SciCombinator

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Concept: Mutant

171

Jasmonates are phytohormones derived from oxygenated fatty acids that regulate a broad range of plant defense and developmental processes. In Arabidopsis, hypocotyl elongation under various light conditions was suppressed by exogenously supplied methyl jasmonate (MeJA). Moreover, this suppression by MeJA was particularly effective under red light condition. Mutant analyses suggested that SCF(COI1)-mediated proteolysis was involved in this function. However, MeJA action still remained in the coi1 mutant, and (+)-7-iso-JA-L-Ile, a well-known active form of jasmonate, had a weaker effect than MeJA under the red light condition, suggesting that unknown signaling pathway are present in MeJA-mediated inhibition of hypocotyl elongation. EMS mutant screening identified two MeJA-insensitive hypocotyl elongation mutants, jasmonate resistance long hypocotyl 1 (jal1) and jal36, which had mutations in the phytochrome B (PHYB) gene. These analyses suggested that inhibition of hypocotyl elongation by jasmonates is enhanced under red light in phyB dependent manner.

Concepts: Fatty acid, Mutant, Plant physiology, Plant hormones, Jasmonate, Jasmonic acid, Methyl jasmonate, Wild Child

169

The model grass Brachypodium distachyon (Brachypodium) is an excellent system for studying the basic biology underlying traits relevant to the use of grasses as food, forage and energy crops. To add to the growing collection of Brachypodium resources available to plant scientists, we further optimized our Agrobacterium tumefaciens-mediated high-efficiency transformation method and generated 8,491 Brachypodium T-DNA lines. We used inverse PCR to sequence the DNA flanking the insertion sites in the mutants. Using these flanking sequence tags (FSTs) we were able to assign 7,389 FSTs from 4,402 T-DNA mutants to 5,285 specific insertion sites (ISs) in the Brachypodium genome. More than 29% of the assigned ISs are supported by multiple FSTs. T-DNA insertions span the entire genome with an average of 19.3 insertions/Mb. The distribution of T-DNA insertions is non-uniform with a larger number of insertions at the distal ends compared to the centromeric regions of the chromosomes. Insertions are correlated with genic regions, but are biased toward UTRs and non-coding regions within 1 kb of genes over exons and intron regions. More than 1,300 unique genes have been tagged in this population. Information about the Western Regional Research Center Brachypodium insertional mutant population is available on a searchable website (http://brachypodium.pw.usda.gov) designed to provide researchers with a means to order T-DNA lines with mutations in genes of interest.

Concepts: DNA, Gene, Genetics, Mutation, Molecular biology, Mutant, Noncoding DNA, Brachypodium distachyon

167

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

Concepts: DNA, Gene, Developmental biology, Mutant, Transcription factor, Transcription factors, Msh homeobox 2, MSX1

167

Primary erythromelalgia (PE) is an autosomal dominant neurological disorder characterized by severe burning pain and erythema in the extremities upon heat stimuli or exercise. Mutations in human SCN9A gene, encoding the α-subunit of the voltage-gated sodium channel, Na(v)1.7, were found to be responsible for PE. Three missense mutations of SCN9A gene have recently been identified in Taiwanese patients including a familial (I136V) and two sporadic mutations (I848T, V1316A). V1316A is a novel mutation and has not been characterized yet. Topologically, I136V is located in DI/S1 segment and both I848T and V1316A are located in S4-S5 linker region of DII and DIII domains, respectively. To characterize the elelctrophysiological manifestations, the channel conductance with whole-cell patch clamp was recorded on the over-expressed Chinese hamster overy cells. As compared with wild type, the mutant channels showed a significant hyperpolarizing shift in voltage dependent activation and a depolarizing shift in steady-state fast inactivation. The recovery time from channel inactivation is faster in the mutant than in the wild type channels. Since warmth can trigger and exacerbate symptoms, we then examine the influence of tempearture on the sodium channel conduction. At 35°C, I136V and V1316A mutant channels exhibit a further hyperpolarizing shift at activation as compared with wild type channel, even though wild type channel also produced a significant hyperpolarizing shift compared to that of 25°C. High temperature caused a significant depolarizing shift in steady-state fast inactivation in all three mutant channels. These findings may confer to the hyperexcitability of sensory neurons, especially at high temperature. In order to identifying an effective treatment, we tested the IC(50) values of selective sodium channel blockers, lidocaine and mexiletine. The IC(50) for mexiletine is lower for I848T mutant channel as compared to that of the wild type and other two mutants which is comparable to the clinical observations.

Concepts: DNA, Mutation, Mutant, Action potential, Electrophysiology, Point mutation, Missense mutation, Erythromelalgia

165

In LEOPARD Syndrome (LS) patients, mutations in the protein tyrosine phosphatase Shp2 cause hypertrophic cardiomyopathy (HCM). The pro-hypertrophic effects of mutant Shp2 are mediated downstream by hyperactivation of mammalian target of rapamycin (mTOR). Our goal was to further define the signaling cascade that is essential for the underlying pathomechanism, thus expanding the list of potential future therapeutic targets. Using cultured neonatal rat cardiomyocytes (NRCM) with adenoviral gene delivery and pharmacological inhibitors, we found that hypertrophy induced by a particularly aggressive LS mutation in Shp2 depends on hyperactivation of Akt and focal adhesion kinase (FAK) as well as mTOR. Dissecting domain-specific functions of Shp2 using double and truncation mutants, we determined that the hypertrophic effects of mutant Shp2 depend on the two SH2 domains and on an intact catalytic center. The latter finding prompted us to test the efficacy of a Shp2 inhibitor targeted directly at the catalytic pocket. This compound, PHPS1, effectively prevented mutant Shp2-induced hypertrophy. In summary, we identified three novel targets for pharmacological therapy of LS-associated cardiac hypertrophy. Of particular importance is the finding that intervention directly at the mutant Shp2 protein is effective, since this would facilitate custom-tailored therapeutic approaches for patients carrying LS mutations in Shp2.

Concepts: Signal transduction, Enzyme, Mutant, Tyrosine kinase, Hypertrophic cardiomyopathy, Focal adhesion, PTEN, EC 3.1.3

150

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing “dark-flash” visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

Concepts: DNA, Psychology, Gene, Mutation, Evolution, DNA repair, Mutant, Conserved sequence

146

Previous experimental study measuring the binding affinities of biotin to the wild type streptavidin (WT) and three mutants (S45A, D128A and S45A/D128A double mutant) has shown that the loss of binding affinity from the double mutation is larger than the direct sum of those from two single mutations. The origin of this cooperativity has been investigated in this work through molecular dynamics simulations and the end-state free energy method using the polarized protein-specific charge. The results show that this cooperativity comes from both the enthalpy and entropy contributions. The former contribution mainly comes from the alternations of solvation free energy. Decomposition analysis shows that the mutated residues nearly have no contributions to the cooperativity. Instead, N49 and S88, which are located at the entry of the binding pocket and interact with the carboxyl group of biotin, make the dominant contribution among all the residues in the first binding shell around biotin.

Concepts: DNA, Mutation, Energy, Mutant, Chemistry, Entropy, Gibbs free energy, Affinity

41

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa L. ssp. japonica), which completes its life cycle in 9 weeks. We sequenced 1,504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.

Concepts: DNA, Gene, Genetics, Mutation, Evolution, Mutant, Point mutation, Rice

31

The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the C-terminal domain of the p63 gene can cause ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility and severe, long-lasting skin erosions. Despite deep knowledge of p63 functions, little is known about mechanisms underlying disease pathology and possible treatments. Here, we show that multiple AEC-associated p63 mutations, but not those causative of other diseases, lead to thermodynamic protein destabilization, misfolding, and aggregation, similar to the known p53 gain-of-function mutants found in cancer. AEC mutant proteins exhibit impaired DNA binding and transcriptional activity, leading to dominant negative effects due to coaggregation with wild-type p63 and p73. Importantly, p63 aggregation occurs also in a conditional knock-in mouse model for the disorder, in which the misfolded p63 mutant protein leads to severe epidermal defects. Variants of p63 that abolish aggregation of the mutant proteins are able to rescue p63’s transcriptional function in reporter assays as well as in a human fibroblast-to-keratinocyte conversion assay. Our studies reveal that AEC syndrome is a protein aggregation disorder and opens avenues for therapeutic intervention.

Concepts: DNA, Protein, Gene, Genetics, Mutation, Transcription, DNA repair, Mutant

29

Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (needle in the k-stack), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in nonreference rice cultivars and to two mutants of the nonmodel species Arabis alpina. In both species, comparing pooled F2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, for any species amenable to mutagenesis, NIKS enables forward genetics without requiring segregating populations, genetic maps and reference sequences.

Concepts: DNA, Gene, Genetics, Mutation, Allele, Evolution, Mutant, Classical genetics