Mutant-like Phenotypes Research Articles

An epiallele of a gene encoding a PfkB-type carbohydrate kinase affects plant architecture in maize.

Plant architecture greatly contributes to grain yield, but the epigenetic regulation of plant architecture remains elusive. Here, we identified the maize (Zea mays L.) mutant plant architecture 1 (par1), which shows reduced plant height, shorter and narrower leaves, and larger leaf angles than the wild type. Interestingly, par1 is an epiallele harboring a de novo CACTA insertion in the intron of the Par1 gene. High DNA methylation levels of the CACTA insertion are associated with strong Par1 expression and normal phenotypes. In contrast, low DNA methylation levels of this insertion are associated with weak Par1 expression and a mutant-like phenotype. The Par1 gene encodes a PfkB-type carbohydrate kinase that converts nucleosides to nucleoside monophosphates both in vitro and in vivo. Additional analyses showed that genes differentially expressed in the par1 mutant are enriched in jasmonic acid (JA) metabolism, and levels of JA metabolites were significantly higher in the mutant than in the wild type. Treatment with either nucleoside monophosphates or a synthetic inhibitor of JA biosynthesis reduced JA levels and partially rescued the mutant phenotype. In summary, we identified an epiallele of a gene encoding a PfkB-type carbohydrate kinase that might affect nucleoside monophosphate and JA levels, thus affecting maize growth.

EPEN-24. 5FU SENSITIVITY IS MEDIATED THROUGH TRISOMY UCK2 EXPRESSION IN 1Q+ PFA EPN

Abstract BACKGROUND Nearly all children with gain of chromosome 1q (1q+) PFA ependymoma (EPN) will die, highlighting the urgent need to develop new treatment strategies for these patients. It has recently been shown that 1q+ creates a p53 mutant-like phenotype through trisomy expression of MDM4, a repressor of p53 signaling. This same study found toxic uracil analogs could reverse trisomy MDM4 effect on p53 and this was mediated through another 1q-trisomy gene UCK2. We and other have previously identified 5-fluorouracil (5FU) as an ependymoma specific inhibitor. In this study, we present the mechanism for 5FU sensitivity. METHODS In this study we utilize 1q+ PFA in vitro models to test the efficacy of combination radiation and 5FU in a preclinical setting. We measured p53 signaling through proteomic array and qRT-PCR for p21, a p53 response gene. RESULTS 5FU enhances radiotherapy in 1q+ PFA cell lines. Specifically, 5FU increases p53 activity mediated by the extra copy of UCK2 located on chromosome 1q in 1q+ PFA. Experimental down regulation of UCK2 resulted in decreased 5FU sensitivity in 1q+ PFA cells. Though not statistically significant, we did see prolong survival and decreased tumor burden in 1q+ PFA orthotopic patient-derived xenograft models treated with focal 2Gyx5days radiation and 5FU. CONCLUSION These results are the first to identify a 1q+ specific therapy approach in PFA EPN. Existing phase 1 studies in pediatrics have already established safety and dose of 5FU, providing a rapid translation into phase 2 trials for children both up front and at recurrence for 1q+ PFA.

ALTERED MERISTEM PROGRAM1 sustains cellular differentiation by limiting HD-ZIP III transcription factor gene expression.

Plants show remarkable developmental and regenerative plasticity through the sustained activity of stem cells in meristems. Under certain conditions, pluripotency can even be reestablished in cells that have already entered differentiation. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis (Arabidopsis thaliana) causes a set of hypertrophic phenotypes, indicating a defect in the suppression of pluripotency. A role of AMP1 in the miRNA-mediated inhibition of translation has previously been reported; however, how this activity is related to its developmental functions is unclear. Here, we examined the functional interaction between AMP1 and the Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors, which are miRNA-controlled determinants of shoot meristem specification. We found that the HD-ZIP III transcriptional output is enhanced in the amp1 mutant and that plant lines with increased HD-ZIP III activity not only developed amp1 mutant-like phenotypes but also showed a synergistic genetic interaction with the mutant. Conversely, the reduction of HD-ZIP III function suppressed the shoot hypertrophy defects of the amp1 mutant. We further provide evidence that the expression domains of HD-ZIP III family members are expanded in the amp1 mutant and that this misexpression occurs at the transcriptional level and does not involve the function of miRNA165/166. Finally, amp1 mutant-specific phenotypes cannot be mimicked by a general inhibition of miRNA function in the AMP1 expression domain. These findings lead us to a model in which AMP1 restricts cellular pluripotency upstream of HD-ZIP III proteins, and this control appears to be not directly mediated by the canonical miRNA pathway.

Identification of Excellent Prognosis IDH Wildtype Glioblastomas Using Genomic and Metabolic Profiling

High grade gliomas (HGGs) are aggressive brain tumors with altered cellular metabolism. HGGs can carry mutations in the tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase 1 (IDH1), conferring distinct biology and improved patient prognosis compared to IDH wildtype (wt) tumors. Using metabolomic analyses of tumor tissue, we previously showed that IDH wt and IDH mutant (IDH mut) tumors have unique metabolomic signatures that correlate with different survival outcomes. Among this cohort of 69 HGG samples, we identified two unique patient tumors that metabolically clustered with IDH mut tumors, but lacked both the IDH mutation and its product 2-hydroxyglutarate. We aimed to discover unique mutations in these two tumors that may impart an IDH mutant-like phenotype in the absence of an IDH1 or IDH2 mutation. Whole exome sequencing (WES) was performed on frozen tumor samples from two patients diagnosed as glioblastoma (GBM), IDH wt via Agilent v5 + IncRNA platform. Alignment to the hg38 genome and variant calling were completed using an accelerated implementation of GATK's BWA and MuTect2 algorithms from Sentieon. Variants were filtered based on supporting reads and variant allele thresholds, with synonymous variants and common SNPs removed. High-confidence variants were further filtered by membership in the four KEGG pathways associated with IDH1 and IDH2. Identified variants were corroborated with metabolomics data from the two unique IDH wt tumors compared with classical GBM IDH wt, oligodendrogliomas IDH mut and astrocytomas IDH mut to identify putative drivers of an IDH mutant-like metabolomic phenotype in these unique IDH wt tumors. Despite the lack of an IDH mutation, one patient survived 45.6 months and the other patient remains alive at last follow up 64 months post diagnosis, much longer than the 16-18-month median survival typical of patients with GBM IDH wt. WES of outlier IDH wt tumor samples revealed 65 unique mutations in the queried KEGG pathways, of which 34 had a variant allele frequency > = 0.15. These variants were processed in Gprofiler, confirming expected enrichment of the carboxylic acid metabolic biologic process, a functional gene set consisting of TCA genes, among these variants (p = 0.002, 3.6-fold enrichment). Accordingly, metabolite levels of intermediates of the TCA cycle, including malate and isocitrate were decreased in the outlier tumor samples compared to classic GBMs IDH wt (p<0.001). Presence of genetic alterations in key variants of the carboxylic acid metabolic biologic process (including ME1, GYP4F3, PTGIS, PFKL, PSPH, AKR1A1, HK2, NOS1) correlated with improved overall survival among GBM patients in the TCGA (p = 0.04). Laboratory validation of these findings in preclinical GBM models is ongoing. Disruption of the TCA cycle independent of an IDH mutation is associated with favorable survival in GBM. Pharmacologic inhibition of these pathways may be a promising strategy to improve GBM outcomes.

Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis

Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.

The rice narrow leaf2 and narrow leaf3 loci encode WUSCHEL‐related homeobox 3A (OsWOX3A) and function in leaf, spikelet, tiller and lateral root development

· In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains. · We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains. · Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. · OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transport-related genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots. OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.

New phenotypes generated by the G57R mutation of BUD23 in Saccharomyces cerevisiae

BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism.

Phenotypic inheritance induced by hairpin RNA in &amp;lt;italic&amp;gt;Drosophila&amp;lt;/italic&amp;gt;

Phenotypic inheritance induced by RNA has been documented in mouse and Caenorhabditis elegans. Here we report a similar inheritance in Drosophila. Mutant phenotypes of eye defects and antenna duplication generated from the crossing of one RNA interference (RNAi) transgenic line harboring one hairpin RNA transgene with a GAL4 driver line were inherited independently of the GAL4 driver. Hairpin RNA injection experiments demonstrated that the hairpin RNA could induce heritable mutant-like phenotypes on the eye and antenna. The penetrance of mutant phenotypes was reduced when the mutants were crossed to ago1 and piwi mutants. Our data suggest that hairpin RNA can induce phenotypic inheritance in Drosophila.

The Mammalian Cos2 Homolog Kif7 Plays an Essential Role in Modulating Hh Signal Transduction during Development

The Hedgehog (Hh) signaling pathway regulates development in animals ranging from flies to humans. Although its framework is conserved, differences in pathway components have been reported. A kinesin-like protein, Costal2 (Cos2), plays a central role in the Hh pathway in flies. Knockdown of a zebrafish homolog of Cos2, Kif7, results in ectopic Hh signaling, suggesting that Kif7 acts primarily as a negative regulator of Hh signal transduction. However, in vitro analysis of the function of mammalian Kif7 and the closely related Kif27 has led to the conclusion that neither protein has a role in Hh signaling. Using Kif7 knockout mice, we demonstrate that mouse Kif7, like its zebrafish and Drosophila homologs, plays a role in transducing the Hh signal. We show that Kif7 accumulates at the distal tip of the primary cilia in a Hh-dependent manner. We also demonstrate a requirement for Kif7 in the efficient localization of Gli3 to cilia in response to Hh and for the processing of Gli3 to its repressor form. These results suggest a role for Kif7 in coordinating Hh signal transduction at the tip of cilia and preventing Gli3 cleavage into a repressor form in the presence of Hh.

Calcium Regulation of Sodium Hypersensitivities of sos3 and athkt1 Mutants

T-DNA disruption mutations in the AtHKT1 gene have previously been shown to suppress the salt sensitivity of the sos3 mutant. However, both sos3 and athkt1 single mutants show sodium (Na+) hypersensitivity. In the present study we further analyzed the underlying mechanisms for these non-additive and counteracting Na+ sensitivities by characterizing athkt1-1 sos3 and athkt1-2 sos3 double mutant plants. Unexpectedly, mature double mutant plants grown in soil clearly showed an increased Na+ hypersensitivity compared with wild-type plants when plants were subjected to salinity stress. The salt sensitive phenotype of athkt1 sos3 double mutant plants was similar to that of athkt1 plants, which showed chlorosis in leaves and stems. The Na+ content in xylem sap samples of soil-grown athkt1 sos3 double and athkt1 single mutant plants showed dramatic Na+ overaccumulation in response to salinity stress. Salinity stress analyses using basic minimal nutrient medium and Murashige-Skoog (MS) medium revealed that athkt1 sos3 double mutant plants show a more athkt1 single mutant-like phenotype in the presence of 3 mM external Ca2+, but show a more sos3 single mutant-like phenotype in the presence of 1 mM external Ca2+. Taken together multiple analyses demonstrate that the external Ca2+ concentration strongly impacts the Na+ stress response of athkt1 sos3 double mutants. Furthermore, the presented findings show that SOS3 and AtHKT1 are physiologically distinct major determinants of salinity resistance such that sos3 more strongly causes Na+ overaccumulation in roots, whereas athkt1 causes an increase in Na+ levels in the xylem sap and shoots and a concomitant Na+ reduction in roots.

Isolation and characterization of an AGAMOUS homologue from cocoa

We report the cloning of a cDNA from TcAG, an AG ( Arabidopsis thaliana MADS-box C-type transcription factor gene AGAMOUS) homologue from cocoa ( Theobroma cacao L.). TcAG was in the cocoa flower expressed primarily in stamens and ovaries, comparable to AG in Arabidopsis. Additionally, we found that TcAG is also expressed in the fruit (pod) wall and during its entire development, as well as in the fruit pulp. Ectopic expression of TcAG in transgenic A. thaliana plants resulted in a range of weak to strong apetala2 ( ap2) mutant-like phenotypes as well as early flowering and curly leaves, as observed in other studies of plants overexpressing a functional AG homologue. The severity of the phenotypes correlated positively with the TcAG transcript level in the transgenic plants.

A Temperature-sensitive Mechanism That Regulates Post-translational Stability of a Plastidial ω-3 Fatty Acid Desaturase (FAD8) in Arabidopsis Leaf Tissues

Trienoic fatty acids (TAs) are the major constituents in plant membrane lipids. In Arabidopsis, two plastidial isozymes of omega-3 fatty acid desaturase, FAD7 and FAD8, are the major contributors for TA production in leaf tissues. Despite a high degree of structural relatedness, activities of these two isozymes are regulated differentially in response to temperature. Elevated temperatures lead to decreases in leaf TA level due to temperature sensitivity of FAD8 activity. A series of FAD7-FAD8 chimeric genes, each encoding a functional plastidial omega-3 desaturase, were introduced into the Arabidopsis fad7fad8 double mutant. Constructs with or without a c-Myc epitope tag were tested. Functionality of each chimeric gene in response to temperature was assayed by Northern and Western analyses and by examining the fatty acid composition. All transformants harboring a chimeric gene containing the FAD8-derived C-terminal coding region (44 amino acids) showed a marked decrease in TA level when exposed to high temperature, similarly as transgenic lines complemented with the native form of FAD8. The reduction of TA level was accompanied by a decrease in the amount of omega-3 desaturase protein but not necessarily by a decrease in its transcript level. Analysis of the decay of c-Myc-tagged products after inhibiting protein synthesis revealed that the FAD8-derived C-terminal region acts in an autoregulatory fashion to destabilize the protein at high temperature. This suggests that the regulation of post-translational stability of FAD8 provides an important regulatory mechanism for modifying its activity in response to temperature, mediating a decrease in TA level at elevated temperatures.

EMBRYONIC FLOWER2, a novel polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis.

In higher plants, developmental phase changes are regulated by a complex gene network. Loss-of-function mutations in the EMBRYONIC FLOWER genes (EMF1 and EMF2) cause Arabidopsis to flower directly, bypassing vegetative shoot growth. This phenotype suggests that the EMF genes play a major role in repression of the reproductive program. Positional cloning of EMF2 revealed that it encodes a zinc finger protein similar to FERTILIZATION-INDEPENDENT SEED2 and VERNALIZATION2 of Arabidopsis. These genes are characterized as structural homologs of Suppressor of zeste 12 [Su(z)12], a novel Polycomb group gene currently identified in Drosophila. In situ hybridization studies have demonstrated that EMF2 RNA is found in developing embryos, in both the vegetative and the reproductive shoot meristems, and in lateral organ primordia. Transgenic suppression of EMF2 produced a spectrum of early-flowering phenotypes, including emf2 mutant-like phenotype. This result confirms the role of EMF2 in phase transitions by repressing reproductive development.

On Phenocopies, Their Developmental Physiology and Genetic Meaning

To sum up: Recognizing that certain facts remain which at present defy interpretation and which call for further analysis, I believe that the evidence which has thus far been brought forward from various sources justifies the following interpretation of the genesis of phenocopies. As mutant genes produce a dislocation in the integrated functions of the normal genotype, so do certain external agents, whether physical or chemical, intervene in gene-determined developmental events by preventing them at one point or another from accomplishing their appointed ends, leading thereby to a mutant-like phenotype. The point of interference varies according to the existing genotype, the developmental stage at intervention, kind and quantity of the external agent, and still other factors. But I believe that in all instances the resulting phenocopies are the result of external pressure which has a suppressing, retarding or disorienting effect on one or more gene-controlled components of the normal or mutant genotype in question. It is probable that with sufficient or the proper kind of pressure many gene-controlled events can be made to appear as being the result of sub-threshold alleles, but I do not believe that the existence of such isoalleles as determiners of phenocopies has been proven or that it is likely. I believe that the phenotypic homologies between mutants and phenocopies, whether they be only superficial or intrinsic, represent two different ways in which weaknesses of ontogenetic integration are revealed. The fact that, in our experience, those mutant phenotypes which exist as several independent gene substitutions are also the ones which can most readily be produced as phenocopies, and that the affected events depend on complex developmental interactions presumably enhances the likelihood of "derailments" to occur. The deficiencies in developmental integration and stability which we have in mind must, of course, be viewed as a failure of evolution to provide me chanisms which could cope with the genic or environmental sources of dislocation. There is abundant evidence, however, for the widespread occurrence of specific modifiers, as constituents of the residual genotype, which in a similar manner alleviate mutant and phenocopic development. This is important in view of the many facts which suggest that these modifiers are in reality an integral, if varying, part of the normal genic endowment of organisms. Taking all evidence into account it seems to me that the most exciting aspect of the study of phenocopies is the opportunity it may provide of shedding light, if only indirectly, on the developmental functions of that awesome skeleton in the closets of genetical science-the normal genotype.