The induction and measurement of DNA damage in nuclei of plant tissues is a new area of study with the alkaline single cell gel electrophoresis/comet assay. Methods to isolate plant cell nuclei cause high levels of DNA damage which are detected by the comet assay. We developed a method to isolate nuclei from leaf tissue of Nicotiana tabacum (a1+/a1; a2+/a2) in a modified Sörensen buffer that resulted in constant, low tail moment values for the negative controls. After treating intact tobacco plants with 1-8 mM ethyl methanesulfonate (EMS) we obtained a direct concentration-response with an average median tail moment of 65.9+/-4.4 micro(m) for plants exposed to the highest EMS concentration as compared to the median control tail moment value of 4.1+/-0.8. We found that the highest resolution was obtained with electrophoretic conditions of 0.74 V/cm at 300 mA for 20 min. Multiple leaves could be analyzed per plant within each treatment group and the tail moments were not significantly different. Tobacco seedlings were treated with EMS in the same manner as used for the comet assay and mutations were induced in the leaf primordia. The mean mutant frequency for the control was 1.46+/-0.20 mutant sectors/leaf. The mutant frequency increased in a concentration dependent manner; the mutant frequency induced by 8 mM EMS was 37.89+/-2.37 mutant sectors/leaf. The comet tail moment values and the leaf mutant frequency were highly correlated (r=0.98). The genetic response factor was calculated by the ratio of the difference in the response within the linear portion of each concentration-response curve divided by the slope of the curve. The genetic response factor for the tail moment was 7.82 while the value for mutation induction was 7.76. In this paper we describe a sensitive method with high resolution to apply the alkaline comet assay to plant leaves. The comet assay response was compared to that of induced point mutation. With this sensitive method for nuclei isolation from plant leaves, the alkaline SCGE assay could be incorporated into in situ plant environmental monitoring.

• MeSH
• Point Mutation * MeSH
• DNA, Plant drug effects   genetics   isolation & purification   MeSH
• Electrophoresis, Agar Gel methods   MeSH
• Ethyl Methanesulfonate toxicity   MeSH
• Plants, Toxic * MeSH
• Plant Leaves MeSH
• Mutagens toxicity   MeSH
• DNA Damage * MeSH
• Nicotiana cytology   drug effects   genetics   MeSH
• Mutagenicity Tests MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• DNA, Plant MeSH
• Ethyl Methanesulfonate MeSH
• Mutagens MeSH

BACKGROUND AND AIMS: The actin cytoskeleton forms a dynamic network in plant cells. A single-point mutation in the DER1 (deformed root hairs1) locus located in the sequence of ACTIN2, a gene for major actin in vegetative tissues of Arabidopsis thaliana, leads to impaired root hair development (Ringli C, Baumberger N, Diet A, Frey B, Keller B. 2002. ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis. Plant Physiology129: 1464-1472). Only root hair phenotypes have been described so far in der1 mutants, but here we demonstrate obvious aberrations in the organization of the actin cytoskeleton and overall plant development. METHODS: Organization of the actin cytoskeleton in epidermal cells of cotyledons, hypocotyls and roots was studied qualitatively and quantitatively by live-cell imaging of transgenic lines carrying the GFP-FABD2 fusion protein and in fixed cells after phalloidin labelling. Patterns of root growth were characterized by FM4-64 vital staining, light-sheet microscopy imaging and microtubule immunolabelling. Plant phenotyping included analyses of germination, root growth and plant biomass. KEY RESULTS: Speed of germination, plant fresh weight and total leaf area were significantly reduced in the der1-3 mutant in comparison with the C24 wild-type. Actin filaments in root, hypocotyl and cotyledon epidermal cells of the der1-3 mutant were shorter, thinner and arranged in more random orientations, while actin bundles were shorter and had altered orientations. The wavy pattern of root growth in der1-3 mutant was connected with higher frequencies of shifted cell division planes (CDPs) in root cells, which was consistent with the shifted positioning of microtubule-based preprophase bands and phragmoplasts. The organization of cortical microtubules in the root cells of the der1-3 mutant, however, was not altered. CONCLUSIONS: Root growth rate of the der1-3 mutant is not reduced, but changes in the actin cytoskeleton organization can induce a wavy root growth pattern through deregulation of CDP orientation. The results suggest that the der1-3 mutation in the ACT2 gene does not influence solely root hair formation process, but also has more general effects on the actin cytoskeleton, plant growth and development.

• MeSH
• Actins genetics   metabolism   MeSH
• Arabidopsis genetics   growth & development   metabolism   MeSH
• Plant Roots growth & development   metabolism   MeSH
• Mutation * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACT2 protein, Arabidopsis MeSH Browser
• Actins MeSH
• Arabidopsis Proteins MeSH

We present a fast detection of M467T, the major mutation causing cystinuria, by capillary electrophoresis version of single-strand conformation polymorphism (SSCP). The DNA fragment (317 bp) carrying the point mutation was amplified by polymerase chain reaction (PCR) on the exon 8 of the SLC3A1 gene, which encodes for the transmembrane glycoprotein rBAT, a part of the active cystine and dibasic amino acids transporter. The complementary strands of the fragment were labeled by fluorescein and TAMRA, respectively. Thus, the electromigration of both strands was recorded independently as a laser-induced fluorescence (LIF) signal, what enabled an effective optimization of separation conditions. The injected sample was denatured by immersing the inlet of the separation capillary into a vial with 0.1 M solution of NaOH prior to analysis. Under optimum conditions, the SSCP analysis in poly(vinyl alcohol) (PVA)-coated silica-fused capillary of an effective length of 15 cm, filled with 4% linear polyacrylamide (LPA) solution, was accomplished in approximately 6 min. The experimentally observed mobility shifts of single-stranded DNA (ssDNA) fragments were compared to the appearance of their calculated two-dimensional conformations using Version 3.0 of MFOLD software. The number of nucleotides involved in the duplex regions of theoretical structures correlates well with their real migration order in the sieving medium.

• MeSH
• Point Mutation MeSH
• Cystinuria diagnosis   genetics   MeSH
• Electrophoresis, Capillary methods   MeSH
• Exons MeSH
• Humans MeSH
• Membrane Glycoproteins chemistry   genetics   MeSH
• Mutation, Missense * MeSH
• Models, Molecular MeSH
• Polymerase Chain Reaction MeSH
• Polymorphism, Single-Stranded Conformational * MeSH
• Protein Structure, Secondary MeSH
• Carrier Proteins chemistry   genetics   MeSH
• Amino Acid Transport Systems, Basic * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Glycoproteins MeSH
• SLC7A9 protein, human MeSH Browser
• Carrier Proteins MeSH
• Amino Acid Transport Systems, Basic * MeSH

STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.

• MeSH
• Point Mutation * MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Membrane Proteins chemistry   genetics   metabolism   MeSH
• Molecular Structure MeSH
• Nucleotides, Cyclic chemistry   metabolism   MeSH
• Protein Domains MeSH
• Binding Sites MeSH
• Hydrogen Bonding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Nucleotides, Cyclic MeSH
• STING1 protein, human MeSH Browser

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

• Keywords
• TUPS, cytochrome c, intramolecular electron transfer, time-resolved spectroscopy, triplet excited state,
• MeSH
• Point Mutation MeSH
• Cysteine chemistry   genetics   MeSH
• Cytochromes c chemistry   genetics   MeSH
• Heme chemistry   MeSH
• Kinetics MeSH
• Horses MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Oxidation-Reduction MeSH
• Pyrenes chemistry   MeSH
• Electron Transport MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cysteine MeSH
• Cytochromes c MeSH
• Heme MeSH
• Pyrenes MeSH
• thiouredopyrenetrisulfonate MeSH Browser

Spinal muscular atrophy (SMA) is caused by homozygous deletion of the SMN1 gene in approximately 96% of cases. Four percent of SMA patients have a combination of the deletion or conversion on one allele and an intragenic mutation on the second one. We performed analysis of point mutations in a set of our patients with suspicion of SMA and without homozygous deletion of the SMN1 gene. A quantitative test determining SMN1 copy number (using real-time PCR and/or MLPA analysis) was performed in 301 patients and only 1 SMN1 copy was detected in 14 of them. When these 14 patients were screened for the presence of point mutations we identified 6 mutations, p.Y272C (in three patients) and p.T274I, p.I33IfsX6, and p.A188S (each in one case). The mutations p.I33IfsX6 and p.A188S were found in two SMAI patients and were not detected previously. Further, evaluation of the relationship between mutation type, copy number of the SMN2 gene and clinical findings was performed. Among our SMA patients with a SMN1 homozygous deletion, we found a family with two patients: the son with SMAII possesses 3 SMN2 copies and the nearly asymptomatic father has a homozygous deletion of SMN1 exon 7 and carries 4 SMN2 copies. Generally, our results illustrate that an increased SMN2 gene copy number is associated with a milder SMA phenotype.

• MeSH
• Point Mutation genetics   MeSH
• Gene Deletion MeSH
• DNA genetics   MeSH
• Adult MeSH
• Exons genetics   MeSH
• Phenotype MeSH
• Gene Dosage * MeSH
• Homozygote MeSH
• Humans MeSH
• Adolescent MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Child, Preschool MeSH
• Survival of Motor Neuron 1 Protein MeSH
• Survival of Motor Neuron 2 Protein MeSH
• Cyclic AMP Response Element-Binding Protein genetics   MeSH
• SMN Complex Proteins MeSH
• Nerve Tissue Proteins genetics   MeSH
• RNA-Binding Proteins genetics   MeSH
• Muscular Atrophy, Spinal genetics   MeSH
• Nucleic Acid Amplification Techniques MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Survival of Motor Neuron 1 Protein MeSH
• Survival of Motor Neuron 2 Protein MeSH
• Cyclic AMP Response Element-Binding Protein MeSH
• SMN Complex Proteins MeSH
• Nerve Tissue Proteins MeSH
• RNA-Binding Proteins MeSH
• SMN1 protein, human MeSH Browser
• SMN2 protein, human MeSH Browser

• MeSH
• Leukemia, Myeloid, Acute * diagnosis   genetics   MeSH
• Point Mutation MeSH
• GTP Phosphohydrolases genetics   MeSH
• Humans MeSH
• Membrane Proteins genetics   MeSH
• Mutation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• GTP Phosphohydrolases MeSH
• Membrane Proteins MeSH
• NRAS protein, human MeSH Browser

Several tools, differing in their technical and practical parameters, are available for the detection of point mutations as well as small deletions and insertions. In this article, a dictionary featuring over fifty methods for detection of mutation is presented. The distinguishing principle for each method is briefly explained. Sorting of and discussion on the methods give the reader a brief introduction to the field of genotyping.

• MeSH
• Point Mutation * MeSH
• Genotype MeSH
• Polymorphism, Single Nucleotide * MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Dictionary MeSH

BACKGROUND: Familial hemiplegic migraine (FHM) is a rare autosomal dominant subtype of migraine with aura. Missense mutations in the chromosome 19 CACNA1A calcium channel gene have been found in approximately half of the families. The T666M mutation, replacing a threonine by a methionine at residue number 666, is the most frequent mutation, reported in 14 independent FHM families; other mutations have so far been described in only 1 or 2 families each. The clinical features of T666M families have been reported, but the course is unknown. OBJECTIVE: To present a detailed description of the clinical features of new FHM families in which we identified the T666M mutation in our CACNA1A screening program. METHODS: As part of our ongoing genetic screening, mutation analysis of the CACNA1A gene was performed by single-strand conformational polymorphism analysis in 33 probands of families with FHM. RESULTS: We identified the T666M mutation in 5 unrelated FHM families. In 3 of the families, patients displayed cerebellar ataxia. In 1 family, some affected members with the mutation had attacks with confusion but without hemiparesis. In 1 family, patients had progressive cognitive dysfunction. CONCLUSIONS: The T666M mutation is the most frequent CACNA1A mutation in FHM; it was found in 5 of 33 FHM families at our laboratory, and in 19 of 39 families with a known mutation reported in the literature (including the present study). Screening for the T666M mutation should therefore be the first step when screening families with FHM. There is a remarkable clinical heterogeneity among families with the T666M mutation.

• MeSH
• Point Mutation * MeSH
• Adult MeSH
• Phenotype MeSH
• Haplotypes MeSH
• Hemiplegia etiology   genetics   MeSH
• Humans MeSH
• Migraine Disorders complications   genetics   MeSH
• Polymorphism, Single-Stranded Conformational MeSH
• Pedigree MeSH
• Calcium Channels genetics   MeSH
• Family Health MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Germany MeSH
• United Kingdom MeSH
• United States MeSH
• Names of Substances
• CACNA1A protein, human MeSH Browser
• Calcium Channels MeSH

• MeSH
• Point Mutation MeSH
• Dyneins genetics   MeSH
• Mutagenesis, Insertional MeSH
• Humans MeSH
• Mutation, Missense MeSH
• RNA Splice Sites genetics   MeSH
• Molecular Sequence Data MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Myosin VIIa MeSH
• Myosins genetics   MeSH
• Codon, Nonsense MeSH
• Polymorphism, Single-Stranded Conformational MeSH
• Pedigree MeSH
• Amino Acid Sequence MeSH
• Sequence Deletion MeSH
• Sequence Homology, Amino Acid MeSH
• Sequence Alignment MeSH
• Tandem Repeat Sequences MeSH
• Usher Syndromes ethnology   genetics   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Geographicals
• Czech Republic ethnology   MeSH
• Italy ethnology   MeSH
• Morocco ethnology   MeSH
• Spain ethnology   MeSH
• Turkey ethnology   MeSH
• Names of Substances
• Dyneins MeSH
• RNA Splice Sites MeSH
• MYO7A protein, human MeSH Browser
• Myosin VIIa MeSH
• Myosins MeSH
• Codon, Nonsense MeSH