen after tillering stage, leaves didn't wither, and no effect on the plants exsert spikes and seed. When the highest temperature in day exceeded 32 degree C in seedling stage, lesion mimic of all mutant expect xsl19 disappeared. Under 32 degree C, lesion mimic would appear gradually, and symptoms weren't inhibited by high temperature after 5 leaf stage. The plant heights of all lesion mimic mutants were 47.56-63.54 cm in the tillering stage, and that of CK was 83.75 cm; but the dwarf phenomenon of mutants only appeared before tillering stage, and didn't affect plant heights finally; the heading dates of mutants were the same to the CK, the ear length of all mutants were 9.43-15.19 cm, and that of CK was 16.41 cm; the total grain quantity per spike of all mutants were 88.17-165.33, and those of xsl19 and CK were 49.50 and 76.17. The results showed all lesion mimic mutants except xsl19 had short spikes and total grain quantity per spike increasing. All lesion mimic mutants were susceptible to Magnaporthe grisea, and they had no relationship with resistance. (authors)

Single-cell analysis reveals a link between CD3- and CD59-mediated signaling pathways in Jurkat T cells

International Nuclear Information System (INIS)

Lipp, A. M.

2012-01-01

Elevation of intracellular free calcium concentration ([Ca2+]i) is a key signal during T cell activation and is commonly used as a read-out parameter for stimulation of T cell signaling. Upon T cell stimulation a variety of calcium signals is produced by individual cells of the T cell population and the type of calcium signal strongly influences cell fate decisions. The heterogeneous nature of T cells is masked in ensemble measurements, which highlights the need for single-cell measurements. In this study we used single-cell calcium measurements in Jurkat cells to investigate signaling pathways, which are triggered by different proteins, namely CD3 and CD59. By application of an automated cluster algorithm the presented assay provides unbiased analysis of a large data set of individual calcium time traces generated by the whole cell population. By using this method we could demonstrate that the Jurkat population generates heterogeneous calcium signals in a stimulus-dependent manner. Furthermore, our data revealed the existence of a link between CD3- and CD59-mediated signaling pathways. Single-cell calcium measurements in Jurkat cells expressing different levels of the T cell receptor (TCR) complex indicated that CD59-mediated calcium signaling is critically dependent on TCR surface expression levels. In addition, triggering CD59-mediated calcium signaling resulted in down-regulation of TCR surface expression levels, which is known to happen upon direct TCR triggering too. Moreover, by using siRNA-mediated protein knock-downs and protein knock-out Jurkat mutants we could show that CD3- and CD59-mediated calcium signaling require identical key proteins. We therefore explored by which mechanism CD59-mediated signaling couples into TCR-mediated signaling. Fluorescence recovery after photobleaching (FRAP) experiments and live-cell protein-protein interaction assays provided no evidence of a direct physical interaction between CD3- and CD59-mediated signaling pathways

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

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Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

2016-01-01

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

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Carlos Bueno

Full Text Available Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

International Nuclear Information System (INIS)

Hugly, S.; McCourt, P.; Somerville, C.; Browse, J.; Patterson, G.W.

1990-01-01

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18 degree C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury - leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of 14 CO 2 into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury

DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

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Marx Kenneth A

2006-03-01

Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

Prevalence of Catalase (-21 A/T Gene Variant in South Indian (Tamil Population

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A. Lourdhu Mary

2014-01-01

Full Text Available Catalase, an endogenous antioxidant enzyme, is responsible for regulating reactive species levels. Several epidemiologic studies have suggested that single nucleotide polymorphism in catalase gene may be associated with many diseases. The genotype of CAT (-21 A/T point mutation in promoter region of catalase gene was determined by polymerase chain based restriction fragment length polymorphism analysis in the DNA of 100 healthy volunteers. The frequency of CAT (-21 A/T gene polymorphism AA, AT, and TT genotypes was found to be 7, 23, and 70 percent, respectively. The mutant “T” allele frequency was found to be 0.82 among the south Indian (Tamil population. Chi square analysis showed that the study population lies within the Hardy-Weinberg equilibrium. The wild type genotype (AA was found to be very low (7% and the mutant genotype (AT/TT was found to be more prevalent (93% among the south Indian population. This suggests that the high prevalence of mutant genotype may increase the susceptibility to oxidative stress associated diseases.

Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation.

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Wang, Aoli; Yan, Xiao-E; Wu, Hong; Wang, Wenchao; Hu, Chen; Chen, Cheng; Zhao, Zheng; Zhao, Peng; Li, Xixiang; Wang, Li; Wang, Beilei; Ye, Zi; Wang, Jinhua; Wang, Chu; Zhang, Wei; Gray, Nathanael S; Weisberg, Ellen L; Chen, Liang; Liu, Jing; Yun, Cai-Hong; Liu, Qingsong

2016-10-25

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress.

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Stetina, Mandy; Behr, Jürgen; Vogel, Rudi F

2014-07-01

As a result of its strong adaptation to wheat and rye sourdoughs, Lactobacillus sanfranciscensis has the smallest genome within the genus Lactobacillus. The concomitant absence of some important antioxidative enzymes and the inability to synthesize glutathione suggest a role of cystine transport in maintenance of an intracellular thiol balance. Diamide [synonym 1,1'-azobis(N,N-dimethylformamide)] disturbs intracellular and membrane thiol levels in oxidizing protein thiols depending on its initial concentration. In this study, RNA sequencing was used to reveal the transcriptional response of L. sanfranciscensis DSM 20451(T) (wild type [WT]) and its ΔtcyB mutant with a nonfunctional cystine transporter after thiol stress caused by diamide. Along with the different expression of genes involved in amino acid starvation, pyrimidine synthesis, and energy production, our results show that thiol stress in the wild type can be compensated through activation of diverse chaperones and proteases whereas the ΔtcyB mutant shifts its metabolism in the direction of survival. Only a small set of genes are significantly differentially expressed between the wild type and the mutant. In the WT, mainly genes which are associated with a heat shock response are upregulated whereas glutamine import and synthesis genes are downregulated. In the ΔtcyB mutant, the whole opp operon was more highly expressed, as well as a protein which probably includes enzymes for methionine transport. The two proteins encoded by spxA and nrdH, which are involved in direct or indirect oxidative stress responses, are also upregulated in the mutant. This work emphasizes that even in the absence of definitive antioxidative enzymes, bacteria with a small genome and a high frequency of gene inactivation and elimination use small molecules such as the cysteine/cystine couple to overcome potential cell damage resulting from oxidative stress. Copyright © 2014, American Society for Microbiology. All Rights

Phenotypic and Proteomic Analysis of the Aspergillus fumigatus ΔPrtT, ΔXprG and ΔXprG/ΔPrtT Protease-Deficient Mutants

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Einav Shemesh

2017-12-01

Full Text Available Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the A. fumigatus transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted xprG, alone and in combination with prtT, a transcription factor previously shown to regulate extracellular proteolysis. xprG deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs to degrade substrate proteins. Deletion of both xprG and prtT resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the xprG/prtT null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of A. fumigatus during infection based on extensive gene redundancy.

Cross-section measurement of single-top t-channel production at ATLAS

International Nuclear Information System (INIS)

Herrberg-Schubert, Ruth Hedwig Margarete

2014-01-01

This study presents the cross-section measurement of electroweak single-top quark production in the t-channel with a semi-leptonically decaying top quark. The study is based on 4.7 fb -1 of proton-proton collision data recorded with the ATLAS detector at the Large Hadron Collider in the year 2011. Selected events contain two highly energetic jets, one of which is identified as originating from a beauty quark, as well as a highly energetic electron or muon and transverse missing energy. The case of three and four jets is also considered but eventually discarded since their inclusion degrades the precision of the result. The event reconstruction is done with a chi-square-based kinematic fit using W boson and top quark mass constraints. The chi-square value in each event serves to classify the event as a signal-like or background-like process. The cross-section is extracted by performing a template-based maximum likelihood fit to the distribution that displays the best discriminatory power: This distribution is chosen such that the shape differences between signal and background with respect to the typical forward light jet kinematics of the t-channel are exploited. An observation of the single-top t-channel process with a significance of 5.7 σ is obtained, and the cross-section is measured to be 111 +29 -28 pb. Assuming vertical stroke V tb vertical stroke 2 >> vertical stroke V td vertical stroke 2 + vertical stroke V ts vertical stroke 2 as well as a (V-A), CP-conserving interaction, and allowing for the presence of anomalous couplings at the W-t-b vertex, the associated value of the CKM matrix element times an anomalous form factor is determined as vertical stroke V tb f L 1 vertical stroke =1.30 +0.13 -0.16 . The corresponding lower limit in the standard model scenario 0≤ vertical stroke V tb vertical stroke ≤1 amounts to 0.77 tb vertical stroke at 95% confidence level.

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

KAUST Repository

Belfield, E.J.; Gan, X.; Mithani, A.; Brown, C.; Jiang, C.; Franklin, K.; Alvey, E.; Wibowo, A.; Jung, M.; Bailey, K.; Kalwani, S.; Ragoussis, J.; Mott, R.; Harberd, N.P.

2012-01-01

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

KAUST Repository

Belfield, E.J.

2012-04-12

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

Enhancement of Gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant

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Jonathan R. Aspe

2014-02-01

Full Text Available Background: Current therapeutic options for advanced pancreatic cancer have been largely disappointing with modest results at best, and though adjuvant therapy remains controversial, most remain in agreement that Gemcitabine should stand as part of any combination study. The inhibitor of apoptosis (IAP protein Survivin is a key factor in maintaining apoptosis resistance, and its dominant-negative mutant (Survivin-T34A has been shown to block Survivin, inducing caspase activation and apoptosis. Methods: In this study, exosomes, collected from a melanoma cell line built to harbor a tetracycline-regulated Survivin-T34A, were plated on the pancreatic adenocarcinoma (MIA PaCa-2 cell line. Evaluation of the presence of Survivin-T34A in these exosomes followed by their ability to induce Gemcitabine-potentiative cell killing was the objective of this work. Results: Here we show that exosomes collected in the absence of tetracycline (tet-off from the engineered melanoma cell do contain Survivin-T34A and when used alone or in combination with Gemcitabine, induced a significant increase in apoptotic cell death when compared to Gemcitabine alone on a variety of pancreatic cancer cell lines. Conclusion: This exosomes/Survivin-T34A study shows that a new delivery method for anticancer proteins within the cancer microenvironment may prove useful in targeting cancers of the pancreas.

Single-shot spiral imaging at 7 T.

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Engel, Maria; Kasper, Lars; Barmet, Christoph; Schmid, Thomas; Vionnet, Laetitia; Wilm, Bertram; Pruessmann, Klaas P

2018-03-25

The purpose of this work is to explore the feasibility and performance of single-shot spiral MRI at 7 T, using an expanded signal model for reconstruction. Gradient-echo brain imaging is performed on a 7 T system using high-resolution single-shot spiral readouts and half-shot spirals that perform dual-image acquisition after a single excitation. Image reconstruction is based on an expanded signal model including the encoding effects of coil sensitivity, static off-resonance, and magnetic field dynamics. The latter are recorded concurrently with image acquisition, using NMR field probes. The resulting image resolution is assessed by point spread function analysis. Single-shot spiral imaging is achieved at a nominal resolution of 0.8 mm, using spiral-out readouts of 53-ms duration. High depiction fidelity is achieved without conspicuous blurring or distortion. Effective resolutions are assessed as 0.8, 0.94, and 0.98 mm in CSF, gray matter and white matter, respectively. High image quality is also achieved with half-shot acquisition yielding image pairs at 1.5-mm resolution. Use of an expanded signal model enables single-shot spiral imaging at 7 T with unprecedented image quality. Single-shot and half-shot spiral readouts deploy the sensitivity benefit of high field for rapid high-resolution imaging, particularly for functional MRI and arterial spin labeling. © 2018 International Society for Magnetic Resonance in Medicine.

Immortalization of human neural stem cells with the c-myc mutant T58A.

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Lidia De Filippis

Full Text Available Human neural stem cells (hNSC represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs--which do not transform in vitro and are not tumorigenic in vivo--the v-myc gene contains several mutations and recombination elements, whose role(s and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC. T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%. Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in

Use of a temperature-sensitive p53 mutant to evaluate mechanisms of 5-fluorodeoxyuridine-mediated radiosensitization

International Nuclear Information System (INIS)

Naida, J.D.; Davis, M.A.; Lawrence, T.S.

1996-01-01

Purpose/Objective: Evidence exists that fluorodeoxyuridine (FdUrd)-mediated radiosensitization occurs in HT29 human colon carcinoma cells (which are p53 mutant) when these cells progress past the G 1 /S boundary in the presence of the drug. It has been demonstrated that wild type p53 levels increase following fluoropyrimidine treatment and that G 1 arrest is associated with increased p53 levels. We hypothesized that the restoration of wild type p53 function might restore G 1 /S arrest after FdUrd treatment, and that this would prevent FdUrd-mediated radiosensitization. Similarly, we hypothesized that cells containing wild type p53 would not be radiosensitized by FdUrd. Materials and Methods: Two clones of HT29 human colon cancer cells (ts29-A and ts29-G) containing murine temperature-sensitive p53 were constructed using electroporation and Geneticin selection. Incubation of these cells at the permissive temperature of 32 deg. C produces wild type p53 function and at the non permissive temperature of 38 deg. C causes mutant p53 function. A G418 resistant control cell line was also constructed (HT29neo). Cells were incubated at either 32 deg. C or 38 deg. C for 24 hours prior to irradiation and with FdUrd (100 nM) or medium only during the last 14 hours of the temperature shift. To assess progression into S phase, single-parameter (propidium iodide (PI)) and two-parameter (PI and bromodeoxyuridine) flow cytometry were performed at the end of drug exposure. A standard clonogenic assay was used. Results: We found that when ts29-A and ts29-G cells were incubated at the non-permissive (inactive p53 conformation) temperature, they progressed into S phase following exposure to FdUrd and were radiosensitized (enhancement ratio 1.5) to a degree similar to that seen in parental HT29 cells. Cells incubated at the permissive (wild-type p53 conformation) temperature demonstrated G 1 arrest, S phase depletion, and G2 arrest. In addition, FdUrd-mediated radiosensitization was

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

International Nuclear Information System (INIS)

Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

1983-01-01

A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

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Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

2018-01-01

We previously constructed three recombinant phyA mutant strains (PP-NP m -8, PP-NP ep -6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NP ep -6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NP ep -6A, PP-NP m -8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NP ep -6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and utilization of cleistogamy gene cl7(t) in rice (Oryza sativa L.).

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Ni, Da-Hu; Li, Juan; Duan, Yong-Bo; Yang, Ya-Chun; Wei, Peng-Cheng; Xu, Rong-Fang; Li, Chun-Rong; Liang, Dan-Dan; Li, Hao; Song, Feng-Shun; Ni, Jin-Long; Li, Li; Yang, Jian-Bo

2014-05-01

Gene transformation is an important method for improvement of plants into elite varieties. However, the possibility of gene flow between genetically modified (GM) crops and similar species is a serious public issue that may potentially endanger ecological stability. Cleistogamy is expected to be an ideal genetic tool for preventing transgene propagation from GM crops. A rice mutant, cl7(t), was created by ethyl methanesulfonate mutagenesis. The mutant exhibited cleistogamy, and had closed spikelets, reduced plant height, and altered morphology of the leaves, panicle, and seeds. Anatomical investigations revealed that the cl7(t) mutant contained more vascular bundles and thicker stems than the wild type, which increased the mechanical strength of its internodes, and anti-lodging ability. Further studies demonstrated that the force required to open the lemma and palea was higher in the cl7(t) mutant, and there was weak swelling ability in the lodicules, which leads to cleistogamy. Allelic analyses and complementation tests indicated that cl7(t) was a novel allele of dep2, a mutant that was previously reported to have similar panicle morphology. Sequence analysis showed that cl7(t) had a single nucleotide substitution (C to A) in the third exon that leads to a Ser substitution with a stop codon, giving a truncated DEP2 protein. Quantitative RT-PCR and in situ hybridization tests demonstrated that there was lower CL7(t) expression level in the spikelets and weaker CL7(t) signals in the lodicules of the cl7(t) mutant compared with wild type, which implies that CL7(t) might participate in the development of lodicules. To improve the agronomic traits of cl7(t) to fit the needs of field production, the cl7(t) mutant was crossed with an intermediate-type rice variety named Guanghui102, which bears some important agronomic traits, including increased grain numbers and high rate of seed setting. Through multi-generational pedigree selection, cleistogamy lines with improved

The yeast rapid tRNA decay pathway competes with elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one or more of several modifications.

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Dewe, Joshua M; Whipple, Joseph M; Chernyakov, Irina; Jaramillo, Laura N; Phizicky, Eric M

2012-10-01

The structural and functional integrity of tRNA is crucial for translation. In the yeast Saccharomyces cerevisiae, certain aberrant pre-tRNA species are subject to nuclear surveillance, leading to 3' exonucleolytic degradation, and certain mature tRNA species are subject to rapid tRNA decay (RTD) if they are appropriately hypomodified or bear specific destabilizing mutations, leading to 5'-3' exonucleolytic degradation by Rat1 and Xrn1. Thus, trm8-Δ trm4-Δ strains are temperature sensitive due to lack of m(7)G(46) and m(5)C and the consequent RTD of tRNA(Val(AAC)), and tan1-Δ trm44-Δ strains are temperature sensitive due to lack of ac(4)C(12) and Um(44) and the consequent RTD of tRNA(Ser(CGA)) and tRNA(Ser(UGA)). It is unknown how the RTD pathway interacts with translation and other cellular processes, and how generally this pathway acts on hypomodified tRNAs. We provide evidence here that elongation factor 1A (EF-1A) competes with the RTD pathway for substrate tRNAs, since its overexpression suppresses the tRNA degradation and the growth defect of strains subject to RTD, whereas reduced levels of EF-1A have the opposite effect. We also provide evidence that RTD acts on a variety of tRNAs lacking one or more different modifications, since trm1-Δ trm4-Δ mutants are subject to RTD of tRNA(Ser(CGA)) and tRNA(Ser(UGA)) due to lack of m(2,2)G(26) and m(5)C, and since trm8-Δ, tan1-Δ, and trm1-Δ single mutants are each subject to RTD. These results demonstrate that RTD interacts with the translation machinery and acts widely on hypomodified tRNAs.

X-ray-sensitive mutants of Chinese hamster ovary cell line

International Nuclear Information System (INIS)

Jeggo, P.A.; Kemp, L.M.

1983-01-01

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D 10 values 5-10-fold of wild-type D 10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D 10 values less than 2-fold of wild-type D 10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks. (Auth.)

Categories and inheritance of resistance to Nilaparvata lugens (Hemiptera: Delphacidae) in mutants of indica rice 'IR64'.

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Sangha, Jatinder Singh; Chen, Yolanda H; Palchamy, Kadirvel; Jahn, Gary C; Maheswaran, M; Adalla, Candida B; Leung, Hei

2008-04-01

Varietal mutants can be useful for developing durable resistance, understanding categories of resistance, and identifying candidate genes involved in defense responses. We used mutants of rice 'IR64' to isolate new sources of resistance to the planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). We compared two mutants that showed a gain and loss of resistance to N. lugens, to determine the categories of resistance to this pest. Under choice tests, female planthoppers avoided settling and laid fewer eggs on the resistant mutant 'D518' than on the susceptible mutant D1131, susceptible check 'TN1', and wild-type IR64, indicating that antixenosis was the resistance category. Similarly, under no-choice conditions, planthoppers laid 29% fewer eggs in D518 than in IR64, but they oviposited more in 'D1131' and TN1. Honeydew excretion was greater on D1131 seedlings but slightly lower on D518 than on IR64. Nymphal survival and adult female weight did not differ among rice cultivars. D518 showed higher tolerance of N. lugens infestations than IR64. Genetic analysis of the F1, F2, and F3 populations derived from D518 x IR64 revealed that resistance in D518 is dominant and controlled by a single gene. Despite the variation in resistance to N. lugens, both mutants and IR64 performed similarly in the field. The mutant D518 is a new source of durable resistance to N. lugens, mainly due to enhanced antixenosis to female hoppers for settling and oviposition.

Partial suppression of the respiratory defect of qrs1/her2 glutamyl-tRNA amidotransferase mutants by overexpression of the mitochondrial pentatricopeptide Msc6p.

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Moda, Bruno S; Ferreira-Júnior, José Ribamar; Barros, Mario H

2016-08-01

Recently, a large body of evidences indicates the existence in the mitochondrial matrix of foci that contain different proteins involved in mitochondrial RNA metabolism. Some of these proteins have a pentatricopeptide repeat motif that constitutes their RNA-binding structures. Here we report that MSC6, a mitochondrial pentatricopeptide protein of unknown function, is a multi copy suppressor of mutations in QRS1/HER2 a component of the trimeric complex that catalyzes the transamidation of glutamyl-tRNAQ to glutaminyl-tRNAQ. This is an essential step in mitochondrial translation because of the lack of a specific mitochondrial aminoacyl glutaminyl-tRNA synthetase. MSC6 over-expression did not abolish translation of an aberrant variant form of Cox2p detected in QRS1/HER2 mutants, arguing against a suppression mechanism that bypasses Qrs1p function. A slight decrement of the mitochondrial translation capacity as well as diminished growth on respiratory carbon sources media for respiratory activity was observed in the msc6 null mutant. Additionally, the msc6 null mutant did not display any impairment in RNA transcription, processing or turnover. We concluded that Msc6p is a mitochondrial matrix protein and further studies are required to indicate the specific function of Msc6p in mitochondrial translation.

Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.

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McClintock, Maria K; Kaznessis, Yiannis N; Hackel, Benjamin J

2016-02-01

Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. © 2015 Wiley Periodicals, Inc.

Characterization of a mutant of Escherichia coli B/R defective in mutation frequency decline

International Nuclear Information System (INIS)

George, D.L.

1974-01-01

A mutant of Escherichia coli B/r designated mfd is very deficient in the ability to exhibit mutation frequency decline (MFD), the characteristic loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with ultraviolet light (uv). This mutant is known to excise pyrimidine dimers very slowly, although it is as uv-resistant as its mfd + B/r parent strain. We have found that the mfd mutant performs the initial incision step of excision repair normally, but repairs the resulting single-strand breaks much more slowly than the mfd + strain. In spite of the slow dimer excision in the mfd mutant, single-strand DNA breaks do not accumulate during postirradiation incubation, implying that incision and excision are well corrdinated. the prolonged postirradiation lag in cell division and DNA synthesis which accompany slow excision in the mfd strain indicates that resumption of these processes of optimal rates is linked to the timing of excision repair. The normal uv-resistance of the mfd mutant also suggests such coordination and shows that the rate of excision repair is independent of its ultimate efficiency in the removal of potentially lethal uv-induced damage. (U.S.)

Chemotaxis-defective mutants of the nematode Caenorhabditis elegans.

Science.gov (United States)

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.

Behaviour of UV-sensitive mutants of Proteus mirabilis to repair incision breaks

International Nuclear Information System (INIS)

Stoerl, K.; Mund, C.

1977-01-01

In U.V.-sensitive mutants of P. mirabilis with the phenotype HCR, REC and EXR single-strand breaks appeared immediately after UV-irradiation. The behaviour of REC- and EXR-mutants was similar to the wildtype. The number of incision breaks observed by sedimentation analysis in these strains was very low. They could be joined during the excision repair process. From the ability of REC- and EXR-strains to rejoin most of the induced single-strand breaks it can be concluded that these strains have approximately the same capacity for excision repair as the wildtype. HCR-mutants of P. mirabilis produced single-strand breaks after UV-irradiation in contrast to HCR-mutants of E. coli. Therefore we suggest that HCR-mutants of P. mirabilis are not completely inhibited in the incision step. The single-strand breaks introduced in the DNA at the beginning of the repair process were not rejoined during further incubation. Experiments with toluenized cells led to the same results. The newly synthesized daughter DNA-strands of UV-irradiated HCR-mutants were of low molecular weight in comparison with those from unirradiated control cells during the repair period. This result is in agreement with the incapability of HCR-mutants to remove the pyrimidine dimers from the parental template strand. (author)

Genetic analysis of plant height in induced mutants of aromatic rice

International Nuclear Information System (INIS)

Kole, P.C.

2005-01-01

Inheritance of plant height in five gamma-ray induced mutants of aromatic rice cultivar Gobindabhog was studied through 6 x 6 diallel cross and segregation analyses. Diallel analysis revealed presence of additive and non-additive gene action with the preponderance of the latter. Proportion of dominant and recessive alleles was distributed unequally among the parents. The direction of dominance was towards tallness. The number of groups of genes was found to be three. The segregation analysis indicated the role of a single major recessive gene for height reduction in three mutants and, in another mutant, a single major recessive gene with negative modifiers. The other semi-dwarf mutant had two major recessive genes with almost equal effect in height reduction. The mutant allele(s) of the latter two mutants were non-allelic to sd sub(1) gene, which could be used as an alternative source of Dee Gee Woo Gen to widen the genetic diversity in semi-dwarfism [it

Role of the DGAT gene C79T single-nucleotide polymorphism in French obese subjects.

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Coudreau, Sylvie Kipfer; Tounian, Patrick; Bonhomme, Geneviève; Froguel, Philippe; Girardet, Jean-Philippe; Guy-Grand, Bernard; Basdevant, Arnaud; Clément, Karine

2003-10-01

Acyl-coenzyme A, diacylglycerol acyltransferase (DGAT), is a key enzyme involved in adipose-cell triglyceride storage. A 79-bp T-to-C single-nucleotide polymorphism (SNP) on the 3' region of the DGAT transcriptional site has been reported to increase promoter activity and is associated with higher BMI in Turkish women. To validate the possible role of this genetic variant in obesity, as well as the variant's possible cellular-functional significance, we performed an association study between the T79C change and several obesity-related phenotypes in 1357 obese French adults and children. The prevalence of the T79C SNP was similar between obese adults and children when each group was compared with the controls. (CC genotype carrier frequencies were 0.25 to 0.29 in the obese groups and 0.21 in controls; p > 0.05.) In each of the obese adult and child groups studied, the T79C variant was not found to be associated with any of the obesity-related phenotypes tested. Although the T79C SNP of the DGAT gene was studied in several groups of white subjects, the association between this SNP and obesity-related phenotypes, previously described, was not confirmed in our population.

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

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Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-07-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

Global regulatory roles of the cAMP/PKA pathway revealed by phenotypic, transcriptomic and phosphoproteomic analyses in a null mutant of the PKA catalytic subunit in Candida albicans.

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Cao, Chengjun; Wu, Mei; Bing, Jian; Tao, Li; Ding, Xuefen; Liu, Xiaoyun; Huang, Guanghua

2017-07-01

The conserved cAMP-dependent protein kinase (PKA) plays critical roles in the regulation of morphological transitions and virulence in the human fungal pathogen Candida albicans. It has long been thought that the PKA catalytic subunit is essential for cell viability in this fungus. Paradoxically, the single adenylyl cyclase-encoding gene, CYR1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. Here, a double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1, t2t1), which encode two isoforms of the PKA catalytic subunit was successfully generated, suggesting that this subunit is not essential for cell viability. Inactivation of the PKA catalytic subunit blocked filamentation and dramatically attenuated white-to-opaque switching, but promoted sexual mating. Comparative transcriptomic analyses demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared with the WT strain, the general transcriptional activity and metabolism were significantly decreased in both the t2t1 and cyr1/cyr1 mutants. Using combined phosphoproteomic and bioinformatic analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. The study sheds new insights into the global regulatory features of the cAMP/PKA pathway in C. albicans. © 2017 John Wiley & Sons Ltd.

Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

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Maristella Maggi

Full Text Available Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively. Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

Decreased Expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T Cells and Peripheral Blood from Tuberculosis Patients

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Schattling, Stefanie; Kohns, Malte; Sander-Jülch, Claudia; Walzl, Gerhard; Hesseling, Anneke; Mayatepek, Ertan; Fleischer, Bernhard; Marx, Florian M.; Jacobsen, Marc

2013-01-01

The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4+ T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease. PMID:23613882

Promise(s of mesenchymal stem cells as an in vitro model system to depict pre-diabetic/diabetic milieu in WNIN/GR-Ob mutant rats.

Directory of Open Access Journals (Sweden)

Soundarya L Madhira

Full Text Available BACKGROUND: Development of model systems have helped to a large extent, in bridging gap to understand the mechanism(s of disease including diabetes. Interestingly, WNIN/GR-Ob rats (Mutants, established at National Centre for Laboratory Animals (NCLAS of National Institute of Nutrition (NIN, form a suitable model system to study obesity with Type 2 diabetes (T2D demonstrating several secondary complications (cataract, cardiovascular complications, infertility, nephropathy etc. The present study has been carried out to explore the potent application(s of multipotent stem cells such as bone marrow mesenchymal stem cells (BM-MSCs, to portray features of pre-diabetic/T2D vis-à-vis featuring obesity, with impaired glucose tolerance (IGT, hyperinsulinemia (HI and insulin resistance (IR seen with Mutant rats akin to human situation. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of BM-MSCs (third passage from Mutants, its lean littermate (Lean and parental control (Control were characterized for: proliferation markers, disease memory to mark obesity/T2D/HI/IR which included phased gene expression studies for adipogenic/pancreatic lineages, inflammatory markers and differentiation ability to form mature adipocytes/Insulin-like cellular aggregates (ILCAs. The data showed that BM-MSCs from Mutant demonstrated a state of disease memory, depicted by an upregulated expression of inflammatory markers (IL-6, TNFα; increased stem cell recruitment (Oct-4, Sox-2 and proliferation rates (CD90+/CD29+, PDA, 'S' phase of cell cycle by FACS and BrdU incorporation; accelerated preadipocyte induction (Dact-1, PPARγ2 with a quantitative increase in mature adipocyte formation (Leptin; ILCAs, which were non-responsive to high glucose did confer the Obese/T2D memory in Mutants. Further, these observations were in compliance with the anthropometric data. CONCLUSIONS: Given the ease of accessibility and availability of MSCs, the present study form the basis to report for

Genetic requirements for high constitutive SOS expression in recA730 mutants of Escherichia coli.

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Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

2011-09-01

The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5'-3' exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5'-3' exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Evaluation of short stature mutants of Basmati-370 for yield and grain quality characteristics

International Nuclear Information System (INIS)

Awan, M.A.; Ahmad, M.; Cheema, A.A.

1982-01-01

Three short stature mutants were induced in an indica rice cultivar by gamma irradiation. The mutants were assessed for their yielding ability and grain quality characteristics. All the mutants out yielded the parent variety, Basmati-370. The increase in yield of the mutants ranged from 19.37% to 29.66%. DM-2 gave the highest yield (3587.96 kg/ha) among the mutants. As regards physical, cooking and eating quality characteristics, there was no significant difference in water absorption, volume expansion ratios and stickiness among the mutants and Basmati-370. However, Basmati-370 was scored best for flavour as this variety had strong aroma as compared to its mutants which were scored for moderately strong aroma. (authors)

Defective glycinergic synaptic transmission in zebrafish motility mutants

Directory of Open Access Journals (Sweden)

Hiromi Hirata

2010-01-01

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

Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

Science.gov (United States)

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

2009-01-01

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

Fifth Single-Shell Tank Integrity Project Expert Panel Meeting August 28-29, 2014

Energy Technology Data Exchange (ETDEWEB)

Martin, Todd M. [Washington River Protection Solutions, LLC, Richland, WA (United States; Gunter, Jason R. [Washington River Protection Solutions, LLC, Richland, WA (United States); Boomer, Kayle D. [Washington River Protection Solutions, LLC, Richland, WA (United States)

2015-01-07

On August 28th and 29th, 2014 the Single-Shell Tank Integrity Project (SSTIP) Expert Panel (Panel) convened in Richland, Washington. This was the Panel’s first meeting since 2011 and, as a result, was focused primarily on updating the Panel on progress in response to the past recommendations (Single-Shell Tank Integrity Expert Panel Report, RPP-RPT-45921, Rev 0, May 2010). This letter documents the Panel’s discussions and feedback on Phase I activities and results.

Excision repair in MUT-mutants of Proteus mirabilis after UV-irradiation

International Nuclear Information System (INIS)

Stoerl, K.; Mund, C.

1977-01-01

The behaviour of MUT-mutants of P.mirabilis to perform certain steps of excision repair after U.V.-irradiation is described. MUT-mutants introduce single-strand breaks in the DNA immediately after U.V.-irradiation, but their ability to excise pyrimidine dimers from the DNA is very diminished. Moreover, they are not able to accomplish the excision repair by rejoining of the single-strand breaks. The connection between the incomplete excision repair and the mutator phenotype of these mutants is discussed. (author)

Spectrum of MTHFR gene SNPs C677T and A1298C: a study among 23 population groups of India.

Science.gov (United States)

Saraswathy, Kallur Nava; Asghar, Mohammad; Samtani, Ratika; Murry, Benrithung; Mondal, Prakash Ranjan; Ghosh, Pradeep Kumar; Sachdeva, Mohinder Pal

2012-04-01

Elevated homocysteine is a risk factor for many complex disorders. The role of methylenetetrahydrofolate reductase (MTHFR) gene in methylation of homocysteine makes it one of the most important candidate genes for these disorders. Considering the heterogeneity in its distribution in world populations, we screened MTHFR C677T and A1298C single nucleotide polymorphisms in a total of 23 Indian caste, tribal and religious population groups from five geographical regions of India and belonging to four major linguistic groups. The frequencies of MTHFR 677T and 1298C alleles were found to be 10.08 and 20.66%, respectively. MTHFR homozygous genotype 677TT was absent in eight population groups and homozygous 1298CC was absent in two population groups. 677T allele was found to be highest among north Indian populations with Indo-European tongue and 1298C was high among Dravidian-speaking tribes of east India and south India. The less common mutant haplotype 677T-1298C was observed among seven population groups and overall the frequency of this haplotype was 0.008, which is similar to that of African populations. cis configuration of 677T and 1298C was 0.94%. However, we could not find any individual with four mutant alleles which supports the earlier observation that presence of more than two mutant alleles may decrease the viability of foetus and possibly be a selective disadvantage in the population.

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper.

Science.gov (United States)

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.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

Science.gov (United States)

Inga, Alberto; Nahari, Dorit; Velasco-Miguel, Susana; Friedberg, Errol C; Resnick, Michael A

2002-08-22

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Human T-Cell Leukemia Virus I Tax Protein Sensitizes p53-Mutant Cells to DNA Damage

Science.gov (United States)

Mihaylova, Valia T.; Green, Allison M.; Khurgel, Moshe; Semmes, Oliver J.; Kupfer, Gary M.

2018-01-01

Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53–containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective. PMID:18559532

Lipid-mediated interactions tune the association of glycophorin A helix and its disruptive mutants in membranes

NARCIS (Netherlands)

Sengupta, Durba; Marrink, Siewert J.

2010-01-01

The specific and non-specific driving forces of helix association within membranes are still poorly understood. Here, we use coarse-grain molecular dynamics simulations to study the association behavior of glycophorin A and two disruptive mutants, T87F and a triple mutant of the GxxxG motif

Purification of Escherichia coli L-asparaginase mutants by a native polyacrylamide gel electrophoresis.

Science.gov (United States)

Wei, Yujun; Chen, Jianhua; Jia, Ruibo; Wang, Min; Wu, Wutong

2008-07-01

The antigenicity of L-asaparaginase (L-ASP) has been problematic for the treatment of leukemia for many years. In order to establish a relationship between the antigenic epitope of L-asparaginase and its antigenicity, several L-asparaginase mutants (mL-ASPs) are constructed and expressed. To effectively purify these enzyme mutants for further investigation, a native preparative polyacrylamide gel electrophoresis is developed. The simplicity and reproducibility of this approach permits the purification of different mutants from the crude enzyme extracts, with a sufficient activity to perform immunological and biological studies. Furthermore, the newly developed method is efficient and cost-effective compared with other methods, such as column chromatography and affinity chromatography. As a result, the enzyme mutants with specific activity of 300 approximately 400 U/mg are obtained by the single-step purification with a high degree of purity.

An early maturing rice mutant released as a variety

International Nuclear Information System (INIS)

Azam, M.A.; Imtiaz Uddin, Md.

2001-01-01

In the content of food grain production deficiency (about 1.0-1.5 million tons of rice per year according to the Bangladesh Bureau of Statistics, 1998) an induced mutation programme was undertaken in 1985. One moderate early maturing and high yielding rice mutant line (BINA6-84-4-115) has been developed by irradiating F 2 seeds of the cross 'BR4' x 'Iratom 38'. Three treatments viz., 250, 300 and 350 Gy were given to the F 2 seeds. Finally, this line was selected in M 6 generation for advanced yield trial. The line was evaluated in comparative trials with another mutant line BINA6-84-4-163. These two mutant lines had been selected earlier from 300 Gy originated lines. The two check varieties, 'BR 11' and 'BR 22' were also included in the trial, which was conducted in two consecutive T. aman seasons (July to December) during 1994 and 1995 at five locations in Bangladesh. From the results, it was evident that the mutant BINA6-84-4-115 did not differ much with the other mutant lines or check varieties in respect to plant height, number of effective tillers and panicle length but it was 10-18 days earlier than the other 3 entries. It produced a similar yield as the check BR 11 in 1994 and a higher yield than the check BR 11 and BR 22 in 1995. This mutant line gave the highest yield per day among all the entries. In addition to this, the grains are long, fine and possess a high L/B ratio, which are of high commercial value. This line has been released by the National Seed Board of Bangladesh in 1998 as a commercial variety under the name 'BINADHAN-4' for cultivation throughout Bangladesh

Selection of mutants resistant to black spot disease by chronic irradiation of gamma-rays in Japanese pear 'Osanijisseiki'

International Nuclear Information System (INIS)

Masuda, Tetsuo; Yoshioka, Toji; Kotobuki, Kazuo; Sanada, Tetsuro; Inoue, Kosuke; Murata, Kenji; Kitagawa, Kenichi; Tabira, Hiroki; Yoshida, Akira

1997-01-01

'Osanijisseiki', a self-compatible, spontaneous bud sport of the Japanese pear 'Nijisseiki' is an excellent cultivar with a smooth skin. However, this cultivar is susceptible to Japanese pear black spot disease caused by Alternaria alternata Japanese pear pathotype. To obtain resistant mutants from 'Osanijisseiki', nursery plants of 'Osanijisseiki' have been irradiated chronically with gamma-rays in the Gamma Field of the Institute of Radiation Breeding, NAR, MAFF, since 1986. Screening tests using AK toxin, a host-specific toxin produced by A. alternata Japanese pear pathotype, were performed form 1988 to 1993. Four branches of young trees planted at a distance of 40 m from the 60 Co source were selected as being resistant mutants in 1991 (IRB 502-13T and IRB 502-14T) and 1993 (IRB 502-17T and IRB 502-18T). Sensitivity of the four resistant mutants to AK-toxin and susceptibility to the pathogen were compared with other of susceptible and resistant cultivars. The results showed that these four mutants possessed intermediate resistance. Furthermore, a mutant, IRB 502-13T, had the same characteristics as the original 'Osanijisseiki', except for the difference in toxin sensitivity. The characteristics of the other mutants, IRB 502 14-T, IRB 502-17T, and IRB 502-18T, care being examined. (author)

A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

Science.gov (United States)

Rodríguez, Irene; Lázaro, José M.; Blanco, Luis; Kamtekar, Satwik; Berman, Andrea J.; Wang, Jimin; Steitz, Thomas A.; Salas, Margarita; de Vega, Miguel

2005-01-01

Recent crystallographic studies of φ29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a φ29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of φ29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity. PMID:15845765

Accurate prediction of stability changes in protein mutants by combining machine learning with structure based computational mutagenesis.

Science.gov (United States)

Masso, Majid; Vaisman, Iosif I

2008-09-15

Accurate predictive models for the impact of single amino acid substitutions on protein stability provide insight into protein structure and function. Such models are also valuable for the design and engineering of new proteins. Previously described methods have utilized properties of protein sequence or structure to predict the free energy change of mutants due to thermal (DeltaDeltaG) and denaturant (DeltaDeltaG(H2O)) denaturations, as well as mutant thermal stability (DeltaT(m)), through the application of either computational energy-based approaches or machine learning techniques. However, accuracy associated with applying these methods separately is frequently far from optimal. We detail a computational mutagenesis technique based on a four-body, knowledge-based, statistical contact potential. For any mutation due to a single amino acid replacement in a protein, the method provides an empirical normalized measure of the ensuing environmental perturbation occurring at every residue position. A feature vector is generated for the mutant by considering perturbations at the mutated position and it's ordered six nearest neighbors in the 3-dimensional (3D) protein structure. These predictors of stability change are evaluated by applying machine learning tools to large training sets of mutants derived from diverse proteins that have been experimentally studied and described. Predictive models based on our combined approach are either comparable to, or in many cases significantly outperform, previously published results. A web server with supporting documentation is available at http://proteins.gmu.edu/automute.

Genetic analysis and molecular detection of the corn endosperm mutants induced by space flight

International Nuclear Information System (INIS)

Zhang Caibo; Zhou Yuanyuan; Wang Hanyu; Wang Hongwei; Wang Shengqing; Rong Tingzhao; Cao Moju

2013-01-01

In this study, two maize inbred lines 08-641 and 18-599 were carried into cosmic space by recoverable satellite 'Shijian 8', grain shrunken transparently and opaquely mutants were selected as experimental materials and their soluble sugar content in kernel were measured by annthrone colorimetry. The content of soluble sugar in mutant st1 kernels began to rise in 10 days after pollination, to reach the peak in 25 days and significantly higher than the contrast 08-641, while in mutant sol kernels it began to rise in 10 days after pollination, to reach the peak in 20 days and significantly higher than the contrast 18-599. The results of genetic analysis and allelism test showed that the trait in both mutants was all controlled by a single recessive gene, the mutant st1 was allelic to the su1 and the mutant sol was allelic to the sh2. DNA sequence alignment found 2 single-base mutations in 2 and 13 exon of su1 gene in the mutant st1 and 3 single-base mutations in 2, 5 and 16 exon of sh2 gene in mutant so1 leading to the change in amino acid sequences. So it is inferred that starch biosynthesis in the mutants may be blocked by these mutations, which lead to the increase of soluble sugar content in kernel. (authors)

Mutant p53 protein in serum could be used as a molecular marker in human breast cancer.

Science.gov (United States)

Balogh, G A; Mailo, D A; Corte, M M; Roncoroni, P; Nardi, H; Vincent, E; Martinez, D; Cafasso, M E; Frizza, A; Ponce, G; Vincent, E; Barutta, E; Lizarraga, P; Lizarraga, G; Monti, C; Paolillo, E; Vincent, R; Quatroquio, R; Grimi, C; Maturi, H; Aimale, M; Spinsanti, C; Montero, H; Santiago, J; Shulman, L; Rivadulla, M; Machiavelli, M; Salum, G; Cuevas, M A; Picolini, J; Gentili, A; Gentili, R; Mordoh, J

2006-04-01

p53 wild-type is a tumor suppressor gene involved in DNA gene transcription or DNA repair mechanisms. When damage to DNA is unrepairable, p53 induces programmed cell death (apoptosis). The mutant p53 gene is the most frequent molecular alteration in human cancer, including breast cancer. Here, we analyzed the genetic alterations in p53 oncogene expression in 55 patients with breast cancer at different stages and in 8 normal women. We measured by ELISA assay the serum levels of p53 mutant protein and p53 antibodies. Immunohistochemistry and RT-PCR using specific p53 primers as well as mutation detection by DNA sequencing were also evaluated in breast tumor tissue. Serological p53 antibody analysis detected 0/8 (0%), 0/4 (0%) and 9/55 (16.36%) positive cases in normal women, in patients with benign breast disease and in breast carcinoma, respectively. We found positive p53 mutant in the sera of 0/8 (0.0%) normal women, 0/4 (0%) with benign breast disease and 29/55 (52.72%) with breast carcinoma. Immunohistochemistry evaluation was positive in 29/55 (52.73%) with mammary carcinoma and 0/4 (0%) with benign breast disease. A very good correlation between p53 mutant protein detected in serum and p53 accumulation by immunohistochemistry (83.3% positive in both assays) was found in this study. These data suggest that detection of mutated p53 could be a useful serological marker for diagnostic purposes.

Development of a 5.1 T conduction-cooled YBCO coil composed of a stack of 12 single pancakes

Energy Technology Data Exchange (ETDEWEB)

Miyazaki, Hiroshi, E-mail: hiroshi17.miyazaki@toshiba.co.jp [Toshiba Corporation, Power Systems Company, 2-4 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iwai, Sadanori; Tosaka, Taizo; Tasaki, Kenji; Hanai, Satoshi; Urata, Masami; Ioka, Shigeru; Ishii, Yusuke [Toshiba Corporation, Power Systems Company, 2-4 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan)

2013-01-15

Highlights: ► We confirmed that performance of YBCO coil was improved by using APC wire. ► We made a conduction-cooled YBCO coil composed of a stack of 12 single pancakes. ► The coil had a central magnetic field as high as 5.1 T at 10 K. ► We also tested the coil operation in a 4 T background magnetic field. -- Abstract: We fabricated and tested a 5 T-class conduction-cooled high-temperature superconducting (HTS) coil composed of a stack of 12 single pancake coils wound with YBCO-coated conductors. The length of each single pancake coil was 25 m, and the inner diameter of the coil was 50 mm. The voltage–current characteristics were measured in liquid nitrogen and under conduction-cooled conditions at 10–60 K. At 10 K, the central magnetic field of the 12 stacked pancake coils was as high as 5.1 T at 305 A. We also tested the coil operation in a 4 T background magnetic field.

Decreased expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4⁺ T cells and peripheral blood from tuberculosis patients.

Science.gov (United States)

Kleinsteuber, Katja; Heesch, Kerrin; Schattling, Stefanie; Kohns, Malte; Sander-Jülch, Claudia; Walzl, Gerhard; Hesseling, Anneke; Mayatepek, Ertan; Fleischer, Bernhard; Marx, Florian M; Jacobsen, Marc

2013-01-01

The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4(+) T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4(+) T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease.

Growth of single T cells and single thymocytes in a high cloning efficiency filler-cell free microculture system.

Science.gov (United States)

Chen, W F; Ewing, T; Scollay, R; Shortman, K

1988-01-01

A high cloning-efficiency microculture system is described in which single T cells, stimulated to divide by phorbol ester and calcium ionophore, grow rapidly under the influence of purified growth factors in the absence of other cells. The kinetics of clonal growth has been monitored over a five day period by phase-contrast microscopy. Mature peripheral T cells, and mature subpopulations from the thymus, responded with a cloning efficiency over 80%; they required IL-2 as a minimum but several other factors enhanced growth. Ly2+L3T4- thymocytes (mean doubling time 10.4 hr) grew more rapidly than Ly2-L3T4+ thymocytes (mean doubling time 15.2 hr). Early (Ly2-L3T4-) thymocytes responded with a cloning efficiency of 60%; their efficient growth was dependent on both IL-1 and IL-2. The typical Ly2+L3T4+ cortical thymocyte did not grow under these conditions.

Dual Inhibition of EGFR with Afatinib and Cetuximab in Kinase Inhibitor-Resistant EGFR-Mutant Lung Cancer with and without T790M Mutations

NARCIS (Netherlands)

Janjigian, Yelena Y.; Smit, Egbert F.; Groen, Harry J. M.; Horn, Leora; Gettinger, Scott; Camidge, D. Ross; Riely, Gregory J.; Wang, Bushi; Fu, Yali; Chand, Vikram K.; Miller, Vincent A.; Pao, William

EGFR-mutant lung cancers responsive to reversible EGFR inhibitors (gefitinib/erlotinib) develop acquired resistance, mediated by second-site EGFR T790M mutation in >50% of cases. Preclinically, afatinib (irreversible ErbB family blocker) plus cetuximab (anti-EGFR monoclonal antibody) overcomes

Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-stranded break repair model for T-DNA integration.

Science.gov (United States)

Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

2017-06-01

Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-stranded break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-stranded break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration, which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosomes 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

The Frequency of Factor V Leiden, Prothrombin G20210A and Methylenetetrahydrofolate Reductase C677T Mutations in Migraine Patients

Directory of Open Access Journals (Sweden)

Ruhsen Öcal

2010-12-01

Full Text Available OBJECTIVE: Migraine is an independent risk factor for ischemic stroke, but its pathophysiology is still unclear. Genetic factors that predispose patients to thrombosis have been studied in patients with migraine to highlight the pathogenesis, but the results remain controversial. In this study, the frequencies of factor V Leiden (FVL, prothrombin (Pt G20210A and methylenetetrahydrofolate reductase (MTHFR C677T mutations were investigated. METHODS: One hundred and sixty patients aged of 15 to 55 years with no history of systemic disease and who had been diagnosed as migraine according to the International Headache Society (IHS diagnostic criteria at Baskent University Hospital Neurology Outpatient Clinics were investigated for FVL, Pt G20210A and MTHFR C677T mutations from their genomic DNA, and the results were compared with those of healthy controls. RESULTS: One hundred and fifty five (96.9% of 160 migraine patients were homozygote normal, 5 (3.1% were heterozygote and none of them were homozygote mutant for FVL. The control group had 9.8% heterozygote individuals but the difference between the percentages was not statistically significant (p> 0.05. There were no homozygote mutant individuals in the Turkish population study in normal subjects like our study. Thirty nine (24.4% of 160 migraine patients were heterozygote and 8 (5% were homozygote mutant for MTHFR C677T. The control group had 37 (34.9% heterozygote and 6 (5.6% homozygote mutant individuals. The difference between the percentages was not statistically significant (p= 0.15. Three (1.9% of 160 migraine patients were heterozygote and 5 (2.9% of the control group were heterozygote mutant for Pt G20210A mutation. The control group had 37 (34.9% heterozygote and 6 (5.6% homozygote mutant individuals. The difference between the percentages was not statistically significant (p= 0.420. CONCLUSION: Our study indicates that FVL, Pt G20210A and MTHFR C677T gene mutations, which are considered

The Frequency of Factor V Leiden, Prothrombin G20210A and Methylenetetrahydrofolate Reductase C677T Mutations in Migraine Patients

Directory of Open Access Journals (Sweden)

Ruhsen Öcal

2010-12-01

Full Text Available OBJECTIVE: Migraine is an independent risk factor for ischemic stroke, but its pathophysiology is still unclear. Genetic factors that predispose patients to thrombosis have been studied in patients with migraine to highlight the pathogenesis, but the results remain controversial. In this study, the frequencies of factor V Leiden (FVL, prothrombin (Pt G20210A and methylenetetrahydrofolate reductase (MTHFR C677T mutations were investigated. METHODS: One hundred and sixty patients aged of 15 to 55 years with no history of systemic disease and who had been diagnosed as migraine according to the International Headache Society (IHS diagnostic criteria at Baskent University Hospital Neurology Outpatient Clinics were investigated for FVL, Pt G20210A and MTHFR C677T mutations from their genomic DNA, and the results were compared with those of healthy controls. RESULTS: One hundred and fifty five (96.9% of 160 migraine patients were homozygote normal, 5 (3.1% were heterozygote and none of them were homozygote mutant for FVL. The control group had 9.8% heterozygote individuals but the difference between the percentages was not statistically significant (p> 0.05. There were no homozygote mutant individuals in the Turkish population study in normal subjects like our study. Thirty nine (24.4% of 160 migraine patients were heterozygote and 8 (5% were homozygote mutant for MTHFR C677T. The control group had 37 (34.9% heterozygote and 6 (5.6% homozygote mutant individuals. The difference between the percentages was not statistically significant (p= 0.15. Three (1.9% of 160 migraine patients were heterozygote and 5 (2.9% of the control group were heterozygote mutant for Pt G20210A mutation. The control group had 37 (34.9% heterozygote and 6 (5.6% homozygote mutant individuals. The difference between the percentages was not statistically significant (p= 0.420. CONCLUSION: Our study indicates that FVL, Pt G20210A and MTHFR C677T gene mutations, which are considered

KRAS Mutant Status, p16 and β-catenin Expression May Predict Local Recurrence in Patients Who Underwent Transanal Endoscopic Microsurgery (TEMS) for Stage I Rectal Cancer.

Science.gov (United States)

Sideris, Michail; Moorhead, Jane; Diaz-Cano, Salvador; Bjarnason, Ingvar; Haji, Amyn; Papagrigoriadis, Savvas

2016-10-01

Transanal endoscopic microsurgery (TEMS) is emerging as an alternative treatment for rectal cancer Stage I. There remains a risk of local recurrence. The Aim of the study was to study the effect of biomarkers in local recurrence for Stage I rectal cancer following TEMS plus or minus radiotherapy. This is a case control study where we compared 10 early rectal cancers that had recurred, against 19 cases with no recurrence, total 29 patients (age=28.25-86.87, mean age=67.92 years, SD=14.91, Male, N=18, Female, N=11). All patients underwent TEMS for radiological Stage I rectal cancer (yT1N0M0 or yT2N0M0) established with combination of magnetic resonance imaging (MRI) and endorectal ultrasound. We prospectively collected all data on tumour histology, morphological features, as well as follow-up parameters. Molecular analysis was performed to identify their status on BRAF, KRAS, p16 O 6 -methylguanine-DNA methyltransferase (MGMT) and β-catenin. Out of 29 specimens analyzed, 19 were KRAS wild type (65.9%) and 10 mutant (34.5%). Recurrence of the tumour was noted in 10 cases (34.5%) from which 60% were pT1 (N=6) and 40% pT2 (N=4). There was a statistically significant association between KRAS mutant status and local recurrence (N=6, p=0.037). P16 expression greater than 5% (mean=10.8%, min=0, max=95) is linked with earlier recurrence within 11.70 months (N=7, p=0.004). Membranous β-catenin expression (N=12, 48%) was also related with KRAS mutant status (p=0.006) but not with survival (p>0.05). BRAF gene was found to be wild type in all cases tested (N=23). KRAS/p16/β-catenin could be used as a combined biomarker for prediction of local recurrence and stratification of the risk for further surgery. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A thermo-sensitive purple leaf rice mutant--PLM12 and its genetical study

International Nuclear Information System (INIS)

Wu Guanting; Wang Xianyu; Jin Wei

2001-01-01

PLM12 was a thermo-sensitive purple leaf mutant selected from Indica rice variety Luqingzao 1 treated with pingyangmycin in combination with γ-rays, and for display of its mutant character, a relatively high temperature was required. Compared with its original parent, many major agronomic traits of PLM12 changed to varied extents. Based on spikelet cutting experiment, it was believed that significant decreases in number of filled grains per panicle, fertility, and 1000-grain weight in PLM12 resulted mainly from a great decline in photosynthetic capacity and serious lack of photosynthate in purple leaves. It was indicated by genetic analysis that expression of the mutant phenotype in PLM12 was conditioned by a single recessive major nuclear gene and modified by several minor genes

Characterization of a Thermo-Inducible Chlorophyll-Deficient Mutant in Barley

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Rong Wang

2017-11-01

Full Text Available Leaf color is an important trait for not only controlling crop yield but also monitoring plant status under temperature stress. In this study, a thermo-inducible chlorophyll-deficient mutant, named V-V-Y, was identified from a gamma-radiated population of the barley variety Vlamingh. The leaves of the mutant were green under normal growing temperature but turned yellowish under high temperature in the glasshouse experiment. The ratio of chlorophyll a and chlorophyll b in the mutant declined much faster in the first 7–9 days under heat treatment. The leaves of V-V-Y turned yellowish but took longer to senesce under heat stress in the field experiment. Genetic analysis indicated that a single nuclear gene controlled the mutant trait. The mutant gene (vvy was mapped to the long arm of chromosome 4H between SNP markers 1_0269 and 1_1531 with a genetic distance of 2.2 cM and a physical interval of 9.85 Mb. A QTL for grain yield was mapped to the same interval and explained 10.4% of the yield variation with a LOD score of 4. This QTL is coincident with the vvy gene interval that is responsible for the thermo-inducible chlorophyll-deficient trait. Fine mapping, based on the barley reference genome sequence, further narrowed the vvy gene to a physical interval of 0.428 Mb with 11 annotated genes. This is the first report of fine mapping a thermo-inducible chlorophyll-deficient gene in barley.

Potent inhibition of HIV-1 replication by a Tat mutant.

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Luke W Meredith

Full Text Available Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

Histological and genetic studies on the male sterile mutants of tomato induced by gamma radiation and chemical mutagens

Energy Technology Data Exchange (ETDEWEB)

Choi, J K [Office of Rural Development, Suwon (Republic of Korea) Horticultural Experiment Station

1976-03-01

The object of this experiment was to test the radiosensitivity of the M/sub 1/ generation, and to determine the inheritance and morphological characteristics of the male-sterile mutants in tomatoes. The results obtained are as follows: Radiation damage varied with the water content of the seeds. The least damage occurred in the range of 11.34 to 14.29 percent moisture. Four tomato cultivars were gamma irradiated. The cultivar Sekai-ichi was the most sensitive while the other responded similarly to each other. Chemical mutagen treatments caused greater damage to germination, to delayed flowering and to pollen fertility than did radiation. Pollen fertility and seeds per fruit in seedling gamma irradiated plants decreased as the dosage and dose rate increased. Five male-sterile mutants were found. Each was controlled by a single recessive gene. All five male-steriles were nonallelic. Floral sized of the male-sterile mutants were slightly smaller. Ovule fertility varied, usually being lower than normal type. Male sterility varied, usually being lower than normal type. Male sterility in 109-1ms strain was caused by unbalanced, insufficient nutrients being made available to the pollen-mother cells from the tapetal tissued. (author).

Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation

Energy Technology Data Exchange (ETDEWEB)

Messing, K.; Ferraris, J.; Bradley, W.E.; Swartz, J.; Seifert, A.M. (Universite du Quebec a Montreal (Canada))

1989-10-01

The frequency of hypoxanthine phosphoribosyl transferase (HPRT) mutants among peripheral T-lymphocytes of radiotherapy technicians primarily exposed to 60Co was measured by the T-cell cloning method. Mutant frequencies of these technicians in 1984 and 1986 were significantly higher than those of physiotherapy technicians who worked in a neighboring service, and correlated significantly with thermoluminescence dosimeter readings recorded during the 6 mo preceding mutant frequency determination. Correlations decreased when related to dose recorded over longer time intervals. HPRT mutant frequency determination in peripheral lymphocytes is a good measure of recently received biologically effective radiation dose in an occupationally exposed population.

Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation

International Nuclear Information System (INIS)

Messing, K.; Ferraris, J.; Bradley, W.E.; Swartz, J.; Seifert, A.M.

1989-01-01

The frequency of hypoxanthine phosphoribosyl transferase (HPRT) mutants among peripheral T-lymphocytes of radiotherapy technicians primarily exposed to 60Co was measured by the T-cell cloning method. Mutant frequencies of these technicians in 1984 and 1986 were significantly higher than those of physiotherapy technicians who worked in a neighboring service, and correlated significantly with thermoluminescence dosimeter readings recorded during the 6 mo preceding mutant frequency determination. Correlations decreased when related to dose recorded over longer time intervals. HPRT mutant frequency determination in peripheral lymphocytes is a good measure of recently received biologically effective radiation dose in an occupationally exposed population

Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.

Science.gov (United States)

Daugeron, Marie-Claire; Lenstra, Tineke L; Frizzarin, Martina; El Yacoubi, Basma; Liu, Xipeng; Baudin-Baillieu, Agnès; Lijnzaad, Philip; Decourty, Laurence; Saveanu, Cosmin; Jacquier, Alain; Holstege, Frank C P; de Crécy-Lagard, Valérie; van Tilbeurgh, Herman; Libri, Domenico

2011-08-01

The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t(6)A(37)) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t(6)A(37) formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

Science.gov (United States)

Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

The APPLE Trial: Feasibility and Activity of AZD9291 (Osimertinib) Treatment on Positive PLasma T790M in EGFR-mutant NSCLC Patients. EORTC 1613.

Science.gov (United States)

Remon, Jordi; Menis, Jessica; Hasan, Baktiar; Peric, Aleksandra; De Maio, Eleonora; Novello, Silvia; Reck, Martin; Berghmans, Thierry; Wasag, Bartosz; Besse, Benjamin; Dziadziuszko, Rafal

2017-09-01

The AZD9291 (Osimertinib) Treatment on Positive PLasma T790M in EGFR-mutant NSCLC Patients (APPLE) trial is a randomized, open-label, multicenter, 3-arm, phase II study in advanced, epidermal growth factor receptor (EGFR)-mutant and EGFR tyrosine kinase inhibitor (TKI)-naive non-small-cell lung cancer (NSCLC) patients, to evaluate the best strategy for sequencing gefitinib and osimertinib treatment. Advanced EGFR-mutant NSCLC patients, with World Health Organization performance status 0-2 who are EGFR TKI treatment-naive and eligible to receive first-line treatment with EGFR TKI will be randomized to: In all arms, a plasmatic ctDNA T790M test will be performed by a central laboratory at the Medical University of Gdansk (Poland) but will be applied as a predictive marker for making treatment decisions only in arm B. The primary objective is to evaluate the best strategy for sequencing of treatment with gefitinib and osimertinib in advanced NSCLC patients with common EGFR mutations, and to understand the value of liquid biopsy for the decision-making process. The progression-free survival rate at 18 months is the primary end point of the trial. The activity of osimertinib versus gefitinib to prevent brain metastases will be evaluated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Therapeutic Targeting of Spliceosomal-Mutant Acquired Bone Marrow Failure Disorders

Science.gov (United States)

2017-05-01

spliceosomal mutant cells . This effort has also highlighted a requirement for innate immune signaling in SF3B1-mutant MDS and has implicated a few specific...relative to single-mutant cells (Figure 5A). As innate immune signaling has been implicated in MDS pathogenesis (Basiorka et al., 2016; Fang et al...Sato et al., 2005; Tang et al., 2008; Vink et al., 2013; Xin et al., 2017). Here, we observed that SF3B1K700E/+ human lymphoid leukemia cells (NALM-6

Dynamic fluorescence spectroscopy on single tryptophan mutants of EIImtl in detergent micelles : Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay

NARCIS (Netherlands)

Swaving Dijkstra, Dolf; Broos, J.; Visser, Antonie J.W.G.; van Hoek, A.; Robillard, George

1997-01-01

The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C

Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

Directory of Open Access Journals (Sweden)

Hassan Abdel-Latif A. Mohamed

2006-06-01

Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

Male sterile mutant in Vigna radiata

International Nuclear Information System (INIS)

Pande, Kalpana; Raghuvanshi, S.S.

1987-01-01

Single and combined treatment of γ-rays and 0.25 per cent EMS were tried on Vigna radiata variety K851. A male sterile mutant was isolated in M 2 generation. Experiments indicated male sterility to be recessive and monogenic in nature. 6 figures. (author)

Phage Pl mutants with altered transducing abilities for Escherichia coli

International Nuclear Information System (INIS)

Wall, J.D.; Harriman, P.D.

1974-01-01

A search was made for mutants of the coliphage P1 with altered transducing frequencies. A method was developed for the rapid assay of transducing frequencies in single plaques using prophage lambda as the transduced bacterial marker. This procedure selects for mutants altered in their ability to package host DNA. Mutants with 5 to 10 times higher or 10 to 20 times lower frequencies than those of wild-type P1 were found. Not only are the markers used for the detection of the mutants affected, but all other markers are similarly affected (not always to the same extent). One of the high transducing frequency mutants is a suppressible amber, indicating that loss of a function increases P1's ability to package host DNA preferentially. (U.S.)

Bacteriophage T4 gene 32 participates in excision repair as well as recombinational repair of UV damages

International Nuclear Information System (INIS)

Mosig, G.

1985-01-01

Gene 32 of phage T4 has been shown previously to be involved in recombinational repair of UV damages but, based on a mutant study, was thought not to be required for excision repair. However, a comparison of UV-inactivation curves of several gene 32 mutants grown under conditions permissive for progeny production in wild-type or polA- hosts demonstrates that gene 32 participates in both kinds of repair. Different gene 32 mutations differentially inactivate these repair functions. Under conditions permissive for DNA replication and progeny production, all gene 32 mutants investigated here are partially defective in recombinational repair, whereas only two of them, P7 and P401, are also defective in excision repair. P401 is the only mutant whose final slope of the inactivation curve is significantly steeper than that of wild-type T4. These results are discussed in terms of interactions of gp32, a single-stranded DNA-binding protein, with DNA and with other proteins

PedonnanceofE3rly MatUring MutantS Derived from ''SuPa'~ Rice ...

African Journals Online (AJOL)

Vienna, Austria in 1994. The dry seeds were in-adiated with gamma rays using three doses (170, 210. --iifid 24OC;Y).frOm C.obalt 60 (lCO) in order shorten the plant height and maturity period. From the resulting mutant. PoPulations ortgindtiriifroni modified single seed descent method, five Jery early maturing lines plus the ...

Oxidant resistance in a yeast mutant deficient in the Sit4 phosphatase

DEFF Research Database (Denmark)

López-Mirabal, H Reynaldo; Winther, Jakob R; Kielland-Brandt, Morten C

2008-01-01

Resistance to thiol oxidation can arise from mutations altering redox homeostasis. A Saccharomyces cerevisiae sit4-110 mutant is here described, which was isolated as resistant to the thiol-specific oxidant dipyridyl disulfide (DPS) and which contains a single-residue substitution in the SIT4 gene...

Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

Energy Technology Data Exchange (ETDEWEB)

Cura, Vincent; Olieric, Natacha; Guichard, Alexandre [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Wang, En-Duo [State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031 (China); Moras, Dino [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Eriani, Gilbert [Architecture et Réactivité de l’ARN, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 Rue René Descartes, 67084 Strasbourg (France); Cavarelli, Jean, E-mail: cava@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

2005-10-01

The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques.

Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

International Nuclear Information System (INIS)

Cura, Vincent; Olieric, Natacha; Guichard, Alexandre; Wang, En-Duo; Moras, Dino; Eriani, Gilbert; Cavarelli, Jean

2005-01-01

The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques

EMS mutant spectra generated by multi-parameter flow cytometry

Energy Technology Data Exchange (ETDEWEB)

Keysar, Stephen B. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Fox, Michael H., E-mail: michael.fox@colostate.edu [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States)

2009-12-01

The CHO A{sub L} cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59{sup -} regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59{sup -} clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

Auditory Cortex tACS and tRNS for Tinnitus: Single versus Multiple Sessions

Directory of Open Access Journals (Sweden)

Laura Claes

2014-01-01

Full Text Available Tinnitus is the perception of a sound in the absence of an external acoustic source, which often exerts a significant impact on the quality of life. Currently there is evidence that neuroplastic changes in both neural pathways are involved in the generation and maintaining of tinnitus. Neuromodulation has been suggested to interfere with these neuroplastic alterations. In this study we aimed to compare the effect of two upcoming forms of transcranial electrical neuromodulation: alternating current stimulation (tACS and random noise stimulation (tRNS, both applied on the auditory cortex. A database with 228 patients with chronic tinnitus who underwent noninvasive neuromodulation was retrospectively analyzed. The results of this study show that a single session of tRNS induces a significant suppressive effect on tinnitus loudness and distress, in contrast to tACS. Multiple sessions of tRNS augment the suppressive effect on tinnitus loudness but have no effect on tinnitus distress. In conclusion this preliminary study shows a possibly beneficial effect of tRNS on tinnitus and can be a motivation for future randomized placebo-controlled clinical studies with auditory tRNS for tinnitus. Auditory alpha-modulated tACS does not seem to be contributing to the treatment of tinnitus.

Repair of gamma radiation damage in wild type and a radiation sensitive mutant of Deinococcus radiodurans

International Nuclear Information System (INIS)

Mizuma, Nagayo

1989-01-01

In an effort to examine production and repair of radiation-induced single and double strand breaks in the DNA, a repair-deficient wild type and a repair-deficient mutant, UV17, of Deinococcus radiodurans were subjected to Co-60 gamma irradiation at a dose rate of 6.3 kGy/hr for wild type and 3.9 kGy/hr for UV17 mutant. The shoulder of the curve of UV17 mutant was narrow but existed with the intercept of 0.7 kGy and the corresponding value of the wild type was 4.2 kGy. Mutant cells exhibited about 6 fold increases in sensitivity for the shoulder relative to the wild type. The D 37 doses in the wild type and the mutant were 0.57 kGy and 0.25 kGy, respectively. From the survival curves, difference in the sensitivity between two strains was mainly due to difference of repair capacity than the number of radiation sensitive target. Sedimentation rate of the main component in the irradiated cells of UV17 mutant increased almost to the level of unirradiated control by the postincubation at 30deg C for 3 hrs. The results indicated that this sensitive mutant also exhibited an ability to restore single strand breaks after exposure to a sublethal dose of 0.6 kGy. When restitution of double strand breaks was analyzed by sedimentation in a neutral sucrose gradient, the wild type showed restitution to DNA-membrane complex from large part of the breaks. For UV17 mutant, the apparent increase in DNA-membrane complex formation was seen after 3 hours incubation. Large part of the decrease in the activities of peak 2 was recovered in the peak 1 for the wild type. For the mutant, there was little restitution to peak 1. Almost free DNA component in UV17 mutant, therefore, was merely degraded into shorter pieces. Restoration of DNA-membrane complex from free DNA derived from gamma-ray induced double strand scission involved closely in the repair of gamma-induced damage and survival. (N.K.)

Promissory rice mutants (Oryza sativa L.) obtained by Gamma Rays induction in Peru

International Nuclear Information System (INIS)

Heros, E.; Gomez, L.

2015-01-01

To improve the available rice cultivars in Peru under irrigated conditions, it was tried the rice seed Amazonas and Capirona, cultivars adapted to irrigated conditions in highland jungle, both characterized for late maturity (140-150 days). The doses were: 150-250-350 Gy that induced mutants with different characteristics to the original cultivar like: early maturity, shorter, high yield and milling quality. In Amazonas cultivar were selected 72 mutants of the M 2 generation with agronomy value and continue in evaluation only the mutant (M35-20). There were applied the same doses of gamma rays with the cultivar Capirona, there is much radiosensitivity at high doses, they were selected two early mutants (MC 35-21 and MC 35-123-3) with an early life cycle days (15 days) than parental cultivar. Two mutants tolerant to salinity (12 dSm). Six mutants are still under evaluation, two of them (MC 25-23-1 and MC 35-45-4) have better yields with performances of 9.1 t ha -1 versus 6.6 t ha -1 . These mutants show lodging and shattering resistance. (Author)

Atomic resolution structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2

International Nuclear Information System (INIS)

Sekar, K.; Yogavel, M.; Gayathri, D.; Velmurugan, D.; Krishna, R.; Poi, M.-J.; Dauter, Z.; Dauter, M.; Tsai, M.-D.

2005-01-01

The atomic resolution crystal structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A 2 is reported. The structure of the double mutant K53,56M has previously been refined at 1.9 Å resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1 Å data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states

Genetic Analysis and Molecular Mapping of a Novel Chlorophyll-Deficit Mutant Gene in Rice

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Xiao-qun HUANG

2008-03-01

Full Text Available A rice etiolation mutant 824ys featured with chlorophyll deficiency was identified from a normal green rice variety 824B. It showed whole green-yellow plant from the seedling stage, reduced number of tillers and longer growth duration. The contents of chlorophyll, chlorophyll a, chlorophyll b and net photosynthetic rate in leaves of the mutant obviously decreased, as well as the number of spikelets per panicle, seed setting rate and 1000-grain weight compared with its wild-type parent. Genetic analyses on F1 and F2 generations of 824ys crossed with three normal green varieties showed that the chlorophyll-deficit mutant character was controlled by a pair of recessive nuclear gene. Genetic mapping of the mutant gene was conducted by using microsatellite markers and F2 mapping population of 495R/824ys, and the mutant gene of 824ys was mapped on the short arm of rice chromosome 3. The genetic distances from the target gene to the markers RM218, RM282 and RM6959 were 25.6 cM, 5.2 cM and 21.8 cM, respectively. It was considered to be a new chlorophyll-deficit mutant gene and tentatively named as chl11(t.

Inheritance and gene expression of a root-growth inhibiting mutant in rice (Oryza sativa L.)

International Nuclear Information System (INIS)

Kitano, H.; Futsuhara, Y.

1990-01-01

Full text: A root-growth inhibiting mutant was induced in the dwarf mutant line, 'Fukei 71', through ethylene-imine. The mutant is characterised by the excessive inhibition of both seminal and crown roots elongation just after germination, although its shoots grow nearly normal. To study the genetics, the mutant was crossed with its original line 'Fukei 71' and some other normal cultivars. Results show that the root-growth inhibition is controlled by a recessive gene (rt), independent of the dwarf gene, d-50(t) locus in Fukei 71. For elucidating the gene action on root morphogenesis, histological and cytological experiments were carried out using a longitudinal and transverse thin section of seminal and/or crown root tips. Observations suggest that the rt gene affects the normal formation of the epidermal system which is differentiated from the protoderm of the root apical meristem. (author)

High yielding small grain mutant of rice variety Pankaj

Energy Technology Data Exchange (ETDEWEB)

NONE

1987-07-01

Full text: By treatment with EMS a mutant has been produced from the variety Pankaj which has better tillering, longer panicle and more grains per panicle. In multilocation trials at Burdwan, Suri and Rampurhat in West Bengal it yielded significantly more than Pankaj and Mahsuri at all locations, with a mean 5.2t. The mutant named BU 79 would be a suitable substitute for Pankaj and similar long-duration rices. (author)

Quantum mechanical/molecular mechanical calculated reactivity networks reveal how cytochrome P450cam and Its T252A mutant select their oxidation pathways.

Science.gov (United States)

Wang, Binju; Li, Chunsen; Dubey, Kshatresh Dutta; Shaik, Sason

2015-06-17

Quantum mechanical/molecular mechanical calculations address the longstanding-question of a "second oxidant" in P450 enzymes wherein the proton-shuttle, which leads to formation of the "primary-oxidant" Compound I (Cpd I), was severed by mutating the crucial residue (in P450cam: Threonine-252-to-Alanine, hence T252A). Investigating the oxidant candidates Cpd I, ferric hydroperoxide, and ferric hydrogen peroxide (Fe(III)(O2H2)), and their reactions, generates reactivity networks which enable us to rule out a "second oxidant" and at the same time identify an additional coupling pathway that is responsible for the epoxidation of 5-methylenylcamphor by the T252A mutant. In this "second-coupling pathway", the reaction starts with the Fe(III)(O2H2) intermediate, which transforms to Cpd I via a O-O homolysis/H-abstraction mechanism. The persistence of Fe(III)(O2H2) and its oxidative reactivity are shown to be determined by interplay of substrate and protein. The substrate 5-methylenylcamphor prevents H2O2 release, while the protein controls the Fe(III)(O2H2) conversion to Cpd I by nailing-through hydrogen-bonding interactions-the conformation of the HO(•) radical produced during O-O homolysis. This conformation prevents HO(•) attack on the porphyrin's meso position, as in heme oxygenase, and prefers H-abstraction from Fe(IV)OH thereby generating H2O + Cpd I. Cpd I then performs substrate oxidations. Camphor cannot prevent H2O2 release and hence the T252A mutant does not oxidize camphor. This "second pathway" transpires also during H2O2 shunting of the cycle of wild-type P450cam, where the additional hydrogen-bonding with Thr252 prevents H2O2 release, and contributes to a successful Cpd I formation. The present results lead to a revised catalytic cycle of Cytochrome P450cam.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

Science.gov (United States)

Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

2014-04-16

Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

Isolation of New Gravitropic Mutants under Hypergravity Conditions.

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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 .

Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis.

Science.gov (United States)

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.

The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

Science.gov (United States)

Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

2016-06-22

Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.

UV and gamma-ray sensitivity of meiosis-deficient mutants in Podospora anserina

International Nuclear Information System (INIS)

Simonet, J.M.

1976-01-01

Two mutants, mei1 and mei2, were isolated by screening for deficiencies occurring in the meiotic process. The sensitivity of mei1 and mei2 mutant strains to UV irradiation showed a significant increase as compared with that of the wild-type stock, hwhereas the sensitivity to γ-rays remained unchanged. The double-mutant strains were no more sensitive than each single mutant. The data indicate that both mei1 and mei2 loci are probably involved in the same pathway of excision-repair of UV-induced lesions

Measurement of Single-top t-channel Production Using ATLAS Data

CERN Document Server

Holzbauer, Jenny; Schwienhorst, R; Repko, W; Lapidus, L; Pratt, S

2012-01-01

This document reports the measurement of the single-top t-channel cross-section using data from the ATLAS detector, located at the Large Hadron Collider on the border of France and Switzerland. The data used were collected during the first half of 2011, from proton-proton collisions with a 7 TeV center-of-mass collision energy. Single-top is electroweak top-quark production and t-channel is one of the standard model production modes. To isolate this production, selections are applied to find events with a similar final state. A cut-based analysis is used to further isolate the signal using a series of selections in several orthogonal kinematic regions. Finally, a statistical analysis is performed to determine the measured cross-section and the CKM matrix element |Vtb|. The cross-section for top and anti-top production is considered separately and the resulting cross-sections are σ t+ = 59 +18 -16 pb for the positive charge channel and σ t- = 33 +13 -12 pb for the negative charge chan...

Effect of sowing dates on yield and yield components on mutant-cum-hybrid lines of bread wheat

International Nuclear Information System (INIS)

Sial, M.A.; Arain, M.A.; Dahot, M.U.; Laghari, K.A.; Naqvi, M.H.; Markhand, G.S.; Mangrio, S.M.; Mirbahar, A.A.

2010-01-01

Twenty-one stable wheat mutant lines along with four check varieties viz., Sarsabz, Kiran-95, T.J.83 and Khirman were evaluated under normal and late sowing dates. The observations were recorded on phenological, morphological and meteorological parameters. Higher yield and improvement in various yield components were recorded at normal sowing as compared to late sowing. Six mutant lines showed superiority in yield than check varieties at normal sowings while three mutants produced more yield than check varieties except Sarsabz at late sowings. At normal sowing eleven mutant lines matured earlier than all check varieties including short duration variety T.J-83 whereas two mutant lines were earlier than Sarsabz and Kiran-95 and thirteen than T.J-83 and Khirman. (author)

Field Performance of Five Soybean Mutants Under Drought Stress Conditions and Molecular Analysis Using SSR Markers

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Y Yuliasti

2017-08-01

Full Text Available The objectives of this research wereto evaluate (1 the performance of soybean mutant lines under drought stress conditions, and(2 the genetic diversity and relationship among the mutant lines using SSR markers.The field evaluation was conducted during the dry season of 2011 and 2012 at the experimental Farm of Mataram University, West Nusa Tenggara, Indonesia. The field experiment was set up in a randomized block design. Ten mutant lines and two control varieties were evaluated in four replications. Genetic distance among evaluated lines were determined based on allelic diversity analysis using 40 simple sequence repeat (SSR loci. Under drought stress conditions, two mutant lines, Kdl3 and Kdl8,showed a better performance compared to the other ones. The high yielding mutant lines were Kdl3and Kdl8, which yielded 1.75 t ha-1and 1.69 t ha-1, respectively, compared to the parent and national control, Panderman 1.43 t ha-1 and Muria 1.32 t ha-1. These mutant linesrequired 30.75 to 32days to flower and 79.75 to 83.75 day to harvest with relatively short plant height 28.25 and 23.35 cmrespectively. Those mutant characters were better than those of the other three mutants, the original parents, and the control soybean species. Since the evaluated soybean mutant lines yielded more under drought stress conditions than the standard varieties, they can be used and registered as drought-tolerant soybean mutants. Moreover, the evaluated soybean accessions showed a wide genetic distance. The accessions were clustered into two groups according to their genetic background, namelygroup I (the Panderman with three mutant lines and group II (the Muria with two mutant lines. Twenty-three out of 40 evaluated SSR loci, including AW31, BE806, CMAC7L, S080, S126, S57, S171, S224, S285, S294, S393, S294, S383, S511, S511, S520, S540, S547, S551, S571, S577, and S578, provided polymorphic alleles between the parents and their mutants and could be used to differentiate

Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

Science.gov (United States)

Truong, Quang Lam; Cho, Youngjae; Kim, Kiju; Park, Bo-Kyoung; Hahn, Tae-Wook

2015-11-01

Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor.

Science.gov (United States)

Haque, Farzin; Lunn, Jennifer; Fang, Huaming; Smithrud, David; Guo, Peixuan

2012-04-24

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This article reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of 12 protein subunits encircled into a 3.6 nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Da), thymine (167 Da), and benzene (105 Da) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete stepwise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices.

Mutants induced in winter rye (Secale cereale L.): Short straw-mutant No. 2714 and late-senescence mutant

Energy Technology Data Exchange (ETDEWEB)

Muszynski, S; Darlewska, M [Department of Plant Breeding and Seed Science, Warsaw Agricultural University, Warsaw (Poland)

1990-01-01

Full text: Mutants were induced by treating dormant seeds with ionizing radiation (fast neutrons) or chemicals (N-nitroso-N-ethyl urea or sodium azide). Among several mutants obtained, of special value is the short-straw mutant No. 2714 and a late senescent mutant. (author)

Genetic analyses of nonfluorescent root mutants induced by mutagenesis in soybean

International Nuclear Information System (INIS)

Sawada, S.; Palmer, R.G.

1987-01-01

Nonfluorescent root mutants in soybean [Glycine max (L.) Merr.] are useful as markers in genetic studies and in tissue culture research. Our objective was to obtain mutagen-induced nonfluorescent root mutants and to conduct genetic studies with them. Thirteen nonfluorescent mutants were detected among 154016 seedlings derived from soybean lines treated with six mutagens. One of these mutants, derived from Williams treated with 20 kR gamma rays, did not correspond to any of the known (standard) nonfluorescent spontaneous mutants. This is the first mutagen-induced nonfluorescent root mutant in soybean. It was assigned Genetic Type Collection no. T285 and the gene symbol fr5 fr5. The fr5 allele was not located on trisomics A, B, or C and was not linked to five chlorophyll-deficient mutants (y9, y11, y12, y13, and y20-k2) or flower color mutant w1. The remaining nonfluorescent root mutants were at the same loci as known spontaneous mutants; i.e., four had the fr1 allele, five had the fr2 allele, and three had the fr4 allele

Mutants of GABA transaminase (POP2 suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh mutants in Arabidopsis.

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Frank Ludewig

Full Text Available BACKGROUND: The gamma-aminubutyrate (GABA shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD, the mitochondrial enzymes GABA transaminase (GABA-T; POP2 and succinic semialdehyde dehydrogenase (SSADH. We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported. PRINCIPAL FINDINGS: To elucidate the role of succinic semialdehyde (SSA, gamma-hydroxybutyrate (GHB and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants. SIGNIFICANCE: We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

Semi-dwarf mutants in triticale and wheat breeding

International Nuclear Information System (INIS)

Driscoll, C.J.

1984-01-01

The triticale lines Beagle and DR-IRA have been subjected to ionizing irradiation and chemical mutagenesis in order to produce semi-dwarf mutants. Beagle is 100 cm tall and DR-IRA 80 cm under average field conditions. A bulk then pedigree method is currently represented by 158 single plots of M 6 (or in some cases M 7 ) mutants that are from 5 to 35 cm shorter than the control variety. The shortest mutants are 65 cm in height. Forty of these mutants are also earlier flowering than the control varieties. Replicated yield testing will be conducted on confirmed mutants in 1983. Response to gibberellic acid of these mutants will also be determined. The Cornerstone male-sterility mutant (ms1c) on chromosome arm 4Aα has been combined with the GA-insensitive/reduced height gene Gai/Rht1 which is also on chromosome arm 4Aα. The ms1c mutant has also been combined with Gai/Rht2 on chromosome 4D and with both Gai/Rht1 and Gai/Rht2. The combination ms1c and Gai/Rht1 has been chosen as the basis of a composite cross. Thirteen varieties were tested with GA 3 and seven (Warigal, Aroona, Oxley, Banks, Avocet, Matipo and Toquifen) which contain Gai/Rht1 were crossed with ms1c Gai/Rht1 and entered into an interpollinating F 2 . The entire composite is homozygous for this semi-dwarf allele and selection will be practiced for increased height on a GA-insensitive background. (author)

Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

International Nuclear Information System (INIS)

Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

2009-01-01

Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

Identification of dominant male sterile mutants in rice (Oryza sativa L.)

International Nuclear Information System (INIS)

Zhu Xudong; Rutger, J.N.

2000-01-01

Genetic male sterile mutants 1783 and 1789 were selected from US variety Orion and Kaybonnet seeds treated by gamma irradiation. Investigation of fertility characterization of pollen and spikelets of these mutants were made by progeny tests in 1783 M 7 and 1789 M 6 generations. The results showed that genetic male sterile mutants 1783 and 1789 with the fertility segregating of 1 sterile: 1 fertile were controlled by a single dominant gene. The pollen staining of mutants characterized partial sterility. Open-pollinated seed set was about 30% and bagged seed set was only 0.3%-3.5%. It is concluded that dominant genetic male sterile is a useful tool in improvement of population for rice breeding

2018-04-29T22:58:14Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

article/87573 2018-04-29T22:58:14Z asr:ART Adolescent Sexuality and Culture: South African Mothers` Perspective Mudhovozi, P Ramarumo, M Sodi, T Adolescent, sexuality, mothers, HIV and AIDS, sexual behaviour, culture. This study explores ...

Tank characterization report for single-shell tank 241-T-104

International Nuclear Information System (INIS)

DiCenso, A.T.; Simpson, B.C.

1994-01-01

In August 1992, Single-Shell Tank 241-T-104 was sampled to determine proper handling of the waste, to address corrosivity and compatibility issues, and to comply with requirements of the Washington Administrative Code (Ecology, 1991). This Tank Characterization Report presents an overview of that tank sampling and analysis effort, and contains observations regarding waste characteristics. It also addresses expected concentration and bulk inventory data for the waste contents based on this latest sampling data and background tank information. The purpose of this report is to describe and characterize the waste in Single-Shall Tank 241-T-104 (hereafter, Tank 241-T-104) based on information given from various sources. This report summarizes the available information regarding the waste in Tank 241-T-104, and using the historical information to place the analytical data in context, arranges this information in a useful format for making management and technical decisions concerning this waste tank. In addition, conclusions and recommendations are given based on safety issues and further characterization needs

Productive mutants in lemongrass induced by gamma rays

International Nuclear Information System (INIS)

Gopinathan Nair, V.

1980-01-01

Seeds of the lemongrass variety O.D. 19 were irradiated with gamma rays at a dose range of 5 to 30 krad. M 1 plants with one or a few tillers differing from the standard plants of O.D. 19 were selected, split into single slips and planted as clonal progenies. Mutants were isolated in M 1 V 1 and carried forward. Forty two M 1 V 2 mutant clones differing from O.D. 19 in morphological characters such as vigour, plant height, growth habit, pigmentation and number of tillers have been established. These were evaluated for tiller number, grass yield and oil content. Six clones gave higher grass yield, the highest being 556 gm per plant per cutting as against 360 gm in the standard. Five clones gave higher oil yield, the highest being 0.42% as against 0.23% in the standard. Isolation of viable mutants with high grass yield and essential oil content indicate the scope for evolving productive mutant varieties in this perennial aromatic grass. The eleven M 1 V 2 mutant clones are being critically evaluated by estimating oil yield per hectare per year. (author)

Analysis of interactions of Salmonella type three secretion mutants with 3-D intestinal epithelial cells.

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Andrea L Radtke

Full Text Available The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2, double (SPI-1/2 and complete T3SS knockout (SPI-1/SPI-2: flhDC also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms.

Isolation of new gravitropic mutants under hypergravity conditions

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Akiko Mori

2016-09-01

Full Text Available 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 upwards. 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.

Genome-Wide Analysis of a TaLEA-Introduced Transgenic Populus simonii × Populus nigra Dwarf Mutant

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Jing Jiang

2012-03-01

Full Text Available A dwarf mutant (dwf1 was obtained among 15 transgenic lines, when TaLEA (Tamarix androssowii late embryogenesis abundant gene was introduced into Populus simonii × Populus nigra by Agrobacterium tumefaciens-mediated transformation. Under the same growth conditions, dwf1 height was significantly reduced compared with the wild type and the other transgenic lines. Because only one transgenic line (dwf1 displayed the dwarf phenotype, we considered that T-DNA insertion sites may play a role in the mutant formation. The mechanisms underlying this effect were investigated using TAIL-PCR (thermal asymmetric interlaced PCR and microarrays methods. According to the TAIL-PCR results, two flanking sequences located on chromosome IV and VIII respectively, were cloned. The results indicated the integration of two independent T-DNA copies. We searched for the potential genes near to the T-DNA insertions. The nearest gene was a putative poplar AP2 transcription factor (GI: 224073210. Expression analysis showed that AP2 was up-regulated in dwf1 compared with the wild type and the other transgenic lines. According to the microarrays results, a total of 537 genes involved in hydrolase, kinase and transcription factor activities, as well as protein and nucleotide binding, showed significant alterations in gene expression. These genes were expressed in more than 60 metabolic pathways, including starch, sucrose, galactose and glycerolipid metabolism and phenylpropanoids and flavonoid biosyntheses. Our transcriptome and T-DNA insertion sites analyses might provide some useful insights into the dwarf mutant formation.

Analysis of the paired TCR α- and β-chains of single human T cells.

Directory of Open Access Journals (Sweden)

Song-Min Kim

Full Text Available Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV and multiple sclerosis (MS. In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+ T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+ T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.

Cytoembryologic study of gamma-ray induced sterile Pisum sativum L. mutants

International Nuclear Information System (INIS)

Molkhova, E.; Vasileva, M.

1977-01-01

Three new pea mutant forms are described - 1878, Crampled petal Waxless type, and Lathyrus type - which were induced by different gamma-ray ( 60 Co) doses and rates. The flowers of the 1878 and Crampled petal Waxless type mutants were very much deformed, while those of the Lathyrus type had smaller flowers with normal morphology. The three mutant forms were entirely sterile and were propagated by segregation in the progeny of heterozygous sister plants. PMC meiosis and the development of the male gametophyte of the Lathyrus type mutant had a normal course, while in the mutant forms Crampled petal Waxless type and 1878 slight disturbances were observed, but the pollen of all three mutants was not functional. The development of the female gametophyte of the three mutants stops at an early phase and only in the Lathyrus type mutant in single cases embryosacks were formed with differentiated sex apparatus and early stages of embryo and endosperm development were scored, but they also soon degenerate. It is pointed out that sterility of the three pea mutant forms studied depends on factors, which stop at different stages the normal development of the generative organs, of the female gametophyte and of embryogenesis. (author)

Collection of rice mutants and application studies of their agronomic characters

International Nuclear Information System (INIS)

Sun Shuxiang; Jin Wei; Luo Qian; Sheng Ping; Huang Rongmin

1993-01-01

More than 1600 accessions of rice mutant germplasm have been collected since 1980, and 1142 accessions of mutants have been identified according to their agronomy and pattern characters. A part of mutants were compared with their original cultivars in eight main agronomic characters. The results showed that the agronomic characters of mutants induced by ionizing radiations changed to both positive and negative directions compared with their original cultivars. Only 6.3% mutants varied in single agronomic character, and 91.1% mutants varied in two to six agronomic characters. Tenetic analysis and Cellular observations were carried out for two kinds of early mutants. It showed that early mutants 'Yuan Feng Zao' are controlled by two independent and incomplete dominant genes. For the dwarf, the reduction of the number of longitudinal cell layers causes the stem shorter and the increase of the number of horizontal cell layers causes the stem wall thicker. More than 100 preserved accessions of mutants were supplied to breeding units as parents or for genetic studies. Sixteen cultivars (lines) were bred from the parents which played an important role in raising the output of rice production

Effect of the mutation (C3435T) at exon 26 of the MDR1 gene on expression level of MDR1 messenger ribonucleic acid in duodenal enterocytes of healthy Japanese subjects.

Science.gov (United States)

Nakamura, Tsutomu; Sakaeda, Toshiyuki; Horinouchi, Masanori; Tamura, Takao; Aoyama, Nobuo; Shirakawa, Toshiro; Matsuo, Masafumi; Kasuga, Masato; Okumura, Katsuhiko

2002-04-01

The effect of the C3435T mutation at exon 26 of the MDR1 gene on the expression levels of MDR1 messenger ribonucleic acid (mRNA) was evaluated by means of real-time polymerase chain reaction in 51 biopsy specimens of duodenum obtained from 13 healthy Japanese subjects. The mRNA levels of MDR1 were 0.38 +/- 0.15, 0.56 +/- 0.14, and 1.13 +/- 0.42 (mean value +/- SE) in the subjects with the homozygote of wild-type allele (C/C), compound heterozygote with mutant T allele (C/T), and the homozygote of the mutant allele (T/T), respectively, reasonably explaining the lower digoxin serum concentration after administration of a single oral dose to subjects harboring a mutant T allele. Good correlation (r =.797; P CYP3A4 in the individual biopsy specimens. This finding suggested a lower plasma concentration of the substrates for CYP3A4 in subjects harboring the C3435T mutation of the MDR1 gene.

Disturbed secretion of mutant adiponectin associated with the metabolic syndrome.

Science.gov (United States)

Kishida, Ken; Nagaretani, Hiroyuki; Kondo, Hidehiko; Kobayashi, Hideki; Tanaka, Sachiyo; Maeda, Norikazu; Nagasawa, Azumi; Hibuse, Toshiyuki; Ohashi, Koji; Kumada, Masahiro; Nishizawa, Hitoshi; Okamoto, Yoshihisa; Ouchi, Noriyuki; Maeda, Kazuhisa; Kihara, Shinji; Funahashi, Tohru; Matsuzawa, Yuji

2003-06-20

Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

Enhanced production of bacitracin by a mutant strain bacillus licheniformis UV-MN-HN-8 (enhanced bacitracin production by mutagenesis)

International Nuclear Information System (INIS)

Aftab, M.N.; Ikram-ul-Haq; Baig, S.

2010-01-01

The present study is focused on the improvement of Bacillus licheniformis through random mutagenesis to obtain mutant having enhanced production of bacitracin. Many isolates of Bacillus licheniformis were isolated and the isolate GP-40 produced maximum bacitracin production (16 +- 0.72 IU/mL). Treatment of Bacillus licheniformis GP-40 with ultraviolet (UV) radiations increased bacitracin production to 29 +- 0.69 IU/mL. Similarly, treatment of vegetative cells of GP-40 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO/sub 2/) increased bacitracin production to 35 +- 1.35 IU/mL and 29 +- 0.89 IU/mL respectively. Studies regarding the combined effect of UV and chemical treatment on parental cells exhibited significantly higher titers of bacitracin with maximum bacitracin production reached to 47.6 +- 0.92 IU/mL. An increase of 2.97 fold production of bacitracin in comparison to wild type was observed. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably mu (h-/sup 1/)max, Yp/x, qp, Qp and Qx mutant strain B. licheniformis UV-MN-HN-8 was found to be a hyper producer of bacitracin. (author)

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls.

Science.gov (United States)

Saitoh, Aya; Takase, Tomoyuki; Kitaki, Hiroyuki; Miyazaki, Yuji; Kiyosue, Tomohiro

2015-01-01

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.

A Medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule development and symbiotic nitrogen fixation.

Science.gov (United States)

Pislariu, Catalina I; Murray, Jeremy D; Wen, JiangQi; Cosson, Viviane; Muni, RajaSekhara Reddy Duvvuru; Wang, Mingyi; Benedito, Vagner A; Andriankaja, Andry; Cheng, Xiaofei; Jerez, Ivone Torres; Mondy, Samuel; Zhang, Shulan; Taylor, Mark E; Tadege, Million; Ratet, Pascal; Mysore, Kirankumar S; Chen, Rujin; Udvardi, Michael K

2012-08-01

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod-), 51 mutants with totally ineffective nodules (Nod+ Fix-), 17 mutants with partially ineffective nodules (Nod+ Fix+/-), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/- Fix-), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/- Fix+), and 11 supernodulating mutants (Nod++Fix+/-). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN'T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod- lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

2018-03-29T05:36:03Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

article/53157 2018-03-29T05:36:03Z ijbcs:ART Biodiversity and ethnobotanical potentials of plant species of University of Agriculture Makurdi Wildlife Park and Ikwe Games Reserve, Benue State, Nigeria Jimoh, S.O Debisi, L Ikyaagba, E.T Plant ...

From one body mutant to one cell mutant. A progress of radiation breeding in crops

International Nuclear Information System (INIS)

Nagatomi, Shigeki

1996-01-01

An effective method was established to obtain non-chimeral mutants with wide spectrum of flower colors, regenerated from floral organs on which mutated sectors were come out on chronic irradiated plants. By this way, six mutant varieties of flower colors have been selected from one pink flower of chrysanthemum, and cultivated for cut-flower production. By the same method, 3 mutant varieties with small and spray type flowers were selected in Eustoma. Mutant varieties such as a rust disease resistant in sugarcane, 6 dwarfs in Cytisus and pure-white mushroom in velvet shank have been selected successively for short period. (J.P.N.)

A single exercise bout augments adenovirus-specific T-cell mobilization and function.

Science.gov (United States)

Kunz, Hawley E; Spielmann, Guillaume; Agha, Nadia H; O'Connor, Daniel P; Bollard, Catherine M; Simpson, Richard J

2018-04-30

Adoptive transfer of virus-specific T-cells (VSTs) effectively treats viral infections following allogeneic hematopoietic stem cell transplantation (alloHSCT), but logistical difficulties have limited widespread availability of VSTs as a post-transplant therapeutic. A single exercise bout mobilizes VSTs specific for latent herpesviruses (i.e. CMV and EBV) to peripheral blood and augments their ex vivo expansion. We investigated whether exercise exerts similar effects on T-cells specific for a NON-latent virus such as adenovirus, which is a major contributor to infection-related morbidity and mortality after alloHSCT. Thirty minutes of cycling exercise increased circulating adenovirus-specific T-cells 2.0-fold and augmented their ex vivo expansion by ~33% compared to rest without altering antigen and MHC-specific autologous target cell killing capabilities. We conclude that exercise is a simple and economical adjuvant to boost the isolation and manufacture of therapeutic VSTs specific to latent and non-latent viruses from healthy donors. Copyright © 2018. Published by Elsevier Inc.

Examination of WF9 mutant sublines for lodging and fusarium resistance

International Nuclear Information System (INIS)

Balint, A.; Bedo, Z.; Kiss, E.

1977-01-01

The mutants produced from the maize line WF9 in our department between 1961-67 were considered regarding stem-strength and Fusarium resistance. The treatments have essentially improved the stem strength of the original line in a great many (11 cases out of 29), and the lodging rate has considerably decreased. Regarding the Fusarium contamination, the changes are mostly disadvantageous, but we achieved definite positive changes in the cases of four mutants. This improvement is also interesting because it coincides with the improvement of the step strength in all cases, and with the considerable increase in protein content in three of the four sublines. (author)

A new Arabidopsis mutant induced by ion beams affects flavonoid synthesis with spotted pigmentation in testa

International Nuclear Information System (INIS)

Tanaka, A.; Tano, S.; Chantes, T.; Yokota, Y.; Shikazono, N.; Watanabe, H.

1997-01-01

A new stable mutant of Arabidopsis thaliana with a spotted pigment in the seed coat, named anthocyanin spotted testa (ast), was induced by carbon ion irradiation. The spotted pigmentation of ast mutant was observed in immature seeds from 1-2 days after flowering (DAF), at the integument of the ovule, and spread as the seed coat formed. Anthocyanin accumulation was about 6 times higher in ast mutant than in the wild-type at 6 DAF of the immature seeds, but was almost the same in mature dry seeds. A higher anthocyanin accumulation was not observed in the seedlings, leaves or floral buds of ast mutant compared with the wild-type, which suggests that a high accumulation of anthocyanins is specific to the seed coat of the immature ast seeds. Reciprocal crosses between ast mutant and the wild-type indicated that ast is a single recessive gene mutation and segregates as a delayed inheritance. The results of crossing with tt7 and ttg mutants also confirmed that the AST gene is probably a regulatory locus that controls flavonoid biosynthesis. A mapping analysis revealed that the gene is located on chromosome I and is closely linked to the SSLP DNA marker nga280 with a distance of 3.2 cM. AST has been registered as a new mutant of Arabidopsis

Assessing T cell differentiation at the single-cell level

NARCIS (Netherlands)

Gerlach, Carmen

2012-01-01

This thesis describes the development and use of a novel technology for single-cell fate mapping, called cellular barcoding. With this technology, unique and heritable genetic tags (barcodes) are introduced into naïve T cells. Using cellular barcoding, we investigated I) how different

Production and characterization of radiation-sensitive meiotic mutants of Coprinus cinereus

International Nuclear Information System (INIS)

Zolan, M.E.; Tremel, C.J.; Pukkila, P.J.

1988-01-01

We have isolated four gamma-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1;rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the pew viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine.

Science.gov (United States)

Ivanov, Ana; Kameka, Alexander; Pajak, Agnieszka; Bruneau, Luanne; Beyaert, Ronald; Hernández-Sebastià, Cinta; Marsolais, Frédéric

2012-06-01

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.

Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages φ29, λ, and T4

Science.gov (United States)

Smith, Douglas E.; Fuller, Derek N.; Raymer, Dorian M.; Rickgauer, Peter; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Catalano, Carlos E.; Kottadiel, Vishal; Rao, Venigalla B.

2007-09-01

A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

Identification and genetic assay of a high-chlorophyll-content mutant in Rice

International Nuclear Information System (INIS)

Liu Baofu; Chen Xifeng; Jin Yang; Gu Zhimin; Ma Bojun; Zhu Xudong

2011-01-01

A deep rice mutant ZM1120 was screened from the γ-rays irradiation mutation library of Zhonghua 11. Compared to the wild-type control, this mutant were darker (greener) in shoots and leaves, and after sowing 60 and 90 d, the content of chlorophyll were increased by 16.0% and 7.2%, respectively, and the content of carotenoid also increased by 23.1% and 24.2%, respectively. After sowing 90 d the net photosynthetic rate and transpiration rate were increased by 16.3% and 11.4%, respectively. The agronomical traits of this mutant significantly changed, and the traits of plant height, flag-leaf length, flag-leaf width, tiller number per plant, panicle length and setting rate decreased, but the grain length and 1000-grain weight increased by 7.9% and 2.6%. Genetic analysis revealed that the mutation phenotype was controlled by a single recessive nuclear gene, and further cloning and function assay will be useful for understanding the mechanism of photosynthesis and for rice breeding in future. (authors)

Amphitrite ornata dehaloperoxidase (DHP): investigations of structural factors that influence the mechanism of halophenol dehalogenation using "peroxidase-like" myoglobin mutants and "myoglobin-like" DHP mutants.

Science.gov (United States)

Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H

2011-09-27

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ∼10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ∼0.3 and ∼0.8 Å, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.

Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using ;Peroxidase-like; Myoglobin Mutants and ;Myoglobin-like; DHP Mutants

Energy Technology Data Exchange (ETDEWEB)

Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H. (SC)

2012-05-14

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O{sub 2} transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity {approx}10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of 'peroxidase-like' Mb mutants and 'Mb-like' DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned {approx}0.3 and {approx}0.8 {angstrom}, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the 'DHP-like' position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the

Inclusive photoproduction of single charged particles at high pT

International Nuclear Information System (INIS)

Apsimon, R.J.; Flower, P.S.; Hallewell, G.; Morris, J.A.G.; Morris, J.V.; Paterson, C.N.; Sharp, P.H.; Atkinson, M.; Brook, N.; Coyle, P.; Dickinson, B.; Donnachie, A.; Doyle, A.T.; Ellison, R.J.; Foster, J.M.; Hughes-Jones, R.E.; Ibbotson, M.; Kolya, S.D.; Lafferty, G.D.; McCann, H.; McManus, C.; Mercer, D.; Ottewell, P.J.; Reid, D.; Thompson, R.J.; Waterhouse, J.; Barberis, D.; Davenport, M.; Eades, J.; McClatchey, R.; Brodbeck, T.J.; Charity, T.; Clegg, A.B.; Henderson, R.C.W.; Hickman, M.T.; Keemer, N.R.; Newton, D.; O'Connor, A.; Wilson, G.W.; Danaher, S.; Galbraith, W.; Thacker, N.A.; Thompson, L.

1989-01-01

Single charged-particle inclusive cross sections for photon, pion and kaon beams on hydrogen at the CERN-SPS are presented as functions of p T and x F . Data cover the range 0.0 T F T < 1.6 GeV/c for the photon-induced data. Using the hadron-induced data to estimate the hadronic behaviour of the photon, the difference distributions and ratios of cross sections are a measure of the contribution of the point-like photon interactions. The data are compared with QCD calculations and show broadly similar features. (orig.)

Tank characterization report for single-shell tanks 241-T-201, 241-T-202, 241-T-203, and 241-T-204

International Nuclear Information System (INIS)

Simpson, B.C.

1998-01-01

A major function of the Tank Waste Remediation System (TWRS) is to characterize waste in support of waste management and disposal activities at the Hanford Site. Analytical data from sampling and analysis, in addition to other available information about a tank are compiled and maintained in a tank characterization report (TCR). This report and its appendices serve as the TCR for the single-shell tank series consisting of 241-T-201, -T-202, -T-203, and -T-204. The objectives of this report are: (1) to use characterization data in response to technical issues associated with T-200 series tank waste and (2) to provide a standard characterization of this waste in terms of a best-basis inventory estimate. Section 2.0 summarizes the response to technical issues, Section 3.0 shows the best-basis inventory estimate, Section 4.0 makes recommendations about the safety status of the tank and additional sampling needs. The appendices contain supporting data and information. Appendix A contains historical information for 241-T-201 to T-204, including surveillance information, records pertaining to waste transfers and tank operations, and expected tank contents derived from a process knowledge-based computer program. Appendix B summarizes sampling events, sample data obtained before 1989, and the most current sampling results. Appendix C reports the statistical analysis and numerical manipulation of data used in issue resolution. Appendix D contains the evaluation to establish the best-basis for the inventory estimate and the statistical analysis performed for this evaluation. Appendix E is a bibliography that resulted from an in-depth literature search of all known information sources applicable to tanks 241-T-201, -T-202, -T-203, and -T-204. The reports listed in Appendix E are available in the Tank Characterization and Safety Resource Center

Incomplete excision repair process after UV-irradiation in MUT-mutants of Proteus mirabillis

International Nuclear Information System (INIS)

Stoerl, K.

1977-01-01

MUT-mutants of P. mirabilis seem to be able to perform the incision step in the course of excision repair. In contrast to the corresponding wildtype strains with MUT-mutants the number of single-strand breaks formed after UV-irradiation is independent of the UV-dose up to about 720 erg/mm 2 . Incubation in minimal medium over a longer time does not result in completion of excision repair; about 3-6 single-strand breaks in the DNA of these mutants remain open. Likewise, the low molecular weight of the newly synthesized daughter DNA confirms an incompletely proceeding or delayed repair process. As a possible reason for the mutator phenotype an alteration of the DNA-polymerase playing a role in excision and resynthesis steps of excision repair is discussed. (author)

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response.

Science.gov (United States)

Xue, Qiong; Bettini, Emily; Paczkowski, Patrick; Ng, Colin; Kaiser, Alaina; McConnell, Timothy; Kodrasi, Olja; Quigley, Máire F; Heath, James; Fan, Rong; Mackay, Sean; Dudley, Mark E; Kassim, Sadik H; Zhou, Jing

2017-11-21

It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high

Transcriptional analysis of the HeT-A retrotransposon in mutant and wild type stocks reveals high sequence variability at Drosophila telomeres and other unusual features

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Piñeyro David

2011-11-01

Full Text Available Abstract Background Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the HeT-A retrotransposon. The sequence of the HeT-A retrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the HeT-A is entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different HeT-A scenarios. Results A detailed study on the variability of HeT-A reveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of HeT-A subfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the HeT-A RNA. Interestingly, our results also suggest that most HeT-A copies are actively expressed regardless of which telomere and where in the telomere they are located. Conclusions Our study demonstrates how the HeT-A sequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

Science.gov (United States)

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

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Chen Bo-Ruei

2012-05-01

Full Text Available Abstract Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning

Hepatitis B surface gene 145 mutant as a minor population in hepatitis B virus carriers

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Komatsu Haruki

2012-01-01

Full Text Available Abstract Background Hepatitis B virus (HBV can have mutations that include the a determinant, which causes breakthrough infection. In particular, a single mutation at amino acid 145 of the surface protein (G145 is frequently reported in the failure of prophylactic treatment. The aim of this study was to evaluate the frequency of the a determinant mutants, especially the G145 variant, in Japan, where universal vaccination has not been adopted. Methods The present study was a retrospective study. The study cohorts were defined as follows: group 1, children with failure to prevent mother-to-child transmission despite immunoprophylaxis (n = 18, male/female = 8/10, age 1-14 years; median 6 years; group 2, HBV carriers who had not received vaccination or hepatitis B immunoglobulin (n = 107, male/female = 107, age 1-52 years; median 16 years. To detect the G145R and G145A mutants in patients, we designed 3 probes for real-time PCR. We also performed direct sequencing and cloning of PCR products. Results By mutant-specific real-time PCR, one subject (5.6% was positive for the G145R mutant in group 1, while the G145 mutant was undetectable in group 2. The a determinant mutants were detected in one (5.6% of the group 1 subjects and 10 (9.3% of the group 2 subjects using direct sequencing, but direct sequencing did not reveal the G145 mutant as a predominant strain in the two groups. However, the subject who was positive according to the mutant-specific real-time PCR in group 1 had overlapped peaks at nt 587 in the electropherogram. In group 2, 11 patients had overlapped peaks at nt 587 in the electropherogram. Cloning of PCR products allowed detection of the G145R mutant as a minor strain in 7 (group 1: 1 subject, group 2: 6 subjects of 12 subjects who had overlapped peaks at nt 587 in the electropherogram. Conclusions The frequency of the a determinant mutants was not high in Japan. However, the G145R mutant was often present as a minor population in

Analysis of a Partial Male-Sterile Mutant of Arabidopsis thaliana Isolated from a Low-Energy Argon Ion Beam Mutagenized Pool

International Nuclear Information System (INIS)

Xu Min; Bian Po; Wu Yuejin; Yu Zengliang

2008-01-01

A screen for Arabidopsis fertility mutants, mutagenized by low-energy argon ion beam, yielded two partial male-sterile mutants tc243-1 and tc243-2 which have similar phenotypes. tc243-2 was investigated in detail. The segregation ratio of the mutant phenotypes in the M2 pools suggested that mutation behaved as single Mendelian recessive mutations. tc243 showed a series of mutant phenotypes, among which partial male-sterile was its striking mutant characteristic. Phenotype analysis indicates that there are four factors leading to male sterility. a. Floral organs normally develop inside the closed bud, but the anther filaments do not elongate sufficiently to position the locules above the stigma at anthesis. b. The anther locules do not dehisce at the time of flower opening (although limited dehiscence occurs later). c. Pollens of mutant plants develop into several types of pollens at the trinucleated stage, as determined by staining with DAPI (4',6-diamidino-2-phenylindole), which shows a variable size, shape and number of nucleus. d. The viability of pollens is lower than that of the wild type on the germination test in vivo and vitro.

Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

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Chakravartty, Vandana

2012-01-01

Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5′-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues

A high yielding, better quality chickpea mutant variety 'NIFA-95'

International Nuclear Information System (INIS)

Hassan, S.; Javed, M.A.; Khattak, S.U.K.; Iqbal, M.M.

2001-01-01

Chickpea or gram (Cicer arietinum L.) is an important legume crop of Pakistan, grown on over one million hectares annually. The national average yield of the crop is very low (0.5 t/ha) and thus the country had to spent about 2 billion rupees ($ 50 million) on import of pulses. The main causes of low yield are non-availability of genetic sources for resistance to various diseases especially gram blight Ascochyta rabiei (Pass.) Lab., insect pest (Pod borer) and non-adoption of proper production technology by the farmers. This calls for earnest efforts of breeders to evolve high yielding and disease resistant varieties of chickpea for provision of quality seeds to the farming community to increase production of this important crop. Seeds of a highly blight susceptible variety '6153' were irradiated at 200 Gy dose of gamma radiation in 1985 and the promising mutant line CMN-446-4 was selected in M3 generation on the basis of disease resistance, greater number of pods and better plant type. After confirmation of its resistance to blight in M 4 and M 5 , the mutant line was evaluated in various trials at different locations. In the advanced and zonal yield trials during 1993-95, the line CMN-446-4 produced the highest grain yield of 2,600 kg/ha as compared to the rest of the mutants and varieties. The line was also evaluated in the chickpea national uniform yield trial, conducted on over 11 locations in the country during 1993-94. In this trial, the mutant line ranked 3rd by producing an average yield of 1,528 kg/ha as compared to the two check varieties 'Punjab-91' (1,316 kg/ha) and 'Paidar-91' (1,391 kg/ha). The mutant line CMN-446-4 is moderately resistant to gram blight, highly resistant to stored pest (pulse beetle), contains 25.3% more protein as compared to the parental variety 6153 and is also better in nitrogen fixing capacity.The proposal for release of the mutant line CMN-446-4 as a new variety under the name 'NIFA-95' for general cultivation in the rainfed

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

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Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.

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Krishnan, Natraj; Rakshit, Kuntol; Chow, Eileen S; Wentzell, Jill S; Kretzschmar, Doris; Giebultowicz, Jadwiga M

2012-03-01

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila. Copyright © 2011 Elsevier Inc. All rights reserved.

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

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

2016-01-01

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

Upregulation of long non-coding RNA TUG1 promotes bladder cancer cell 5 proliferation, migration and invasion by inhibiting miR-29c.

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Guo, Peng; Zhang, Guohui; Meng, Jialin; He, Qian; Li, Zhihui; Guan, Yawei

2018-01-10

Bladder cancer (BC) is one of the leading causes of cancer-related death in the word. Long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) plays an important role in the development and progression of numerous cancers, including BC. However, the exact role of TUG1 in modulating BC progression is still poorly known. In this study, we found that TUG1 was upregulated and microRNA-29c (miR-29c) was downregulated in BC tissues and cell lines. Overexpression of TUG1 promoted the cell proliferation of T24 and EJ cells, whereas TUG1 knockdown had the opposite effect. Upregulation of TUG1 obviously facilitated the migration and invasion of T24 and EJ cells. In contrast, TUG1 silencing repressed the migration and invasion of T24 and EJ cells. Furthermore, TUG1 knockdown markedly increased the expression of miR-29c in vitro. On the contrary, overexpression of TUG1 remarkably decreased the expression of miR-29c. Transfection with plasmids containing mutant TUG1 has no effect on the expression of miR-29c. There were direct interactions between miR-29c and the binding sites of TUG1. In addition, the inhibitory effects of small interfering RNA specific for TUG1 on BC cell proliferation, migration and invasion were reversed by downregulation of miR-29c. Collectively, our study strongly demonstrates that TUG1 promotes BC cell proliferation, migration and invasion by inhibiting miR-29c, suggesting that lncRNATUG1 may be a promising target for BC gene therapy.

2018-02-21T09:46:29Z https://www.ajol.info/index.php/index/oai oai ...

African Journals Online (AJOL)

article/55713 2018-02-21T09:46:29Z ajrh:ART HIV/AIDS - Related Stigma and Discrimination in Nigeria: Review of Research Studies and future directions for Prevention Strategies Monjok, E Smesny, A Essien, EJ HIV/AIDS, Stigma, ...

Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

Science.gov (United States)

Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

2015-02-01

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

Use of gamma radiation for increasing plant disease control efficacy

International Nuclear Information System (INIS)

Chamswarng, Chiradej; Intanoo, Wanwilai; Piadang, Nattayana

2006-09-01

Irradiation of Trichoderma harzianum with 0.5-8.0 k Gay of gamma ray revealed 41 strains resistant to 10 ppm propiconazole or benomyl fungicides and two strains (23/03-7 and 27/08-1) were resistant to 10 ppm of both fungicides. After these two mutant strains were repeatedly irradiated with gamma ray, 74 mutant strains were obtained. Among these, three mutant strains, used as seed treatment effectively protected tomato seedlings from the infection of Pythium apanidermatum with significantly higher surviving seedlings than the Pythium inoculated control. The higher root colonization of mutant strains was obtains from strains 03/7-113, 03-/7-114 and 08/1-11. Rice seeds (RD 17), previously soaked in water for 24 ht were placed in spore suspension of T. harzianum prepared from 1 kg of fresh culture of 81 mutant isolated derived from single or double irradiation with gamma ray in 50 1 of water for 30 min. Two mutants including 23/03-7 (derived from single irradiation) and 03/7-134 (derived from double irradiation) provided percentages of root colonization by 29.63 and 25.93, while growth-promoted roots were 12.48 and 12.65 cm in length. respectively. These two strains were tested in rice field by treating pre-soaked seeds with Trichoderma suspensions for 30 min and incubated for 24 hr before sowing. Detection of root colonization by T. harzianum at 35, 45 and 102 days after sowing revealed that all Trichoderma strains effectively colonized rice roots at all stages of growth, particularly two mutants completely colonized rice root at 102 days after sowing. After harvesting, a mutant strain 30/7-134 increased rice yield to the maximum level at 29.65% over a control, while the percentage of fertile-seeds and it's seed weight, total seed weight, fertile-seed weight were significantly higher than a control. However, all Trichoderma strains provided the potential increases of rice yield over a control. Strain 03/7-134 significantly reduced percentage of dirty-panicle diseased

Type I enteropathy-associated T-cell lymphoma in the colon of a 29-year-old patient and a brief literature review

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Zhang JC

2016-02-01

Full Text Available Jiu-Cong Zhang, Yong Wang, Xiu-Feng Wang, Fang-Xin Zhang Department of Gastroenterology, Lanzhou General Hospital of Lanzhou Military Command, Lanzhou, People’s Republic of China Abstract: Enteropathy-associated T-cell lymphoma (EATL is a rare gastrointestinal non-Hodgkin’s lymphoma, originating from intraepithelial T-lymphocyte, which is specifically associated with celiac disease. EATL most commonly presents in the sixth and seventh decades of life. We report a unique case of type I EATL in the colon with liver metastasis, which was presented with nonspecific radiological findings and at a very young age (29 years old compared with previously published data. We suggest that EATL should be regarded as part of differential diagnosis in any patient presenting with abdominal pain, diarrhea, weight loss, and malabsorption because delay in treatment can result in an irreversible clinical outcome. Keywords: enteropathy-associated T-cell lymphoma, colon, ulcer, liver metastasis

Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

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Nannenga Brent L

2011-12-01

Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.).

Science.gov (United States)

Hanzawa, Eiko; Sasaki, Kazuhiro; Nagai, Shinsei; Obara, Mitsuhiro; Fukuta, Yoshimichi; Uga, Yusaku; Miyao, Akio; Hirochika, Hirohiko; Higashitani, Atsushi; Maekawa, Masahiko; Sato, Tadashi

2013-11-20

Root system architecture is an important trait affecting the uptake of nutrients and water by crops. Shallower root systems preferentially take up nutrients from the topsoil and help avoid unfavorable environments in deeper soil layers. We have found a soil-surface rooting mutant from an M2 population that was regenerated from seed calli of a japonica rice cultivar, Nipponbare. In this study, we examined the genetic and physiological characteristics of this mutant. The primary roots of the mutant showed no gravitropic response from the seedling stage on, whereas the gravitropic response of the shoots was normal. Segregation analyses by using an F2 population derived from a cross between the soil-surface rooting mutant and wild-type Nipponbare indicated that the trait was controlled by a single recessive gene, designated as sor1. Fine mapping by using an F2 population derived from a cross between the mutant and an indica rice cultivar, Kasalath, revealed that sor1 was located within a 136-kb region between the simple sequence repeat markers RM16254 and 2935-6 on the terminal region of the short arm of chromosome 4, where 13 putative open reading frames (ORFs) were found. We sequenced these ORFs and detected a 33-bp deletion in one of them, Os04g0101800. Transgenic plants of the mutant transformed with the genomic fragment carrying the Os04g0101800 sequence from Nipponbare showed normal gravitropic responses and no soil-surface rooting. These results suggest that sor1, a rice mutant causing soil-surface rooting and altered root gravitropic response, is allelic to Os04g0101800, and that a 33-bp deletion in the coding region of this gene causes the mutant phenotypes.

Kinetics, improved activity and thermostability of endoglucanase and beta glucosidase from a mutant-derivative of aspergillus niger ms82

International Nuclear Information System (INIS)

Sohail, M.; Ahmad, A.; Khan, S.A.; Uddin, F.

2013-01-01

A mutant MS301 of Aspergillus niger MS82 showed 1.5 to 2.5-fold improved endoglucanase and beta-glucosidase activity when grown on crude lignocellulosic substrates under solid-state and submerged conditions. Indicators of thermal stability of enzymes (Tm and T1/2) showed that the wild type and mutant endoglucanase was more heat-resistant compared to beta-glucosidase. However, mutant and parent enzymes shared almost the same values for melting temperatures and half-lives. Endoglucanase and beta-glucosidase from both the strains showed optimum activity under acidic pH. Energy of activation (Ea) of mutant beta-glucosidase was substantially lower than the parent enzyme while Ea of mutant endoglucanase was slightly less than the parent. The lowered Ea values can be attributed to the improved beta-glucosidase activity of the mutant strain. Moreover, the MS301 enzymes were better in hydrolyzing purified and crude cellulosic materials than the parent MS82. (author)

A sorghum (Sorghum bicolor mutant with altered carbon isotope ratio.

Directory of Open Access Journals (Sweden)

Govinda Rizal

Full Text Available Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium's efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor mutant with a low δ13C characteristic. A mutant (named Mut33 with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT. The back-cross (BC1F1 progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 μmol CO2.mol-1air and the maximum rate of photosynthesis was less than 5μmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

International Nuclear Information System (INIS)

Kaefer, E.; Mayor, O.

1986-01-01

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or γ-rays. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. (Auth.)

Radiation induced early maturing mutants in barley

International Nuclear Information System (INIS)

Kumar, R.; Chauhan, S.V.S.; Sharma, R.P.

1978-01-01

In M 2 generation, two early maturing plants were screened from a single spike progeny of a plant obtained from 20 kR of gamma-ray irradiation of a six-rowed barley (Hordeum vulgare L. var. Jyoti). Their true breeding nature was confirmed in M 3 generation. These mutants flower and mature 38 and 22 days earlier than those of control. (auth.)

A broad phenotypic screen identifies novel phenotypes driven by a single mutant allele in Huntington's disease CAG knock-in mice.

Directory of Open Access Journals (Sweden)

Sabine M Hölter

Full Text Available Huntington's disease (HD is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.

2018-04-28T16:45:29Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

article/49910 2018-04-28T16:45:29Z ijonas:ART Iodometric determination of ampicillin in proprietary capsules Ejele, A Anyanwu, N Anunuso, C Ampicillin, capsule formulation, iodometric titration, concentration. The concentration of ampicillin in ...

Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant.

Science.gov (United States)

Brokaw, C J; Luck, D J

1985-01-01

Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.

Characterization of a Weak Allele of Zebrafish cloche Mutant

Science.gov (United States)

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

Pediatric-specific\tAntimicrobial\tResistance\tPatterns\tof\tUrinary\tTract\tInfections: A\tSingle-Centre\tExperience\tfrom\tTurkey

Directory of Open Access Journals (Sweden)

Yasar Kandur

2016-12-01

Full Text Available Objectives: Antimicrobial resistance of the causative microorganisms of pediatric urinary tract infection (UTI is a growing problem.\tThe\taim\tof\tthis\tstudy\tis\tto\tdetermine\tthe\tchanging\tpattern\tof\tantibiotic\tsusceptibility\tin\tUTIs\tin\tan\toutpatient\tsetting. Methods: We\tretrospectively\treviewed\tthe\tmedical\trecords\tof\tpediatric\tpatients\twith\tUTI\twho\twere\tfollowed-up\tin\tour\tcenter between\tJanuary-2014\tand\tMay-2015. Results: One hundred and seventy-one patients (M/F= 53/118;\tmean age 56 ± 47.2\tmonths with UTI were enrolled\tin this study.\tA\ttotal\tof\t231\turinary\tpathogens\twere\tisolated\tfrom\tUTI\tepisodes.\tThe\tmost\tcommon\tcausative\tagent\twas\tEscherichia\tcoli (E. coli (70.6% followed by Klebsiella spp. (16.5%, Proteus spp. (6.5%. One point eight percent of E. coli isolates were resistant to amikacin, 17.8% to gentamicin, 60.7% to TMP-SMX, 66,9% to ampicillin, 52.1% to cefixime, 46% to ceftriaxone, 54.6%\tto\tcefuroxime\tand\t4.9%\tto\tnitrofurantoin. Conclusions: TMP-SMX and nitrofurantoin are poor empirical choices for pediatric patients due high resistance rates and gastrointestinal side effects, respectively. Second and third -generation cephalosporins (cefixime may not be considered as appropriate\tempiric\tantibiotic\talternatives\tanymore\tgiven\ttheir\thigh\tresistance\trates\tin\tthe\tnext\tfew\tyears.\tPhysicians\twho\twork in\tthe\tprimary\thealth\tcare\tshould\tbe\tencouraged\tfor\tthe\tselection\tof\tmore\tappropriate\tantibiotics.

Structural optimization of N1-aryl-benzimidazoles for the discovery of new non-nucleoside reverse transcriptase inhibitors active against wild-type and mutant HIV-1 strains.

Science.gov (United States)

Monforte, Anna Maria; De Luca, Laura; Buemi, Maria Rosa; Agharbaoui, Fatima E; Pannecouque, Christophe; Ferro, Stefania

2018-02-01

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of human immunodeficiency virus (HIV) infection. These regimens are extremely effective in suppressing virus replication. Recently, our research group identified some N 1 -aryl-2-arylthioacetamido-benzimidazoles as a novel class of NNRTIs. In this research work we report the design, the synthesis and the structure-activity relationship studies of new compounds (20-34) in which some structural modifications have been introduced in order to investigate their effects on reverse transcriptase (RT) inhibition and to better define the features needed to increase the antiviral activity. Most of the new compounds proved to be highly effective in inhibiting both RT enzyme at nanomolar concentrations and HIV-1 replication in MT4 cells with minimal cytotoxicity. Among them, the most promising N 1 -aryl-2-arylthioacetamido-benzimidazoles and N 1 -aryl-2-aryloxyacetamido-benzimidazoles were also tested toward a panel of single- and double-mutants strain responsible for resistance to NNRTIs, showing in vitro antiviral activity toward single mutants L100I, K103N, Y181C, Y188L and E138K. The best results were observed for derivatives 29 and 33 active also against the double mutants F227L and V106A. Computational approaches were applied in order to rationalize the potency of the new synthesized inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies.

Science.gov (United States)

Li, Guotian; Jain, Rashmi; Chern, Mawsheng; Pham, Nikki T; Martin, Joel A; Wei, Tong; Schackwitz, Wendy S; Lipzen, Anna M; Duong, Phat Q; Jones, Kyle C; Jiang, Liangrong; Ruan, Deling; Bauer, Diane; Peng, Yi; Barry, Kerrie W; Schmutz, Jeremy; Ronald, Pamela C

2017-06-01

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake ( Oryza sativa ssp japonica ), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations. © 2017 American Society of Plant Biologists. All rights reserved.

High-Protein Soybean Mutants by Using Irradiation Technique

International Nuclear Information System (INIS)

Yathaputanon, C.; Kumsueb, B.; Srisombun, S.

2009-07-01

Full text: Soybean variety improvement for high seed protein using induced mutation was initiated. Approximately 5,000 seeds of soybean variety Chiang Mai 60 were irradiated with gamma rays at the dose of 200 Grays at Kasetsart University. High-protein seed mutants in M2 to M4 generations were selected at Nakhon Ratchasima Field Crops Research Center during 2004-2008. The Pedigree method of selection was used. Kjeldahl method was used to analyze seed protein percentages. The M2 seeds protein content of the M2 generation was 45.2% while that of the original parent was 43.0%. M3s were seeded plant to row. In each row, the best four plants were selected for protein analysis. The average protein content of selected mutant lines was 3.9% while the check variety had average protein content of 42.4%. In the M4 generation, the result showed that the average protein contents of the selected mutant lines and the check variety were 42.8% and 42.0%, respectively. In the 2007-2008 trials, four promising mutants had and average protein content of 428%, while the check variety had and average protein content of 41.1%. The four mutants produced the mean grain yield of 2.20-2.42 t/Ha, which was 10.21% higher than that of Chiang Mai 60. The mutant lines produced both a high grain protein content and a high grain yield. They will be further tested their adaptability in the research centers and farmer fields

New applications of CRISPR/Cas9 system on mutant DNA detection.

Science.gov (United States)

Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

2018-01-30

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

Nonbehavioral Selection for Pawns, Mutants of PARAMECIUM AURELIA with Decreased Excitability

Science.gov (United States)

Schein, Stanley J.

1976-01-01

The reversal response in Paramecium aurelia is mediated by calcium which carries the inward current during excitation. Electrophysiological studies indicate that strontium and barium can also carry the inward current. Exposure to high concentrations of barium rapidly paralyzes and later kills wild-type paramecia. Following mutagenesis with nitrosoguanidine, seven mutants which continued to swim in the `high-barium' solution were selected. All of the mutants show decreased reversal behavior, with phenotypes ranging from extremely non-reversing (`extreme' pawns) to nearly wild-type reversal behavior (`partial' pawns). The mutations fall into three complementation groups, identical to the pwA, pwB, and pwC genes of Kung et al. (1975). All of the pwA and pwB mutants withstand longer exposure to barium, the pwB mutants surviving longer than the pwA mutants. Among mutants of each gene, survival is correlated with loss of reversal behavior. Double mutants (A–B, A–C, B–C), identified in the exautogamous progeny of crosses between `partial' mutants, exhibited a more extreme non-reversing phenotype than either of their single-mutant (`partial' pawn) parents.———Inability to reverse could be expected from an alteration in the calcium-activated reversal mechanism or in excitation. A normal calcium-activated structure was demonstrated in all pawns by chlorpromazine treatment. In a separate report (Schein, Bennett and Katz 1976) the results of electrophysiological investigations directly demonstrate decreased excitability in all of the mutants, a decrease due to an altered calcium activation. The studies of the genetics, the survival in barium and the electro-physiology of the pawns demonstrate that the pwA and pwB genes have different effects on calcium activation. PMID:1001878

Single top quark t W + X production at the LHC a closer look

CERN Document Server

Belyaev, A.

2001-01-01

We have reexamined the tW+X single top-quark production process which is important at the LHC contrary to the Tevatron. The special attention was paid to the treatment of the 2->2[Wt] process and the part of it's next-to-leading correction: 2->3[Wtb] process. We show that 2->3[Wtb] process has to be correctly taken into account with a proper subtraction of the top pair contribution and that it has qualitatively different kinematical distributions from the 2->2[Wt] process. We present the total cross section of the tW+X production to be about 62 pb at QCD scale be taken as a top quark mass, suggest the method of combining Wt and Wtb processes with gauge invariant subtraction of the tt-bar part which allows to reproduce correct kinematical properties and perform a proper event simulation of the tW+X process in the whole kinematical region.

A comprehensive dataset of genes with a loss-of-function mutant phenotype in Arabidopsis.

Science.gov (United States)

Lloyd, Johnny; Meinke, David

2012-03-01

Despite the widespread use of Arabidopsis (Arabidopsis thaliana) as a model plant, a curated dataset of Arabidopsis genes with mutant phenotypes remains to be established. A preliminary list published nine years ago in Plant Physiology is outdated, and genome-wide phenotype information remains difficult to obtain. We describe here a comprehensive dataset of 2,400 genes with a loss-of-function mutant phenotype in Arabidopsis. Phenotype descriptions were gathered primarily from manual curation of the scientific literature. Genes were placed into prioritized groups (essential, morphological, cellular-biochemical, and conditional) based on the documented phenotypes of putative knockout alleles. Phenotype classes (e.g. vegetative, reproductive, and timing, for the morphological group) and subsets (e.g. flowering time, senescence, circadian rhythms, and miscellaneous, for the timing class) were also established. Gene identities were classified as confirmed (through molecular complementation or multiple alleles) or not confirmed. Relationships between mutant phenotype and protein function, genetic redundancy, protein connectivity, and subcellular protein localization were explored. A complementary dataset of 401 genes that exhibit a mutant phenotype only when disrupted in combination with a putative paralog was also compiled. The importance of these genes in confirming functional redundancy and enhancing the value of single gene datasets is discussed. With further input and curation from the Arabidopsis community, these datasets should help to address a variety of important biological questions, provide a foundation for exploring the relationship between genotype and phenotype in angiosperms, enhance the utility of Arabidopsis as a reference plant, and facilitate comparative studies with model genetic organisms.

A low noise single-transistor transimpedance preamplifier for Fourier-transform mass spectrometry using a T feedback network.

Science.gov (United States)

Lin, Tzu-Yung; Green, Roger J; O'Connor, Peter B

2012-09-01

A novel single-transistor transimpedance preamplifier has been introduced for improving performance in Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry. A low noise junction field-effect transistor (JFET), BF862, is used as the main amplification stage of this trans-impedance preamplifier, and a T-shaped feedback network is introduced as both the feedback and the gate biasing solutions. The T feedback network has been studied using an operational amplifier (Op Amp), AD8099. Such a feedback system allows ~100-fold less feedback resistance at a given transimpedance, hence preserving bandwidth, which is beneficial to applications demanding high gain. The single-transistor preamplifier yields a tested transimpedance of ~10(4) Ω (80 dBΩ) in the frequency range between 1 kHz and 1 MHz (mass-to-charge ratio, m/z, of around 180-180k for a 12-T FT-ICR system), with a low power consumption of ~6 mW, which implies that this preamplifier is well suited to a 12-T FT-ICR mass spectrometer. In trading noise performance for higher trans-impedance, an alternative preamplifier design, an AD8099 preamplifier with the T feedback network, has also been studied with a capability of ~10(6) Ω (120 dBΩ) transimpedance in the same frequency range. The resistive components in the T feedback network reported here can be replaced by complex impedances, which allows adaptation of this feedback system to other frequency, transimpedance, and noise characteristics for applications not only in other mass spectrometers, such as Orbitrap, time-of-flight (TOF), and ion trap systems, but also in other charge/current detecting systems such as spectroscopy systems, microscopy systems, optical communication systems, or charge-coupled devices (CCDs).

2018-03-29T07:13:34Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

article/95913 2018-03-29T07:13:34Z rj:ART HIV/AIDS Management and The Mobile Workforce: A Case ... The HIV /AIDS pandemic has a peculiarity with persons employed in the security forces, health care delivery chain and students in hostels.

A temperature-sensitive dcw1 mutant of Saccharomyces cerevisiae is cell cycle arrested with small buds which have aberrant cell walls.

Science.gov (United States)

Kitagaki, Hiroshi; Ito, Kiyoshi; Shimoi, Hitoshi

2004-10-01

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Deltadcw1 Deltadfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Deltadfg5). When DC61 cells were incubated at 37 degrees C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37 degrees C. At 37 degrees C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37 degrees C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37 degrees C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Deltadcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

clustering common bean mutants based on heterotic groupings

African Journals Online (AJOL)

ACSS

2015-02-19

Feb 19, 2015 ... Blair, W.M., Porch, T., Cichy, K., Galeano, H. C,. Lariguet, P., Pankhurst, C. and Broughton, W. 2007a. Induced mutants in common bean. (Phaseolus vulgaris) and their potential use in nutrition quality, breeding and gene discovery. Israel Journal of Plant Sciences. 55:191 - 200. Blair, W.M., Fregene, A.M., ...

Fanconi anemia group A and C double-mutant mice: functional evidence for a multi-protein Fanconi anemia complex.

Science.gov (United States)

Noll, Meenakshi; Battaile, Kevin P; Bateman, Raynard; Lax, Timothy P; Rathbun, Keany; Reifsteck, Carol; Bagby, Grover; Finegold, Milton; Olson, Susan; Grompe, Markus

2002-07-01

Fanconi anemia (FA) is a genetically heterogeneous disorder associated with defects in at least eight genes. The biochemical function(s) of the FA proteins are unknown, but together they define the FA pathway, which is involved in cellular responses to DNA damage and in other cellular processes. It is currently unknown whether all FA proteins are involved in controlling a single function or whether some of the FA proteins have additional roles. The aim of this study was 1) to determine whether the FA group A and group C genes have identical or partially distinct functions, and 2) to have a better model for human FA. We generated mice with a targeted mutation in fanca and crossed them with fancc disrupted animals. Several phenotypes including sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc double -/-, and controls. Fibroblast cells and hematopoietic precursors from fanca/fancc double-mutant mice were not more sensitive to MMC than those of either single mutant. fanca/fancc double mutants had no evidence for an additive phenotype at the cellular or organismal level. These results support a model where both FANCA and FANCC are part of a multi-protein nuclear FA complex with identical function in cellular responses to DNA damage and germ cell survival.

Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner.

Science.gov (United States)

Oresic, Kristina; Mueller, Britta; Tortorella, Domenico

2009-06-01

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.

Dynamic changes of peripheral blood T-lymphocyte DNA-Synthesis in rabbits after fractionated and single exposure to 60Co-γ rays

International Nuclear Information System (INIS)

Wang Zongwu; Chen Tiehe; Yu Zhijie; Han Ling; Pan Yusha; Su Fuqiang

1988-01-01

The experiments in 59 rabbits γ-irradiated with doses of 0, 0.5, 1.0, 2.0, and 3.0 Gy in fractional and single exposure to 60 Co-γ rays were reported, respectively · Dynamics of the changes of DNA-Synthesis in T-lymphocytes of peripheral blood was obserced during 29 days after γ-irradiation. Marked inhibition in DNA-synthesis was found on 1st day after irradiation. Recovery was observed in 3rd day after irradiation. The levels of DNA-synthesis before irradiation was recovered on 7th day after exposure for all groups. For fractionated irradiation, however, an increase, rather than a decrese, of DNA-synthesis was in the group of 1.0 Gy

Adiponectin Single Nucleotide Polymorphism (+276G/T) and Its ...

African Journals Online (AJOL)

The present study was investigating the association between the single nucleotide polymorphism +276 G/T of the adiponectin gene with serum adiponectin level in patients with coronary artery disease (CAD). In this study 100 healthy controls and 100 Egyptian patients with coronary artery disease of both genders ...

Purification, gene cloning, gene expression, and mutants of Dps from the obligate anaerobe Porphyromonas gingivalis.

Science.gov (United States)

Ueshima, Junichi; Shoji, Mikio; Ratnayake, Dinath B; Abe, Kihachiro; Yoshida, Shinichi; Yamamoto, Kenji; Nakayama, Koji

2003-03-01

The periodontopathogen Porphyromonas gingivalis is an obligate anaerobe that is devoid of catalase but exhibits a relatively high degree of resistance to peroxide stress. In the present study, we demonstrate that P. gingivalis contains a Dps homologue that plays an important role in the protection of cells from peroxide stress. The Dps protein isolated from P. gingivalis displayed a ferritin-like spherical polymer consisting of 19-kDa subunits. Molecular cloning and sequencing of the gene encoding this protein revealed that it had a high similarity in nucleotide and amino acid sequences to Dps proteins from other species. The expression of Dps was significantly increased by exposure of P. gingivalis to atmospheric oxygen in an OxyR-dependent manner, indicating that it is regulated by the reactive oxygen species-regulating gene oxyR. The Dps-deficient mutants, including the dps single mutant and the ftn dps double mutant, showed no viability loss upon exposure to atmospheric oxygen for 6 h. In contrast to the wild type, however, these mutants exhibited the high susceptibility to hydrogen peroxide, thereby disrupting the viability. On the other hand, no significant difference in sensitivity to mitomycin C and metronidazole was observed between the wild type and the mutants. Furthermore, the dps single mutant, compared with the wild type, showed a lower viability in infected human umbilical vein endothelial cells.

Magnetic Analysis of a Single-Aperture 11T Nb3Sn Demonstrator Dipole for LHC Upgrades

Energy Technology Data Exchange (ETDEWEB)

Auchmann, B. [CERN; Karppinen, M. [CERN; Kashikhin, V. [Fermilab; Zlobin, A. V. [Fermilab

2012-05-01

The planned upgrade of the LHC collimation system foresees additional collimators to be installed in the dispersion suppressor areas around points 2, 3, and 7. The necessary longitudinal space for the collimators could be provided by replacing some 8.33-T 15-m-long NbTi LHC main dipoles with shorter 11-T Nb3Sn dipoles compatible with the LHC lattice and main systems. To demonstrate this possibility, in 2011 Fermilab and CERN started a joint R&D program with the goal of building a 5.5-m-long tw in-aperture dipole prototype suitable for installation in the LHC by 2014. The first step of this program is the development of a 2-m-long single-aperture demonstration dipole with the nominal field of 11 T at the LHC nominal current of ~11.85 kA and 60-m m bore with ~20% margin. This paper presents the results of magnetic analysis of the single-aperture Nb3Sn demonstrator dipole for the LHC collimation system upgrade.

Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function.

Science.gov (United States)

Kondabagil, Kiran; Dai, Li; Vafabakhsh, Reza; Ha, Taekjip; Draper, Bonnie; Rao, Venigalla B

2014-11-01

The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH(-) nucleophile. The ATPase Walker B motif, thus, may be redefined as "β-strand (4-6 hydrophobic-rich amino acids)-DE-hydrophobic/nonpolar amino acid". Copyright © 2014 Elsevier Inc. All rights reserved.

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

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Roland Züst

Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Studies on leaf mutants of Pea. (Part) I. Morphology, performance and somatic chromosomes

International Nuclear Information System (INIS)

Kaul, M.L.H.; Anjali, A.

1988-01-01

Three recessive non-allelic mutant genes alter foliar morphology of pea when present singly and in combination. Gene acacia replaces tendrils by a terminal leaflet, afila replaces leaflets by tendrils and cochleata replaces stipules by spoon shaped appendages. In combination, these genes drastically alter leaf morphology; plants can be identified only after flowering. The mutant genes influence shoot height, floral organ number, maturity period, grain yield and seed protein production; inter- and intra-genotypic variability in certain metric traits is significant. Influence of cochleata gene over floral form and function is considerable. In terms of seed yield and protein content, breeding value of all the mutants except of acacia is low because these mutant genes represent foreign untuned genes in pea genome. Segregation deficit is maximum in triple gene mutant with highly impaired fertility and low seed production. Somatic chromosome number in all the mutants and recombinants is 14; in morphology the chromosomes do not differ from the initial line, Bonneville. (author). 9 refs., 4 tabs

Characteristic, inheritance and breeding application of rice mutants with greenable albino leaf

International Nuclear Information System (INIS)

Fang Xiantao; Ma Hongli; Zhao Fuyuan; Zhang Qingqi; Zhang Shubiao

2009-01-01

Inheritance and main agronomic traits of photo-thermo-sensitive genic male sterile line with green-revertible albino leaf were investigated. The results indicated that the mutants might be divided into three types: albino regreening type (W2, W3, W4 and W10), albino to kelly type (W9) and abino-regreening-albino-regreening type (W1 and W7). Genetic study indicated that green-revertible albino leaf color trait of the mutants as controlled by a single recessive gene. These mutants had similar agronomic traits and fertility characteristics to the corresponding male sterile line 'Peiai 64S'. The hybrids of these mutants had similar characteristics with original-hybrids in plant type, developing of tillers and plant height. The yield components of the mutant hybrids were different depending on different mutants. The yield potential of hybrids of W1, W2 and W3 were similar to the original-hybrid. The results also indicated that W1, W2 and W3 had breeding application value. (authors)

Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.

Science.gov (United States)

Sigurdardóttir, Anna Gudný; Arnórsdóttir, Jóhanna; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Suhre, Karsten; Kristjánsson, Magnús M

2009-03-01

Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin I. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR, Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by approximately 3 degrees C, both in T(50%) and T(m). Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15DDeltaC) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation

Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting.

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Steven Y C Tong

Full Text Available We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR, high-resolution melting (HRM assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T was 32.98 would have an H275Y assay C(T>30. Analysis of the TaqMan C(T values for 609 consecutive clinical samples predicted that 207 (34% of the samples would result in an HRM assay C(T>30 and therefore not be amenable to the HRM assay.The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m against C(T. Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.

Onjisaponin B derived from Radix Polygalae enhances autophagy and accelerates the degradation of mutant α-synuclein and huntingtin in PC-12 cells.

Science.gov (United States)

Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

2013-11-15

Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae--i.e., polygalacic acid, senegenin and onjisaponin B--onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.

Glycogen synthase kinase 3-{beta} phosphorylates novel S/T-P-S/T domains in Notch1 intracellular domain and induces its nuclear localization

Energy Technology Data Exchange (ETDEWEB)

Han, Xiangzi [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Department of Preventive Medicine, Yanbian University College of Medicine, Yanji (China); Ju, Ji-hyun [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of)

2012-06-29

Highlights: Black-Right-Pointing-Pointer Novel S/T-P-S/T domains were identified in NICD. Black-Right-Pointing-Pointer Phosphorylation of NICD on the S/T-P-S/T domains induced nuclear localization. Black-Right-Pointing-Pointer GSK-3{beta} phosphorylated S and T residues in NICD S/T-P-S/T domains. -- Abstract: We identified two S/T-P-S/T domains (2122-2124, 2126-2128) inducing Notch intracellular domain (NICD) nuclear localization. The GFP-NICD (1963-2145) fusion protein deletion mutant without classical NLS was localized in the nucleus like the full length GFP-NICD. However, quadruple substitution mutant (T2122A T2124A S2126A T2128A) showed increased cytoplasmic localization. GSK-3{beta} enhanced nuclear localization and transcriptional activity of WT NICD but not of quadruple substitution mutant. In vitro kinase assays revealed that GSK-3{beta} phosphorylated S and T residues in NICD S/T-P-S/T domains. These results suggest that the novel S/T-P-S/T domain, phosphorylated by GSK-3{beta} is also involved in the nuclear localization of NICD as well as classical NLS.

2018-04-06T22:48:29Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

article/61361 2018-04-06T22:48:29Z bcse:ART Voltammetric determination of chloramphenicol at electrochemically pretreated glassy carbon electrode Alemu, Hailemichael; Department of Chemistry and Chemical Technology, National University ...

Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4

International Nuclear Information System (INIS)

Gauss, P.; Krassa, K.B.; McPheeters, D.S.; Nelson, M.A.; Gold, L.

1987-01-01

The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single zinc-finger sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. The authors have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. The results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression

Stress Marker Signatures in Lesion Mimic Single and Double Mutants Identify a Crucial Leaf Age-Dependent Salicylic Acid Related Defense Signal.

Science.gov (United States)

Kaurilind, Eve; Brosché, Mikael

2017-01-01

Plants are exposed to abiotic and biotic stress conditions throughout their lifespans that activates various defense programs. Programmed cell death (PCD) is an extreme defense strategy the plant uses to manage unfavorable environments as well as during developmentally induced senescence. Here we investigated the role of leaf age on the regulation of defense gene expression in Arabidopsis thaliana. Two lesion mimic mutants with misregulated cell death, catalase2 (cat2) and defense no death1 (dnd1) were used together with several double mutants to dissect signaling pathways regulating defense gene expression associated with cell death and leaf age. PCD marker genes showed leaf age dependent expression, with the highest expression in old leaves. The salicylic acid (SA) biosynthesis mutant salicylic acid induction deficient2 (sid2) had reduced expression of PCD marker genes in the cat2 sid2 double mutant demonstrating the importance of SA biosynthesis in regulation of defense gene expression. While the auxin- and jasmonic acid (JA)- insensitive auxin resistant1 (axr1) double mutant cat2 axr1 also led to decreased expression of PCD markers; the expression of several marker genes for SA signaling (ISOCHORISMATE SYNTHASE 1, PR1 and PR2) were additionally decreased in cat2 axr1 compared to cat2. The reduced expression of these SA markers genes in cat2 axr1 implicates AXR1 as a regulator of SA signaling in addition to its known role in auxin and JA signaling. Overall, the current study reinforces the important role of SA signaling in regulation of leaf age-related transcript signatures.

Definitive Radiotherapy for T1–2 Hypopharyngeal Cancer: A Single-Institution Experience

International Nuclear Information System (INIS)

Nakajima, Aya; Nishiyama, Kinji; Morimoto, Masahiro; Nakamura, Satoaki; Suzuki, Osamu; Kawaguchi, Yoshifumi; Miyagi, Ken; Fujii, Takashi; Yoshino, Kunitoshi

2012-01-01

Purpose: To analyze the outcome in T1–2 hypopharyngeal cancer (HPC) patients treated with definitive radiotherapy (RT). Patients and Methods: A total of 103 patients with T1–2 hypopharyngeal squamous cell carcinoma treated with radical RT between March 2000 and June 2008 at our institution were analyzed. Pre-RT neck dissection (ND) was performed in 26 patients with advanced neck disease. Chemotherapy was used concurrently with RT in 14 patients. Sixty patients were associated with synchronous or metachronous malignancies. The median follow-up for surviving patients was 41 months. Results: The 3-year overall and cause-specific survival rates were 70% and 79%, respectively. The 3-year local control rates were 87% for T1 and 83% for T2 disease. The ultimate local control rate was 89%, including 7 patients in whom salvage was successful. The ultimate local control rate with laryngeal preservation was 82%. Tumors of the medial wall of the pyriform sinus tended to have lower control rates compared with tumors of the lateral or posterior pharyngeal wall. Among patients with N2b–3 disease, the 3-year regional control rates were 74% for patients with pre-RT ND and 40% for patients without ND. The 3-year locoregional control rates were as follows: Stage I, 100%; Stage II, 84%; Stage III, 67%; Stage IVA, 43%; Stage IVB, 67%. Forty-two patients developed disease recurrence, with 29 (70%) patients developing recurrence within the first year. Of the 103 patients, 6 developed late complications higher than or equal to Grade 3. Conclusions: Definitive RT accomplished a satisfactory local control rate and contributed to organ preservation.

Morphogenesis of bacteriophage phi29 of Bacillus subtilis: cleavage and assembly of the neck appendage protein

International Nuclear Information System (INIS)

Tosi, M.E.; Reilly, B.E.; Anderson, D.L.

1975-01-01

Each of the 12 neck appendages of the Bacillus subtilis bacteriophage phi 29 consists of a single protein molecular weight of about 75,000, and on the mature virion the appendages are assembled to the lower of two collars. The appendage protein is cleaved from a percursor protein, P(J), with a molecular weight of about 88,000. This cleavage is independent of neck assembly, occurring during infection by mutants that cannot synthesize the proteins of the upper and lower collars of the neck. The cleaved form of the appendage protein is efficiently complemented in vitro to particles lacking appendages. Thus, cleavage of the appendage precursor protein apparently does not occur in situ on the maturing virus

2018-03-29T05:10:46Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

article/84944 2018-03-29T05:10:46Z zjer:ART A Comparative Analysis of the Causes of Primary School Dropout in an Urban and Commercial Farm Setting in Norton Area Muchenje, F.; University of Zimbabwe This study sought to compare the ...

Global translational impacts of the loss of the tRNA modification t6A in yeast

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Patrick C. Thiaville

2015-12-01

Full Text Available The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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Carlos A Santiviago

2009-07-01

Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

Characterization of T-DNA insertion mutants with decreased virulence in the entomopathogenic fungus Beauveria bassiana JEF-007.

Science.gov (United States)

Kim, Sihyeon; Lee, Se Jin; Nai, Yu-Shin; Yu, Jeong Seon; Lee, Mi Rong; Yang, Yi-Ting; Kim, Jae Su

2016-10-01

The bean bug, Riptortus pedestris, is a major agricultural pest that reduces crop quality and value. Chemical pesticides have contributed to pest management, but resistance to these chemicals has significantly limited their use. Alternative strategies with different modes of action, such as entomopathogenic fungi, are therefore of great interest. Herein, we explored how entomopathogenic fungi can potentially be used to control the bean bug and focused on identifying virulence-related genes. Beauveria bassiana (JEF isolates) were assayed against bean bugs under laboratory conditions. One isolate, JEF-007, showed >80 % virulence by both spray and contact exposure methods. Agrobacterium tumefaciens-mediated transformation (AtMT) of JEF-007 generated 249 random transformants, two of which (B1-06 and C1-49) showed significantly reduced virulence against Tenebrio molitor and R. pedestris immatures. Both species were used for rapid screening of virulence-reduced mutants. The two transformants had different morphologies, conidial production, and thermotolerance than the wild type. To determine the localization of the randomly inserted T-DNA, thermal asymmetric interlaced (TAIL) PCR was conducted and analysis of the two clones found multiple T-DNA insertions (two in B1-06 and three in C1-49). Genes encoding complex I intermediate-associated protein 30 (CIA30) and the autophagy protein (Atg22) were possibly disrupted by the T-DNA insertion and might be involved in the virulence. This work provides a strong platform for future functional genetic studies of bean bug-pathogenic B. bassiana. The genes putatively involved in fungal virulence should be experimentally validated by knockdown in future studies.

Condensin II mutation causes T-cell lymphoma through tissue-specific genome instability

Science.gov (United States)

Woodward, Jessica; Taylor, Gillian C.; Soares, Dinesh C.; Boyle, Shelagh; Sie, Daoud; Read, David; Chathoth, Keerthi; Vukovic, Milica; Tarrats, Nuria; Jamieson, David; Campbell, Kirsteen J.; Blyth, Karen; Acosta, Juan Carlos; Ylstra, Bauke; Arends, Mark J.; Kranc, Kamil R.; Jackson, Andrew P.; Bickmore, Wendy A.

2016-01-01

Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2nes) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4+CD8+ T-cell stage from which tumors initiate. Premalignant CD4+CD8+ T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis. PMID:27737961

Design and analysis of single- ended robust low power 8T SRAM cell

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Gupta Neha

2016-01-01

Full Text Available This paper is based on the observation of 8T single ended static random access memory (SRAM and two techniques for reducing the sub threshold leakage current, power consumption are examined. In the first technique, effective supply voltage and ground node voltages are changed using a dynamic variable voltage level technique(VVL. In the second technique power supply is scaled down. This 8T SRAM cell uses one word line, two bitlinesand a transmission gate. Simulations and analytical results show that when the two techniques combine the new SRAM cell has correct read and write operation and also the cell contains 55.6% less leakage and the dynamic power is 98.8% less than the 8T single ended SRAM cell. Simulations are performed using cadence virtuoso tool at 45nm technology.

Molecular mechanisms associated with leukemic transformation of MPL-mutant myeloproliferative neoplasms

DEFF Research Database (Denmark)

Beer, Philip A; Ortmann, Christina A; Stegelmann, Frank

2010-01-01

Somatic activating mutations in MPL, the thrombopoietin receptor, occur in the myeloproliferative neoplasms, although virtually nothing is known about their role in evolution to acute myeloid leukemia. In this study, the MPL T487A mutation, identified in de novo acute myeloid leukemia......, was not detected in 172 patients with a myeloproliferative neoplasm. In patients with a prior MPL W515L-mutant myeloproliferative neoplasm, leukemic transformation was accompanied by MPL-mutant leukemic blasts, was seen in the absence of prior cytoreductive therapy and often involved loss of wild-type MPL...

Search for single production of a vector-like T quark decaying into a top quark and a Higgs boson

Energy Technology Data Exchange (ETDEWEB)

Gonzalez, Daniel; Marchesini, Ivan; Nowatschin, Dominik; Schmidt, Alexander; Schumann, Svenja; Tholen, Heiner; Usai, Emanuele [Universitaet Hamburg (Germany). Institut fuer Experimentalphysik

2016-07-01

We search for singly produced vector-like top quark partners (T) in pp-collisions at √(s)=13 TeV with the CMS experiment. Several BSM models, such as composite Higgs and extra dimensions, predict vector-like quarks to be accessible at the LHC. At 13 TeV, single production of vector-like quarks might be enhanced over pair production, depending on the coupling parameters for the individual interactions. In this analysis, we target the decay of the vector-like heavy T quark into a Higgs boson and a top quark, where the top quark decay includes a lepton. Higgs-boson candidates are reconstructed using new methods to resolve the substructure of boosted jets and top-quark candidates are formed by combining leptons, missing transverse energy and jets. With the top-quark and Higgs-boson candidates, we aim for the complete reconstruction of the four-vector of the new particle in question. The largest fraction of the background is contributed through the top-quark pair production process. First results on the search for single vector-like top partners at 13 TeV are presented.

Mutant matrix metalloproteinase-9 reduces postoperative peritoneal adhesions in rats.

Science.gov (United States)

Atta, Hussein; El-Rehany, Mahmoud; Roeb, Elke; Abdel-Ghany, Hend; Ramzy, Maggie; Gaber, Shereen

2016-02-01

Postoperative peritoneal adhesions continue to be a major source of morbidity and occasional mortality. Studies have shown that matrix metalloproteinase-9 (MMP-9) levels are decreased postoperatively which may limits matrix degradation and participate in the development of peritoneal adhesions. In this proof-of-principle study, we evaluated the effect of gene therapy with catalytically inactive mutant MMP-9 on postoperative peritoneal adhesions in rats. Adenovirus encoding mutant MMP-9 (Ad-mMMP-9) or saline was instilled in the peritoneal cavity after cecal and parietal peritoneal injury in rats. Expression of mutant MMP-9 transcript was verified by sequencing. Adenovirus E4 gene expression, adhesion scores, MMP-9, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1 (TGF-β1) expression were evaluated at sacrifice one week after treatment. Both mutant MMP-9 transcripts and adenovirus E4 gene were expressed in Ad-mMMP-9 treated adhesions. Adhesions severity decreased significantly (p = 0.036) in the Ad-mMMP-9-treated compared with saline-treated adhesions. Expression of MMP-9 mRNA and protein were elevated (p = 0.001 and p = 0.029, respectively) in the Ad-mMMP-9-treated adhesions compared with saline-treated adhesions. While tPA levels were increased (p = 0.02) in Ad-mMMP-9 treated adhesions compared with saline-treated adhesions, TGF-β1 and PAI-1 levels were decreased (p = 0.017 and p = 0.042, respectively). No difference in mortality were found between groups (p = 0.64). Mutant MMP-9 gene therapy effectively transduced peritoneal adhesions resulting in reduction of severity of primary peritoneal adhesions. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

Evaluation of yield and N2 fixation of mutant lines of groundnut in Malaysia

International Nuclear Information System (INIS)

Rusli, I.; Harun, A.R.; Rahman, K.A.; Shamsuddin, S.; Rahim, K.A.; Danso, S.K.A.

1998-01-01

The 15 N-dilution technique was used to evaluate N 2 fixation in groundnut (Arachis hypogaea L.) in three field trials of cultivars Matjan and V-13 (parents), their selected mutant lines, and a other local and foreign genotypes. Matjan mutant MJ/40/42 consistently produced the highest pod yields, at above 4 t ha -1 , 14-22% higher yields than the parent. In contrast, none of the V-13 mutants had consistently better yields than the parent. The mutant lines did not show consistent agronomic performance from year to year. Total dry matter yield did not correlate with pod yield, and pod yield did not correlate with amount of N fixed

Studies on induced partially resistant mutants of barley against powdery mildew

International Nuclear Information System (INIS)

Roebbelen, G.; Abdel-Hafez, A.G.; Reinhold, M.; Kwon, H.J.; Neuhaus-Steinmetz, J.P.; Heun, M.

1983-01-01

After mutagenic seed treatment of three partially resistant cultivars of spring barley with EMS and NaN 3 , 45 mutants in a first and 16 in a second experiment were selected in the M 2 -M 4 generations. The screening was done alternatively under natural infection in the field or controlled infection with a single pathotype in the greenhouse. These mutants exhibited a higher resistance and a higher susceptibility, respectively, than the initial cultivars Asse, Bomi and Vada. Some mutants expressed their altered resistance behaviour particularly during certain stages of development. High-level resistance was conditioned by mutation in the ml-o locus in three cases. For several Bomi mutants pathotype specificity with and without reversed ranking was proven as well as pathotype non-specificity in comparison with the reaction of the original cultivar. In 14 cases studied the inheritance of the involved mutants was monogenic recessive. The laevigatum locus responsible for the intermediate mildew resistance of Bomi was not affected by the mutations. Detection of groups of allelic mutants showed that there are at least two regions in the barley genome in which mutations for mildew resistance can occur rather frequently. In total, the past ten years of this mutation research have given convincing evidence that the strategies of mutant screening applied have yielded promising new material both for breeding and for progress in basic understanding of host-pathogen interactions. (author)

Exogenous Expressions of FTO Wild-Type and R316Q Mutant Proteins Caused an Increase in HNRPK Levels in 3T3-L1 Cells as Demonstrated by DIGE Analysis

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Nil Guzel

2017-01-01

Full Text Available Fat mass and obesity-associated protein is an enzyme that oxidatively demethylates DNA. Although there are numerous studies regarding the catalytic function of FTO, the overall existence or absence of FTO on cellular proteome has not been investigated. This study investigated the changes in the soluble proteome of 3T3-L1 cells upon expression of the WT and the mutant (R316Q FTO proteins. Protein extracts prepared from 3T3-L1 cells expressing either the WT or the mutant FTO proteins were used in DIGE experiments. Analysis of the data revealed the number of spots matched to every member and there were 350 ± 20 spots with 30.5% overall mean coefficient of variation. Eleven regulated protein spots were excised from the gels and identified by MALDI-TOF/TOF. One of the identified proteins was heterogeneous nuclear ribonucleoprotein K, which displayed more than 2.6- and 3.7-fold increases in its abundance in the WT and the mutant FTO expressing cells, respectively. Western blot analysis validated these observations. This is the first study revealing the presence of a parallel increase in expressions of FTO and HNRNPK proteins. This increase may codictate the metabolic changes occurring in the cell and may attribute a significance to HNRNPK in FTO-associated transformations.

Enhanced longevity in tau mutant Syrian hamsters, Mesocricetus auratus

NARCIS (Netherlands)

Oklejewicz, Malgorzata; Daan, Serge

The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span

Treatment of visuospatial neglect with biparietal tDCS and cognitive training: a single-case study