scholarly journals VALINE-RESISTANCE, A POTENTIAL MARKER IN PLANT CELL GENETICS. II. OPTIMIZATION OF UV MUTAGENESIS AND SELECTION OF VALINE-RESISTANT COLONIES DERIVED FROM TOBACCO MESOPHYLL PROTOPLASTS

Genetics ◽  
1985 ◽  
Vol 109 (2) ◽  
pp. 409-425 ◽  
Author(s):  
M A Grandbastien ◽  
J P Bourgin ◽  
M Caboche
Keyword(s):  
Spontaneous Mutation ◽  
Wild Type Plant ◽  
Acid Uptake ◽  
Wild Type ◽  
Induced Mutations ◽  
Mutant Plant ◽  
Potential Marker ◽  
Valine Resistance ◽  
Selection Of ◽  
Haploid Plants

ABSTRACT The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.—Using cell densities of approximately 104 cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Valr-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10-6 per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10-5 to 5 × 10-4 resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F1 double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive, resistant and two intermediary resistant types believed to result from the presence of a mutant allele at only one of the two loci involved. The frequencies of UV-induced mutations in cells derived from haploid plants of one of the intermediary types were compared to those observed in wild-type cells. The results are considered in light of the amphidiploid structure of the tobacco genome.

scholarly journals A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression

2019 ◽  
Author(s):  
William A. Ng ◽  
Andrew Ma ◽  
Molly Chen ◽  
Bruce H. Reed
Keyword(s):  
Gene Expression ◽  
Screening Strategy ◽  
Loss Of Function ◽  
Wild Type ◽  
Induced Mutations ◽  
Allelic Series ◽  
Lethal Mutations ◽  
F1 Progeny ◽  
Genetic Cross ◽  
Selection Of

AbstractWe have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.

VALINE-RESISTANCE, A POTENTIAL MARKER IN PLANT CELL GENETICS. I. DISTINCTION BETWEEN TWO TYPES OF VALINE-RESISTANT TOBACCO MUTANTS ISOLATED FROM PROTOPLAST-DERIVED CELLS

Genetics ◽  
1985 ◽  
Vol 109 (2) ◽  
pp. 393-407
Author(s):  
J P Bourgin ◽  
J Goujaud ◽  
C Missonier ◽  
C Pethe
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plant Cell ◽  
Biochemical Characterization ◽  
Acid Uptake ◽  
Recessive Character ◽  
Potential Marker ◽  
Valine Resistance ◽  
Recessive Mutant ◽  
Mutant Lines

ABSTRACT In previous experiments, seven lines of valine-resistant plants were regenerated from protoplast-derived haploid tobacco mesophyll cells which had been UV mutagenized and submitted to selection by toxic concentrations of valine. In this study we described the transmission of valine-resistance to progeny and a preliminary phenotypical and biochemical characterization of the resistant plants.—Two types were thus distinguished among the seven mutant lines. Valine-resistance of the mutants of the first type (three lines) was transmitted as a single Mendelian dominant character (Vr1), whereas valine-resistance of the second type (four lines) was transmitted as a digenic recessive character (vr2 and vr3). Allelism tests revealed that the four recessive mutant lines yielded resistant progeny when intercrossed and, therefore, bear recessive mutant alleles at the same two unlinked loci.—When cultured at a density of 100 cell/ml, protoplast-derived cells of mutants of the first type had a low level of resistance to valine, whereas protoplast-derived cells of mutants of the second type displayed a high level of resistance to valine and to other amino acids.—According to the results of 14 C-labelled amino acid uptake experiments, the amino acid resistance of mutants of the second type, but not valine-resistance of the first type, could be accounted for by reduced uptake of several amino acids. Possible uses of valine-resistance as a marker in plant cell genetics are discussed.

scholarly journals A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression

2020 ◽  
Vol 10 (6) ◽  
pp. 1893-1901
Author(s):  
William A. Ng ◽  
Andrew Ma ◽  
Molly Chen ◽  
Bruce H. Reed
Keyword(s):  
Gene Expression ◽  
Screening Strategy ◽  
Loss Of Function ◽  
Wild Type ◽  
Induced Mutations ◽  
Allelic Series ◽  
Lethal Mutations ◽  
F1 Progeny ◽  
Genetic Cross ◽  
Selection Of

We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.

scholarly journals Putative transmembrane transporter modulates higher-level aggression in Drosophila

2017 ◽  
Vol 114 (9) ◽  
pp. 2373-2378 ◽  
Author(s):  
Budhaditya Chowdhury ◽  
Yick-Bun Chan ◽  
Edward A. Kravitz
Keyword(s):  
Causal Effect ◽  
Nerve Terminals ◽  
Rna Seq ◽  
Wild Type ◽  
Temperature Dependent ◽  
Number Of Genes ◽  
Interesting Family ◽  
Solute Carrier ◽  
Transmembrane Transporter ◽  
Selection Of

By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems.

scholarly journals Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the mdx-4cv Model of Duchenne Muscular Dystrophy

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 595
Author(s):  
Stephen Gargan ◽  
Paul Dowling ◽  
Margit Zweyer ◽  
Jens Reimann ◽  
Michael Henry ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Biochemical Properties ◽  
Mass Spectrometric ◽  
Cellular Stress Response ◽  
Diaphragm Muscle ◽  
Spectrometric Analysis ◽  
Mitochondrial Enzymes ◽  
Wild Type ◽  
Potential Marker

Extraocular muscles (EOMs) represent a specialized type of contractile tissue with unique cellular, physiological, and biochemical properties. In Duchenne muscular dystrophy, EOMs stay functionally unaffected in the course of disease progression. Therefore, it was of interest to determine their proteomic profile in dystrophinopathy. The proteomic survey of wild type mice and the dystrophic mdx-4cv model revealed a broad spectrum of sarcomere-associated proteoforms, including components of the thick filament, thin filament, M-band and Z-disk, as well as a variety of muscle-specific markers. Interestingly, the mass spectrometric analysis revealed unusual expression levels of contractile proteins, especially isoforms of myosin heavy chain. As compared to diaphragm muscle, both proteomics and immunoblotting established isoform MyHC14 as a new potential marker in wild type EOMs, in addition to the previously identified isoforms MyHC13 and MyHC15. Comparative proteomics was employed to establish alterations in the protein expression profile between normal EOMs and dystrophin-lacking EOMs. The analysis of mdx-4cv EOMs identified elevated levels of glycolytic enzymes and molecular chaperones, as well as decreases in mitochondrial enzymes. These findings suggest a process of adaptation in dystrophin-deficient EOMs via a bioenergetic shift to more glycolytic metabolism, as well as an efficient cellular stress response in EOMs in dystrophinopathy.

scholarly journals CAND2/PMTR1 Is Required for Melatonin-Conferred Osmotic Stress Tolerance in Arabidopsis

2021 ◽  
Vol 22 (8) ◽  
pp. 4014
Author(s):  
Lin-Feng Wang ◽  
Ting-Ting Li ◽  
Yu Zhang ◽  
Jia-Xing Guo ◽  
Kai-Kai Lu ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Stress Tolerance ◽  
Wild Type Plant ◽  
Wild Type ◽  
Melatonin Receptor ◽  
Ros Scavenging ◽  
Exogenous Melatonin ◽  
Osmotic Stress Tolerance ◽  
Osmotic Stress Response ◽  
In The Wild

Osmotic stress severely inhibits plant growth and development, causing huge loss of crop quality and quantity worldwide. Melatonin is an important signaling molecule that generally confers plant increased tolerance to various environmental stresses, however, whether and how melatonin participates in plant osmotic stress response remain elusive. Here, we report that melatonin enhances plant osmotic stress tolerance through increasing ROS-scavenging ability, and melatonin receptor CAND2 plays a key role in melatonin-mediated plant response to osmotic stress. Upon osmotic stress treatment, the expression of melatonin biosynthetic genes including SNAT1, COMT1, and ASMT1 and the accumulation of melatonin are increased in the wild-type plants. The snat1 mutant is defective in osmotic stress-induced melatonin accumulation and thus sensitive to osmotic stress, while exogenous melatonin enhances the tolerance of the wild-type plant and rescues the sensitivity of the snat1 mutant to osmotic stress by upregulating the expression and activity of catalase and superoxide dismutase to repress H2O2 accumulation. Further study showed that the melatonin receptor mutant cand2 exhibits reduced osmotic stress tolerance with increased ROS accumulation, but exogenous melatonin cannot revert its osmotic stress phenotype. Together, our study reveals that CADN2 functions necessarily in melatonin-conferred osmotic stress tolerance by activating ROS-scavenging ability in Arabidopsis.

Whole-Genome Effects of Ethyl Methanesulfonate-Induced Mutation on Nine Quantitative Traits in Outbred Drosophila melanogaster

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1257-1265 ◽  
Author(s):  
Hsiao-Pei Yang ◽  
Ana Y Tanikawa ◽  
Wayne A Van Voorhies ◽  
Joana C Silva ◽  
Alexey S Kondrashov
Keyword(s):  
Drosophila Melanogaster ◽  
Quantitative Traits ◽  
Spontaneous Mutation ◽  
Developmental Time ◽  
Ethyl Methanesulfonate ◽  
Induced Mutations ◽  
Mean Values ◽  
Eye Color ◽  
Recessive Mutations ◽  
The Mean

Abstract We induced mutations in Drosophila melanogaster males by treating them with 21.2 mm ethyl methanesulfonate (EMS). Nine quantitative traits (developmental time, viability, fecundity, longevity, metabolic rate, motility, body weight, and abdominal and sternopleural bristle numbers) were measured in outbred heterozygous F3 (viability) or F2 (all other traits) offspring from the treated males. The mean values of the first four traits, which are all directly related to the life history, were substantially affected by EMS mutagenesis: the developmental time increased while viability, fecundity, and longevity declined. In contrast, the mean values of the other five traits were not significantly affected. Rates of recessive X-linked lethals and of recessive mutations at several loci affecting eye color imply that our EMS treatment was equivalent to ∼100 generations of spontaneous mutation. If so, our data imply that one generation of spontaneous mutation increases the developmental time by 0.09% at 20° and by 0.04% at 25°, and reduces viability under harsh conditions, fecundity, and longevity by 1.35, 0.21, and 0.08%, respectively. Comparison of flies with none, one, and two grandfathers (or greatgrandfathers, in the case of viability) treated with EMS did not reveal any significant epistasis among the induced mutations.

scholarly journals Mutants of downy mildew resistance in Lactuca sativa (lettuce).

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 867-874
Author(s):  
P A Okubara ◽  
P A Anderson ◽  
O E Ochoa ◽  
R W Michelmore
Keyword(s):  
Molecular Marker ◽  
Downy Mildew ◽  
Genomic Dna ◽  
Spontaneous Mutation ◽  
Multigene Families ◽  
Downy Mildew Resistance ◽  
Bremia Lactucae ◽  
Wild Type ◽  
Mildew Resistance ◽  
Recessive Mutations

Abstract As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

scholarly journals A microfluidic device enabling drug resistance analysis of leukemia cells via coupled dielectrophoretic detection and impedimetric counting

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yağmur Demircan Yalçın ◽  
Taylan Berkin Töral ◽  
Sertan Sukas ◽  
Ender Yıldırım ◽  
Özge Zorlu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Drug Resistant ◽  
Wild Type ◽  
Gene Expressions ◽  
Before And After ◽  
The Difference ◽  
Selection Of ◽  
Resistant Cells

AbstractWe report the development of a lab-on-a-chip system, that facilitates coupled dielectrophoretic detection (DEP-D) and impedimetric counting (IM-C), for investigating drug resistance in K562 and CCRF-CEM leukemia cells without (immuno) labeling. Two IM-C units were placed upstream and downstream of the DEP-D unit for enumeration, respectively, before and after the cells were treated in DEP-D unit, where the difference in cell count gave the total number of trapped cells based on their DEP characteristics. Conductivity of the running buffer was matched the conductivity of cytoplasm of wild type K562 and CCRF-CEM cells. Results showed that DEP responses of drug resistant and wild type K562 cells were statistically discriminative (at p = 0.05 level) at 200 mS/m buffer conductivity and at 8.6 MHz working frequency of DEP-D unit. For CCRF-CEM cells, conductivity and frequency values were 160 mS/m and 6.2 MHz, respectively. Our approach enabled discrimination of resistant cells in a group by setting up a threshold provided by the conductivity of running buffer. Subsequent selection of drug resistant cells can be applied to investigate variations in gene expressions and occurrence of mutations related to drug resistance.

scholarly journals Degradation of trinitrotoluene by transgenic nitroreductase in Arabidopsis plants

2018 ◽  
Vol 64 (No. 8) ◽  
pp. 379-385 ◽  
Author(s):  
Zhu Bo ◽  
Han Hongjuan ◽  
Fu Xiaoyan ◽  
Li Zhenjun ◽  
Gao Jianjie ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Transgenic Arabidopsis ◽  
Environmental Pollutant ◽  
Specific Rate ◽  
Wild Type Plant ◽  
Wild Type ◽  
Fresh Weight ◽  
Human Carcinogen ◽  
Specific Rate Constant ◽  
Nitroreductase Activity

The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. TNT is toxic to many organisms, it is known to be a potential human carcinogen, and is persistent in the environment. This study presents a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of NAD(P)H-flavin nitroreductase (NFSB) from the Sulfurimonas denitrificans DSM1251. The resulting transgenic Arabidopsis plants demonstrated significantly enhanced TNT tolerance and a strikingly higher capacity to remove TNT from their media. The highest specific rate constant of TNT disappearance rate was 1.219 and 2.297 mL/g fresh weight/h for wild type and transgenic plants, respectively. Meanwhile, the nitroreductase activity in transgenic plant was higher than wild type plant. All this indicates that transgenic plants show significantly enhanced tolerances to TNT; transgenic plants also exhibit strikingly higher capabilities of removing TNT from their media and high efficiencies of transformation.

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