Mutagenesis Protocol Research Articles

DNA assembly by multi-fragment digestion/ligation and homologous recombination

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.

Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions.

The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

Measuring Stress Phenotypes in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human fungal pathogen capable of surviving in a wide range of environments and hosts. It has been developed as a model organism to study fungal pathogenesis due to its fully sequenced haploid genome and optimized gene deletion and mutagenesis protocols. These methods have greatly aided indetermining the relationship between Cryptococcus genotype and phenotype. Furthermore, the presence of congenic mata and matα strains associated with a defined sexual cycle has helped further understand cryptococcal biology. Several in vitro stress conditions have been optimized to closely mimic the stress that yeast encounter in the environment or within the infected host. These conditions have proven to be extremely useful in elucidating the role of several genes in allowing yeast to adapt and survive in hostile external environments. This chapter describes various in vitro stress conditions that could be used to test the sensitivity of different mutant strains, as well as the protocol for preparing them. We have also included a list of mutants that could be used as a positive control strain when testing the sensitivity of the desired strain to a specific stress.

Understanding Pseudophosphatase Function Through Biochemical Interactions.

Pseudophosphatases have been solidified as important signaling molecules that regulate signal transduction cascades. However, their mechanisms of action remain enigmatic. Reflecting this mystery, the prototypical pseudophosphatase STYX (phospho-serine-threonine/tyrosine-binding protein) was named with allusion to the river of the dead in Greek mythology to emphasize that these molecules are "dead" phosphatases. Although proteins with STYX domains do not catalyze dephosphorylation, this does not preclude their having other functions, including as integral elements of signaling networks. Thus, understanding their roles may mark them as potential novel drug targets. This chapter outlines common strategies used to characterize the functions of pseudophosphatases, using as an example MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine-threonine/tyrosine-binding], which has been linked to tumorigenesis, hepatocellular carcinoma, glioblastoma, apoptosis, and neuronal differentiation. We start with the importance of "restoring" (when possible) phosphatase activity in a pseudophosphatase, so the active mutant may be used as a comparison control throughout immunoprecipitation and mass spectrometry analyses. To this end, we provide protocols for site-directed mutagenesis, mammalian cell transfection, co-immunoprecipitation, phosphatase activity assays, and immunoblotting that we have used to investigate MK-STYX and the active mutant MK-STYXactive. We also highlight the importance of utilizing RNA interference (RNAi) "knockdown" technology to determine a cellular phenotype in various cell lines. Therefore, we outline our protocols for introducing short hairpin RNA (shRNA) expression plasmids into mammalian cells and quantifying knockdown of gene expression with real-time quantitative PCR (qPCR). We also provide a bioinformatic approach to investigating MK-STYX and MK-STYX(active mutant). These bioinformatic approaches can stand alone experimentally but also complement and enhance "wet" bench approaches such as binding assays and/or activity assays. A combination of cellular, molecular, biochemical, proteomic, and bioinformatic techniques has been a powerful tool in identifying novel functions of MK-STYX. Likewise, the information provided here should be a helpful guide to elucidating the functions of other pseudophosphatases.

N-Glycan Insertion for Probing Protein-Protein Interactions and Epitope Mapping.

Introducing an N-linked glycosylation motif into recombinant proteins at specific sites is a useful tool in probing protein-protein interactions and epitope mapping. Due to their large size, a new N-glycan can block protein-protein interactions if it is introduced by site-directed mutagenesis on the same face as a ligand or antibody binding site. Recombinant mutant proteins containing these engineered glycans can then be studied using binding or functional assays to establish if the new glycan causes steric hindrance, prevents an important protein-protein interaction, or blocks (auto)antibody binding. In this book chapter, we provide guides and protocols for inserting engineered glycans, including how to use AlphaFold models to choose amino acid residues on the surface of protein domains that are suitable for mutagenesis into N-linked glycosylation motifs as well as protocols for site-directed mutagenesis and recombinant protein expression of the N-glycan variants.

Comparative Analysis of Physical and Chemical Mutagenesis in Chrysanthemum cv. 'Candid': Assessing Genetic Variation and Breeding Potential.

In this study, we developed a mutagenesis protocol specifically designed for chrysanthemum cv. "Candid" in order to introduce genetic variation. By subjecting chrysanthemum shoots to different doses of physical and chemical mutagens, we successfully generated a total of 24 mutants, each with unique genetic compositions. We observed that the mortality rate was lowest when the shoots were exposed to 10 Gy gamma irradiation and 1.00% EMS. To assess the diversity and relatedness among the mutants, we employed RAPD and SSR markers. The combination of these markers allowed us to construct a dendrogram that effectively categorized the mutant population into distinct clusters based on the specific mutagen treatments. Interestingly, the mutants induced by 10 Gy gamma irradiation exhibited greater genetic diversity in terms of flower colors. On the other hand, mutants created with 1.00% EMS displayed a higher level of variation and yielded more viable mutants. To determine the optimal markers for studying genetic diversity, we analyzed the polymorphic information content (PIC) of different markers. Among the tested markers, OPA-07 (RAPD) and JH47 (SSR) showed the highest PIC values, indicating their effectiveness in capturing genetic variability within the mutant population. Conversely, the PIC values of OPD-07 and JH20 demonstrated the lowest among the markers tested. Our results revealed a percentage of polymorphism ranging from 81.81% to 100% for RAPD markers and 66.66% to 100% for SSR markers. These findings indicate that physical mutation induced by 10 Gy gamma irradiation can be clearly distinguished from chemical mutation induced by EMS at concentrations of 1% and 0.75% in chrysanthemum cv. "Candid.″ Overall, this study provides valuable insights into the genetic composition of the generated mutants and highlights their potential for enhancing chrysanthemum-breeding programs. The identified markers, particularly, OPA-07 and JH47, can serve as valuable tools for future studies aimed at exploring and exploiting the genetic diversity within the chrysanthemum population.

Morphometric frequency and spectrum of gamma-ray-induced chlorophyll mutants identified by phenotype and development of novel variants in lentil (Lens culinaris Medik.).

Genetic variations are a crucial source of germplasm heterogeneity, as they contribute to the development of new traits for plant breeding by offering an allele resource. Gamma rays have been widely used as a physical agent to produce mutations in plants, and their mutagenic effect has attracted much attention. Nonetheless, few studies have examined the whole mutation spectrum in large-scale phenotypic evaluations. To comprehensively investigate the mutagenic effects of gamma irradiation on lentils, biological consequences on the M1 generation and substantial phenotypic screening on the M2 generation were undertaken. Additionally, the study followed the selected mutants into the M3 generation to evaluate the agronomic traits of interest for crop improvement. Seeds of lentil variety Moitree were irradiated with a range of acute gamma irradiation doses (0, 100, 150, 200, 250, 300, and 350 Gy) to induce unique genetic variability. This research focused on determining the GR50 value while considering seedling parameters and examining the status of pollen fertility while comparing the effects of the gamma irradiation dosages. The GR50 value was determined to be 217.2 Gy using the seedling parameters. Pollens from untreated seed-grown plants were approximately 85% fertile, but those treated with the maximum dosage (350 Gy) were approximately 28% fertile. Numerous chlorophyll and morphological mutants were produced in the M2 generation, with the 300 Gy -treated seeds being the most abundant, followed by the 250 Gy -treated seeds. This demonstrated that an appropriate dosage of gamma rays was advantageous when seeking to generate elite germplasm resources for one or multiple traits. Selected mutants in the M3 generation showed improved agronomic traits, including plant height, root length, number of pods per plant, and yield per plant. These investigations will contribute to a comprehensive understanding of the mutagenic effects and actions of gamma rays, providing a basis for the selection and design of suitable mutagens. This will facilitate the development of more controlled mutagenesis protocols for plant breeding and help guide future research directions for crop improvement using radiation-induced mutation breeding techniques.

Enabling low-cost and robust essentiality studies with high-throughput transposon mutagenesis (HTTM).

Transposon-insertion sequencing (TIS) methods couple high density transposon mutagenesis with next-generation sequencing and are commonly used to identify essential or important genes in bacteria. However, this approach can be work-intensive and sometimes expensive depending on the selected protocol. The difficulty to process a high number of samples in parallel using standard TIS protocols often restricts the number of replicates that can be performed and limits the deployment of this technique to large-scale projects studying gene essentiality in various strains or growth conditions. Here, we report the development of a robust and inexpensive High-Throughput Transposon Mutagenesis (HTTM) protocol and validate the method using Escherichia coli strain BW25113, the parental strain of the KEIO collection. HTTM reliably provides high insertion densities with an average of one transposon every ≤20bp along with impressive reproducibility (Spearman correlation coefficients >0.94). A detailed protocol is available at protocol.io and a graphical version is also included with this article.

Conjugation and transposon mutagenesis of Xenorhabdus griffiniae HGB2511, the bacterial symbiont of the nematode Steinernema hermaphroditum (India).

Symbiosis, the beneficial interactions between two organisms, is a ubiquitous feature of all life on Earth, including associations between animals and bacteria. However, the specific molecular and cellular mechanisms which underlie the diverse partnerships formed between animals and bacteria are still being explored. Entomopathogenic nematodes transport bacteria between insect hosts, together they kill the insect, and the bacteria consume the insect and serve as food source for the nematodes. These nematodes, including those in the Steinernema genus, are effective laboratory models for studying the molecular mechanisms of symbiosis because of the natural partnership they form with Xenorhabdus bacteria and their straightforward husbandry. Steinernema hermaphroditum nematodes and their Xenorhabdus griffiniae symbiotic bacteria are being developed as a genetic model pair for studying symbiosis. Our goal in this project was to begin to identify bacterial genes that may be important for symbiotic interactions with the nematode host. Towards this end, we adapted and optimized a protocol for delivery and insertion of a lacZ- promoter-probe transposon for use in the S. hermaphroditum symbiont, X. griffiniae HGB2511 (Cao et al., 2022). We assessed the frequencies at which we obtained exconjugants, metabolic auxotrophic mutants, and active promoter- lacZ fusions. Our data indicate that the Tn 10 transposon inserted relatively randomly based on the finding that 4.7% of the mutants exhibited an auxotrophic phenotype. Promoter-fusions with the transposon-encoded lacZ , which resulted in expression of β-galactosidase activity, occurred in 47% of the strains. To our knowledge, this is the first mutagenesis protocol generated for this bacterial species, and will facilitate the implementation of large scale screens for symbiosis and other phenotypes of interest in X. griffiniae .

Site-Directed Mutagenesis Protocol to Determine the Role of Amino Acid Residues in Polycomb Group (PcG) Protein Function.

Site-directed mutagenesis (SDM) is a technique in molecular biology and protein engineering that is widely used to determine the significance of specific residues involved in post-translational modifications (PTMs), protein structure, function, and stability. Here, we describe a simple and cost-effective polymerase chain reaction (PCR)-based SDM method. This method can be used to introduce point mutation, short addition, or deletions in protein sequences. Using polycomb repressive complex-2 (PRC2)-associated protein JARID2 as an example, we demonstrate how SDM can be used to study structural and consequently functional changes in a protein.

Changes on mutant pot chrysanthemums (Dendranthema × grandiflora Tzelev.)

Chrysanthemum (Dendranthema x grandiflora Tzvelev) is one of the most important ornamental plants in the world that has the richest mutant varieties with numerous colors. The objective of this study is to determine the effective mutagen dose (EMD50) for creating variations by gamma irradiation. It is aimed to get a mutagenesis protocol that could develop new mutants in pot chrysanthemums. To determine the EMD50, rooted cuttings of brownish-red color ‘Brandevil’ variety were irradiated by gamma radiation at 0, 10, 20, 30, 40, 50 Gray (Gy) doses. According to the shoot lengths, EMD50 was calculated as 27 Gy. The mutation frequency was calculated as 4.8%. Some changes were observed for flower numbers per plant, plant heights and widths, shapes and colors of both flowers and leaves. The color changes varied from brownish-red to yellow and orange. Two different colors appeared in the same pot at some genotypes as well as form changes of flowers. The similarity of the mutants was determined by the hierarchical cluster dendrogram involving five groups. Various colors were obtained for leaves and flowers. Remarkable mutations of the selected mutants were multiplied by tissue culture.

Functional Effects of Epilepsy Associated KCNT1 Mutations Suggest Pathogenesis via Aberrant Inhibitory Neuronal Activity

KCNT1 (K+ channel subfamily T member 1) is a sodium-activated potassium channel highly expressed in the nervous system which regulates neuronal excitability by contributing to the resting membrane potential and hyperpolarisation following a train of action potentials. Gain of function mutations in the KCNT1 gene are the cause of neurological disorders associated with different forms of epilepsy. To gain insights into the underlying pathobiology we investigated the functional effects of 9 recently published KCNT1 mutations, 4 previously studied KCNT1 mutations, and one previously unpublished KCNT1 variant of unknown significance. We analysed the properties of KCNT1 potassium currents and attempted to find a correlation between the changes in KCNT1 characteristics due to the mutations and severity of the neurological disorder they cause. KCNT1 mutations identified in patients with epilepsy were introduced into the full length human KCNT1 cDNA using quick-change site-directed mutagenesis protocol. Electrophysiological properties of different KCNT1 constructs were investigated using a heterologous expression system (HEK293T cells) and patch clamping. All mutations studied, except T314A, increased the amplitude of KCNT1 currents, and some mutations shifted the voltage dependence of KCNT1 open probability, increasing the proportion of channels open at the resting membrane potential. The T314A mutation did not affect KCNT1 current amplitude but abolished its voltage dependence. We observed a positive correlation between the severity of the neurological disorder and the KCNT1 channel open probability at resting membrane potential. This suggests that gain of function KCNT1 mutations cause epilepsy by increasing resting potassium conductance and suppressing the activity of inhibitory neurons. A reduction in action potential firing in inhibitory neurons due to excessively high resting potassium conductance leads to disinhibition of neural circuits, hyperexcitability and seizures.

Optimized methods for random and targeted mutagenesis in field pea (Pisum sativum L.).

Field pea is an important pulse crop for its dense nutritional profile and contribution to sustainable agricultural practices. Recently, it has received extensive attention as a potential leading source of plant-based proteins. However, the adoption of peas as a mainstream source of proteins is affected by a relatively moderate protein content, anti-nutritional factors and high levels of off-flavor components that reduce protein quality. Availability of genetic variation for desirable seed quality traits is the foundation for the sustainable development of pea varieties with improved protein content and quality. Mutagenesis has been an important tool in gene functional characterization studies and creating genetic variability for crop breeding. Large-scale mutagenesis of a crop using physical and chemical agents requires diligent selection of the mutagen and optimization of its dose to increase the frequency of mutations. In this study, we present detailed optimized protocols for physical and chemical mutagenesis of pea using gamma irradiation and ethyl methanesulfonate (EMS), respectively. Gamma radiation and EMS titration kill curves were established to identify optimal doses of the two mutagenic agents. Based on germination, survival rate and growth phenotypes, a gamma radiation dose of 225 Gy and EMS concentration of 5 mm were selected as optimal dosages for mutagenesis in field pea. The presented protocol has been modified from previously established mutagenesis protocols in other crop plants. Our results indicate that the optimal mutagen dosage is genotype dependent. CRISPR/Cas-based gene editing provides a precise and rapid method for targeted genetic manipulation in plants. With the recent success of gene editing in pea using CRISPR/Cas, this innovative technology is expected to become an integral component of the gene discovery and crop improvement toolkit in pea. Here, we describe an optimized methods for targeted mutagenesis of pea protoplasts, including mesophyll protoplast extraction, PEG-mediated transformation and gene editing of a LOX gene using CRISPR/Cas system. The general strategies and methods of mutagenesis described here provide an essential resource for mutation breeding and functional genomics studies in pea. These methods also provide a foundation for similar studies in other crops.

A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding

Watermelon, Citrullus lanatus, is the world’s third largest fruit crop. Reference genomes with gaps and a narrow genetic base hinder functional genomics and genetic improvement of watermelon. Here, we report the assembly of a telomere-to-telomere gap-free genome of the elite watermelon inbred line G42 by incorporating high-coverage and accurate long-read sequencing data with multiple assembly strategies. All 11 chromosomes have been assembled into single-contig pseudomolecules without gaps, representing the highest completeness and assembly quality to date. The G42 reference genome is 369 321 829 bp in length and contains 24 205 predicted protein-coding genes, with all 22 telomeres and 11 centromeres characterized. Furthermore, we established a pollen-EMS mutagenesis protocol and obtained over 200 000 M1 seeds from G42 . In a sampling pool, 48 monogenic phenotypic mutations, selected from 223 M1 and 78 M2 mutants with morphological changes, were confirmed. The average mutation density was 1 SNP/1.69 Mb and 1 indel/4.55 Mb per M1 plant and 1 SNP/1.08 Mb and 1 indel/6.25 Mb per M2 plant. Taking advantage of the gap-free G42 genome, 8039 mutations from 32 plants sampled from M1 and M2 families were identified with 100% accuracy, whereas only 25% of the randomly selected mutations identified using the 97103v2 reference genome could be confirmed. Using this library and the gap-free genome, two genes responsible for elongated fruit shape and male sterility (ClMS1) were identified, both caused by a single base change from G to A. The validated gap-free genome and its EMS mutation library provide invaluable resources for functional genomics and genetic improvement of watermelon.

In Vitro Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures.

The alternative sigma factor B (σB) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σB loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σB or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σB). However, σB becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σB loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σB loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σB in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

Biochemical and in silico identification of the active site and the catalytic mechanism of the circadian deadenylase HESPERIN.

The 24-h molecular clock is based on the stability of rhythmically expressed transcripts. The shortening of the poly(A) tail of mRNAs is often the first and rate-limiting step that determines the lifespan of a mRNA and is catalyzed by deadenylases. Herein, we determine the catalytic site of Hesperin, a recently described circadian deadenylase in plants, using a modified site-directed mutagenesis protocol and a custom vector, pATHRA. To explore the catalytic efficiency of AtHESPERIN, we investigated the effect of AMP and neomycin, and used molecular modeling simulations to propose a catalytic mechanism. Collectively, the biochemical and in silico results classify AtHESPERIN in the exonuclease-endonuclease-phosphatase deadenylase superfamily and contribute to the understanding of the intricate mechanisms of circadian mRNA turnover.

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method.

Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

CRISPR–Cas9 Mutagenesis in Xenopus tropicalis for Phenotypic Analyses in the F0 Generation and Beyond

CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Xenopus tropicalis Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR-Cas9 can be used to create either mosaic loss-of-function Xenopus embryos that display F0 generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR-Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.

Determining ethyl methane sulfonate-mediated (EMS) mutagenesis protocol for inducing high biomass yield in fodder barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) has narrow genetic base for selection of promising ideotypes for the following traits: high biomass yield for livestock feed, enhanced agronomic and nutritional traits, and resistance to biotic and abiotic stresses. Ethyl methane sulfonate (EMS) mutagenesis offers opportunities for inducing genetic variation for key traits for development of feed barley ideotypes. The objective of this study was to determine optimal EMS dosage and exposure time to induce genetic variation for selection of high biomass yield six-row feed barley mutants. Five EMS dosages (i.e. 0.1%, 0.3%, 0.5%, 0.7% and 0.9% v/v) and five exposure times (i.e. 0.5 hr, 1 hr, 1.5 hr, 2 hrs and 2.5 hrs) were used on a six-row fodder barley using a 5 x 5 factorial treatment structure in a complete randomized design with three replications. Non-treated seed were used as a comparative control. Data was recorded for percent germination, seedling survival, shoot height, root height, shoot and root biomass. Significant (p < 0.05) EMS dosage x exposure time was observed for assessed traits indicating their influence on phenotypic variation in feed barley. Overall, a declining trend was observed for assessed traits with increased EMS dosage and exposure time. The LD50 value of 0.64% (v/v) EMS dosage was identified as an optimal dose for large-scale mutagenesis protocol to select fodder barley mutants with high biomass yield

Investigation of Biochemical Characterization of NEMO and Its Interaction with IKK/NF‐kB

The transcriptional activity of the NF-kB family of protein factors in response to cellular signals is primarily regulated by two other families of proteins- the IkB inhibitors and IkB kinases (IKK). Two distinct signaling pathways activate IKK kinases leading to the degradation of specific IkB inhibitors and release of specific NF-kB factors. The canonical signaling pathway activates IKK2 and the non-canonical, IKK1. The activation of IKK2 is strongly dependent on an adapter protein, NEMO (NF-kB essential modulator), whereas IKK1 activation also requires NF-kB inducing kinase (NIK). In a resting cell, NF-kB remains inactive by association of the inhibitory IkB proteins. Upon upstream stimulation, NEMO promotes IKK2 phosphorylation that induces IkBα leading to its ubiquitination and consequent degradation by proteasome. Free NF-kB can transloca­te in the nucleus and bind to specific sites of gene promoters to induce target gene expressions. IKK complex is an uncharacterized heterotrimer of three proteins, IKK1, IKK2 and NEMO. The kinase domain of IKK2 (and also IKK1) contains a pair of serines within the kinase ‘activation loop’ that undergoes phosphorylation in response to canonical signaling pathway marking IKK2 activation. NEMO is a IKK regulatory subunit and leads to IKK activity. If cells do not activate NEMO, NF-kB will not be functioned through the main pathway. Disease-causing NEMO mutations indicate an impairment of NF-kB activation. The gene encoding NEMOis in the X chromosome. NEMO has two types of disorders by hemizygous or heterozygous NEMOmutations. Thus, NEMO plays a specific protective role in genetic diseases, which provides molecular targets for new therapies. However, there is little information about the NEMO disease and more research is needed to help further studies. To this end, we will first investigate biochemical characterization of NEMO wild-type (WT) and its mutant. Full-length human NEMO WT has already been subcloned into the pET15b vector in frame with an N-terminal hexahistidine tag. A disease-causing NEMO mutant will be prepared by the Quickchange mutagenesis protocol with base changes incorporated in the oligonucleotide primers. His-tagged NEMO proteins will be expressed in BL21 (DE3) cells and then the soluble proteins will be purified by Ni affinity chromatography. Eluted fractions will be collected and loaded on SDS-PAGE gels, and protein concentrations will be determined by Bradford assay. After that, we will compare the degree of self-association between WT and mutant using gel filtration on Fast protein liquid chromatography (FPLC). Since the IKK complex serves a central role in maintaining the NF-kB pathway, further study will involve NEMO:IKK2(or IKK1) interaction in order to provide more information about disease-causing NEMO mutants and their regulation in IKK/NF-kB activation. This would be helpful for future applications to other studies regarding its role in other associated diseases.