Substitution Of Alanine For Glycine Research Articles

Rapid in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici

As the incidence of fungicide resistance in plant pathogens continues to increase, control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele-specific PCR (ASqPCR) is an ideal technique for the in-field analysis of fungicide resistance as it can quantify the frequency of mutations in fungicide targets. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew. In Australia, strobilurin-resistant Bgt was first discovered in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome b (cytb) gene in the cytochrome bc1 enzyme complex, resulting in a substitution of alanine for glycine at position 143 (G143A). We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures, within 90 min of sample collection. The in situ analysis of samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9–100%. Validation of the analysis with a newly developed laboratory based digital PCR assay found no significant differences between the two methods. We have successfully developed an in-field quantification method, for a strobilurin-resistant allele, by pairing the ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of these type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.

Human HPRT1 gene and the Lesch–Nyhan disease: Substitution of alanine for glycine and inversely in the HGprt enzyme protein

ABSTRACTLesch–Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report three novel independent mutations in the coding region of the HPRT1 gene from genomic DNA of (a) a carrier sister of two male patients with LND: c.569G>C, p.G190A in exon 8; and (b) two LND affected male patients unrelated to her who had two mutations: c.648delC, p.Y216X, and c.653C>G, p.A218G in exon 9. Molecular analysis reveals the heterogeneity of genetic mutation of the HPRT1 gene responsible for the HGprt deficiency. It allows fast, accurate detection of carriers and genetic counseling.

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication

The L6 region of bovine adenovirus type 3 (BAdV-3) encodes a non-structural protein named 100K. Rabbit antiserum raised against BAdV-3 100K recognized a protein of 130 kDa at 12-24 h and proteins of 130, 100, 95 and 15 kDa at 36-48 h after BAdV-3 infection. The 100K species localized to the nucleus and the cytoplasm of BAdV-3-infected cells. In contrast, 100K localized predominantly to the cytoplasm of the transfected cells. However, BAdV-3 infection of cells transfected with 100K-enhanced yellow fluorescent protein-expressing plasmid detected fluorescent protein in the nucleus of the cells, suggesting that other viral proteins may be required for the nuclear localization of 100K. Interaction of BAdV-3 100K with BAdV-3 33K protein did not alter the cytoplasmic localization of 100K. However, co-expression of BAdV-3 100K and BAdV-3 protease localized 100K to the nucleolus of the transfected cells. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (aa 740-745 and 781-786) in transfected or BAdV-3-infected cells. The cleaved C terminus (107 aa) was localized to the nucleolus of the transfected cells. Further analysis suggested that the cleaved C terminus contains a bipartite nuclear localization signal and utilizes import receptor importin-α3 of the classical importin-α/β transport pathway for nuclear transport. Successful isolation of recombinant BAdV-3 expressing mutant 100K (substitution of alanine for glycine in the potential protease cleavage site) suggested that cytoplasmic cleavage of BAdV-3 100K by adenoviral protease is not essential for virus replication.

Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification.

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

Cancer‐associated missense mutations of caspase‐8 activate nuclear factor‐κB signaling

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a 5-year survival rate of ~50%. With the use of a custom cDNA-capture system coupled with massively parallel sequencing, we have now investigated transforming mechanisms for this malignancy. The cDNAs of cancer-related genes (n=906) were purified from a human HNSCC cell line (T3M-1 Cl-10) and subjected to high-throughput resequencing, and the clinical relevance of non-synonymous mutations thus identified was evaluated with luciferase-based reporter assays. A CASP8 (procaspase-8) cDNA with a novel G-to-C point mutation that results in the substitution of alanine for glycine at codon 325 was identified, and the mutant protein, CASP8 (G325A), was found to activate nuclear factor-κB (NF-κB) signaling to an extent far greater than that achieved with the wild-type protein. Moreover, forced expression of wild-type CASP8 suppressed the growth of T3M-1 Cl-10 cells without notable effects on apoptosis. We further found that most CASP8 mutations previously detected in various epithelial tumors also increase the ability of the protein to activate NF-κB signaling. Such NF-κB activation was shown to be mediated through the COOH-terminal region of the second death effector domain of CASP8. Although CASP8 mutations associated with cancer have been thought to promote tumorigenesis as a result of attenuation of the proapoptotic function of the protein, our results now show that most such mutations, including the novel G325A identified here, separately confer a gain of function with regard to activation of NF-κB signaling, indicating another role of CASP8 in the transformation of human malignancies including HNSCC.

Identification of a missense mutation in the melusin-encoding ITGB1BP2 gene in a patient with dilated cardiomyopathy

In a heterogeneous cohort of patients (n=255) with sporadic and familial dilated cardiomyopathy (DCM), we searched for novel disease-associated mutations in the human melusin-encoding ITGB1BP2 gene and found only one missense mutation, which was a substitution of alanine for glycine at position 313 located in the carboxy-terminal spacer region of the molecule. This point mutation (c.938C>G) was identified in a 45-year-old male with familial DCM and severe impairment of left-ventricular function, but was absent in 300 healthy control subjects. However, its functional significance in the context of heart failure is unclear, as this amino acid substitution was predicted to be without disease-causing effects. In this report, we confirm the low prevalence of mutations and single nucleotide polymorphisms in the coding sequence of the human melusin gene in patients with DCM, ruling out the possibility that genetic variations in this myocardially transcribed gene may have a significant impact on the epidemiology of DCM-induced heart failure.

Novel de novo Mutation in the KCNJ2 Gene in a Patient With Andersen-Tawil Syndrome

Andersen-Tawil syndrome is a rare autosomal-dominant disease characterized by episodic muscle weakness, cardiac arrhythmias, and dysmorphic features. Mutations in the KCNJ2 gene (which encodes an inward-rectifying potassium channel protein, Kir2.1) have been reported to be responsible for this disorder. Reported here is a novel de novo mutation in the KCNJ2 gene in a patient with Andersen-Tawil syndrome. This mutation predicts the substitution of alanine for glycine at position 146 (Gly146Ala, c.437G > C) of Kir2.1 and is located at the extracellular pore loop region that serves as a principal ion-selective filter. The patient did not respond to acetazolamide, but experienced an improvement of the paralytic symptoms on treatment with a combination of spironolactone, amiloride, and potassium supplements.

Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

Cell fusion mediated by Epstein–Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL.

Positive Effect of Transplantation of hNT Neurons (NTera 2/D1 Cell-Line) in a Model of Familial Amyotrophic Lateral Sclerosis

Transplantation of hNT Neurons derived from the human teratocarcinoma cell-line (NTera2/D1) has been shown to ameliorate motor dysfunction in a number of injury or disease models in which the deficits are fairly localized. However, these cells have not been used before in a model with more extensive neurodegeneration. The aim of this study is to determine the effects of hNT Neuron transplants on motor neuron function in a mouse model of familial amyotrophic lateral sclerosis (FALS) in which there is a substitution of Alanine for Glycine at position 93 of the human SOD1 gene (G93A). Amyotrophic lateral sclerosis is a fatal degenerative motor neuron disease affecting the spinal cord, brainstem, and cortex. This disease clinically manifests as progressive muscular weakness and atrophy, leading to paralysis and death within 3–5 years of diagnosis. The FALS represents 10–13% of all cases. A range of behavioral tests was used to examine spontaneous locomotor activity, coordination, and muscle strength of mice. Long-term (10–11 weeks) transplantation of hNT Neurons into the L4–L5 segments of the ventral horn spinal cord of FALSG93A mice at 7 weeks of age (before onset of overt behavioral symptoms of disease) delayed the onset of motor dysfunction for at least 3 weeks. The average lifespan of the transplanted mice was 128 days compared to 106 days for media-injected group. The last mouse in the hNT Neuron transplanted group was euthanized at 135 days of age when it display partial paralysis of the hindlimbs. Immunohistochemical analysis of the implanted spinal cords demonstrated the survival of grafted hNT Neurons and showed many healthy-appearing motor neurons near the implant site. These results suggest that hNT Neuron transplantation may be a promising therapeutic strategy for ALS.

Bcl-2 expression by retrograde transport of adenoviral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1 transgenic mice.

We investigated genes expression by retrograde axonal transport of replication-defective adenoviruses carrying genes for LacZ (AdLacZ) and Bcl-2 in motor neurons of transgenic mice expressing mutant human Cu/Zn superoxide dismutase (SOD1) gene containing a substitution of alanine for glycine at position 93. We found that intramuscular injection of AdLacZ into the tongue of mutant SOD1 transgenic mice and their wild-type littermates at various ages results in high expression of the transgene and similar time course of expression in hypoglossal cranial nerve nuclei, suggesting no difference in the behavior of the transgene expression between the two groups. Subsequently, we employed a molecular switching cassette for Bcl-2 designed to express Bcl-2 by Cre-loxP recombination using adenoviral vectors, and examined the COS7 and primary neuronal cells with the mutant SOD1 gene. The overexpression of Bcl-2 in both cells and the neuronal protection against staurosporine-induced apoptosis were observed, after dual infection of adenoviral vectors with cassette for Bcl-2 (AxCALNLBcl-2) and Cre recombinase (AxCANCre). After inoculation of AxCALNLBcl-2 followed by AxCANCre into the tongue of both mutant SOD1 transgenic mice and wild-type littermates, Bcl-2 was detected in both the injection site and the hypoglossal nuclei of brainstems, suggesting that this was the result of retrograde transport of AxCALNLBcl-2 and AxCANCre and expression of Bcl-2 by Cre recombinase in the hypoglossal nuclei. This strategy for delivery of exogenous genes such as Bcl-2 will be useful for studying neuronal death/survival and introducing foreign genes into postmitotic motor neurons, and in gene therapy for motor neuron diseases such as ALS.

Myristoylation Alters Retinoic Acid-Induced Down-Regulation of MARCKS in Immortalized Hippocampal Cells

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent PKC-substrate in the brain, which has been implicated in brain development, cytoskeletal remodeling, calcium/calmodulin signaling, and neuroplasticity. The sequence of the Macs gene codes for a protein that has three highly conserved domains including a 5′ myristoylation region and a 25-amino-acid phosphorylation site domain (PSD), which are involved in anchoring MARCKS to the cellular membrane. In this study, we examined the role of the myristoylation signal in the regulation of MARCKS in transfected rat hippocampal cells (H19-7) following retinoic acid (RA) treatment. A mutant MARCKS lacking the myristoylation signal was engineered by substitution of alanine for glycine at position 2 of the Macs gene and was found to be exclusively expressed in the cytosol fraction of transfected cells. Exposure of the wild-type MARCKS-transfected cells to RA resulted in an apparent shift of MARCKS from the membrane to the cytosol, while the total protein of wild-type MARCKS was not significantly changed. In contrast, RA-exposed cells transfected with the mutant MARCKS revealed a dramatic reduction of expression of MARCKS protein in both cytosol and total protein fractions. These data suggest that the absence of the myristoyl moiety may not only alter the anchoring of the protein to the membrane but also play a novel role in modulating cellular levels of MARCKS protein in response to RA.

Molecular Identification of the Ryanodine Receptor Pore-forming Segment

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca(2+) release measurements, [(3)H]ryanodine binding, and single channel recordings in planar lipid bilayers. We report here that single point mutations introduced into this region of the mouse cardiac ryanodine receptor reduce or abolish high affinity [(3)H]ryanodine binding. Single channel analysis revealed that a single substitution of alanine for glycine 4824 within this region reduced the single channel conductance by 97%, from 798 picosiemens (pS) for the wild type channel to 22 pS. The G4824A mutant channel was modulated by Ca(2+), Mg(2+), ATP, caffeine, ruthenium red, and ryanodine. Co-expression of the wild type and G4824A mutant proteins produced single channels that have intermediate unitary conductances of 516, 256, 176, and 60 pS. These data suggest that this conserved region constitutes an essential part of the ryanodine binding site and the channel conduction pathway of the ryanodine receptor.

Anisotropy of the dielectric properties in diglycine nitrate

Abstract The anisotropy of the dielectric permittivity and spontaneous polarization for pure and α-alanine doped diglycine nitrate has been studied. The actual polar direction is considered to be the normal direction to the 60°X-cut. The deviation of the spontaneous polarization vector in the mirror plane m for the ferroelectric phase has not been observed. The mechanism of partial substitution of alanine for glycine in this crystal is shown to be the same as in triglycine sulfate.

Detection of neutral amino acid substitutions in proteins.

The field of biochemical genetics relies heavily upon the detection by electrophoresis of genetically determined variants of proteins. Most of these variants differ by substitutions that involve charged amino acids. Genetic variants of another large class, ones that involve substitutions among neutral amino acids, are not easily detected and are often ignored. Ampholyte isoelectric focusing in some cases can separate proteins indistinguishable by standard electrophoresis, including genetic variants of mouse hemoglobins that differ only by neutral amino acid substitutions. A revolutionary variation of isoelectric focusing, in which gradients covering a small pH range are fixed into place in a polyacrylamide gel, provides greater resolution of these nearly identical proteins. Mouse hemoglobin tetramers that differ only by the substitution of alanine for glycine in the alpha-globin chains are resolved by several millimeters with the new technique; by comparison, these tetramers are imperfectly resolved on a standard pH 7-9 isoelectric focusing gel. This improved technique of isoelectric focusing was used to identify a variety of previously unreported genetic variants of mouse hemoglobin alpha chains. Immobilized gradients tailored to the requirements of the proteins being analyzed will extend greatly the ranges of protein variations that can be easily recognized for diverse applications, including genetic quality-control analyses and in studies of genetics, mutagenesis, and evolution.