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Key message Novel disease resistance gene paralogues are generated by targeted chromosome cleavage of tandem duplicated NBS-LRR gene complexes and subsequent DNA repair in soybean. This study demonstrates accelerated diversification of innate immunity of plants using CRISPR. Abstract Nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) gene families are key components of effector-triggered immunity. They are often arranged in tandem duplicated arrays in the genome, a configuration that is conducive to recombinations that will lead to new, chimeric genes. These rearrangements have been recognized as major sources of novel disease resistance phenotypes. Targeted chromosome cleavage by CRISPR/Cas9 can conceivably induce rearrangements and thus emergence of new resistance gene paralogues. Two NBS-LRR families of soy have been selected to demonstrate this concept: a four-copy family in the Rpp1 region (Rpp1L) and a large, complex locus, Rps1 with 22 copies. Copy-number variations suggesting large-scale, CRISPR/Cas9-mediated chromosome rearrangements in the Rpp1L and Rps1 complexes were detected in up to 58.8% of progenies of primary transformants using droplet-digital PCR. Sequencing confirmed development of novel, chimeric paralogs with intact open reading frames. These novel paralogs may confer new disease resistance specificities. This method to diversify innate immunity of plants by genome editing is readily applicable to other disease resistance genes or other repetitive loci.

In people with schizophrenia, self-efficacy (i.e. the belief in one's capability to perform particular tasks/skills) is associated with and motivates performance of social, health and independent living behaviours. Less well known is whether self-efficacy is associated with subjective quality of life (sQoL) or whether psychopathology impacts this relationship.Measure whether greater self-efficacy is associated with greater community functioning and sQoL and whether emotional discomfort mediates this relationship.Fifty-two community living people with schizophrenia completed measures of self-efficacy for everyday living and social situations, clinical symptoms, sQoL and community functioning.Greater everyday living and social self-efficacy was significantly correlated with greater sQoL and community functioning and lower emotional discomfort (Holding negative capability self-beliefs may contribute to poorer outcome for people with schizophrenia. Intervention aimed at facilitating recovery should therefore provide opportunities to develop knowledge and skills required for success in desired life roles and the belief that tasks required for success can be performed.

Naturally occurring chromosomal crossovers (CO) during meiosis are a key driver of genetic diversity. The ability to target CO at specific allelic loci in hybrid plants would provide an advantage to the plant breeding process by facilitating trait introgression, and potentially increasing the rate of genetic gain. We present the first demonstration of targeted CO in hybrid maize utilizing the CRISPR Cas12a system. Our experiments showed that stable and heritable targeted CO can be produced in F1 somatic cells using Cas12a at a significantly higher rate than the natural CO in the same interval. Molecular characterization of the recombinant plants demonstrated that the targeted CO were driven by the non-homologous end joining (NHEJ) or HDR repair pathways, presumably during the mitotic cell cycle. These results are a step towards the use of RNA-guided nuclease technology to simplify the creation of targeted genome combinations in progeny and accelerate breeding., Kouranov et al. demonstrate Cas12a-mediated targeted chromosomal crossover (CO) in transgenic hybrid maize plants and produce heritable crossover in F1 plants when Cas12a transgene is segregated away. The authors also report that the frequency of LbCas12a-mediated CO is higher than control natural CO in the same intervals in the progeny of 4 different events.

Saliva is increasingly being targeted for metabolic studies due to its non-invasive collection methods. Tracing levels of certain metabolites within biofluids can provide indications for a myriad of physiological conditions. This study was performed on a panel of eight analytes found in saliva that have shown associations with physiological conditions of human performance, such as stress, inflammation, and circadian rhythm. This dual polarity liquid chromatography tandem mass spectrometric (LCMS/MS) method was developed to accommodate a diverse group of analytes including steroids, alkaloids, and neurotransmitters. Samples collected during field exercises from soldiers were compared to those of civilians and baseline levels of each of these compounds was determined in saliva. Although most analytes showed no significant differences between the two populations, relative cortisol levels were higher for soldiers than for civilians. This developed dual polarity LCMS/MS method can be applied to very diverse groups of salivary analytes simultaneously.

Objectives:The aim of this study was to evaluate the impact of computer-assisted “drill-and-strategy” cognitive remediation (CR) for community-dwelling individuals with schizophrenia on cognition, everyday self-efficacy, and independent living skills.Methods:Fifty-six people with schizophrenia or schizoaffective disorder were randomized into CR or computer game (CG) playing (control), and offered twenty 1-hr individual sessions in a group setting over 10 weeks. Measures of cognition, psychopathology, self-efficacy, quality of life, and independent living skills were conducted at baseline, end-group and 3 months following intervention completion.Results:Forty-three participants completed at least 10 sessions and the end-group assessment. Linear mixed-effect analyses among completers demonstrated a significant interaction effect for global cognition favoring CR (p=.028). CR-related cognitive improvement was sustained at 3-months follow-up. At end-group, 17 (77%) CR completers showed a reliable improvement in at least one cognitive domain. A significant time effect was evident for self-efficacy (p=.028) with both groups improving over time, but no significant interaction effect was observed. No significant effects were found for other study outcomes, including the functional measure.Conclusions:Computer-assisted drill-and-strategy CR in schizophrenia improved cognitive test performance, while participation in both CR and CG playing promoted enhancements in everyday self-efficacy. Changes in independent living skills did not appear to result from CR, however. Adjunctive psychosocial rehabilitation is likely necessary for improvements in real-world community functioning to be achieved. (JINS, 2018, 24, 549–562)

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101–105 %) and high precision (5–8 %) for the detection of these five nerve agent hydrolysis products in serum.

Background Cognitive remediation (CR) therapies are upheld as promising methods of reducing cognitive impairment in schizophrenia. However, controlled trials with blind assessors and active comparison conditions are lacking, along with evidence of generalization of CR to everyday function and self-efficacy. In addition, the role of patient-specific factors such as motivation in predicting adherence and training outcomes has not been investigated. This assessor-blinded, randomized controlled trial compared the impact of ‘drill-and-strategy’ CR with a computer game (CG) control delivered in a group-setting on cognitive function, independent living skills and self-efficacy, and examined the impact of intrinsic motivation on group attendance and treatment response. Methods Fifty-six people with schizophrenia or schizoaffective disorder were randomized into CR or CG, and offered 20 one-hour sessions over 10 weeks. Measures of cognition (MATRICS consensus cognitive battery), psychopathology (Positive and Negative Syndrome Scale), self-efficacy (Revised Self Efficacy Scale) and independent living skills (Independent Living Skills Survey) were administered at baseline, end-group and three-months post-group. Intrinsic motivation (Intrinsic Motivation Inventory-Schizophrenia Research) was measured in-session at baseline and end-group. Results Primary analysis was conducted for participants who completed end-therapy assessment (CR=22; Control=21). Linear mixed-effect analysis found a significant interaction effect for cognition (p=.028). Pairwise comparisons revealed that cognition was better at end-group and three-month follow-up than baseline for CR completers, with no differences between timepoints for controls. Three-quarters (77%) of CR completers showed a reliable improvement in at least one cognitive domain. A significant time effect was also evident for self-efficacy (p=.028), with the combined groups showing higher self-efficacy at end-group than baseline. No changes in independent living skills were observed. Early reports of program value predicted session attendance above baseline cognitive and clinical symptoms. Enhanced program interest and value over time increased the likelihood of reliable cognitive improvement. Discussion Drill-and-strategy CR, delivered as a stand-alone treatment in a group setting, may improve cognition in schizophrenia when compared to active controls. Enhancing motivation may increase the likelihood of achieving meaningful cognitive improvements. This type of CR, however, may not translate to independent living domains, even if enhanced cognition and confidence in completing everyday behaviors is achieved. Independent living skills may need to be targeted directly to achieve meaningful changes in this domain.

In separate, replicated experiments, we experimentally established wash-off coefficients (kw) for the chemical agent VX on grass, utilizing 1 and 3 L VX droplets at 0.017 (1 min), 1, and 4 h after dissemination. A 10 mm (0.39 in.) rain event at 0.017 h after dissemination washed off 95 and 83% of the 1 and 3 L VX droplets, respectively. At 1 h after dissemination, a 10 mm rain event washed off 0.03 and 0.5% of the 1 and 3 L VX droplets, respectively. At 0.017 h after dissemination, the kw values for 1 and 3 L droplets were 0.095 and 0.083 mm1, respectively. At 1 and 4 h after dissemination, the kw values for VX were approximately 3 orders of magnitude less than those at 0.017 h. Grass contaminated with 3 L VX droplets was exposed to multiple (10) 100 L light rain events, followed by multiple (10) surface wipes at 0.017, 0.5, 1, 4, and 24 h after dissemination. The cumulative proportions of 3 L VX droplets washed off by the rain events at 0.017 and 1 h after dissemination were 75.3 and 0.99%, respectively, and the cumulative proportions of VX wiped off after those rain events were 0.8 and 0.3%, respectively. Results from these investigations of agentplant interactions provide input for predictive models and functional information for Go/No-Go decisions that can affect soldiers on agent-contaminated battlefields.

Exposures to organophosphorus nerve agents (OPNA) remain a threat to both civilian and military populations. Verification of exposures typically involves determinations of urinary metabolites or adducted proteins in blood. Urinary alkyl methylphosphonic acid metabolites resulting from hydrolysis of OPNAs provide a convenient marker for OPNA exposure. In a military setting, urine is a relatively easy sample to obtain, and a rapid turnaround for analyses for the identification of metabolites is critical for field commanders. Timely information on use and identity of OPNAs facilitates decisions regarding employment of personal protective equipment and additional strategies to mitigate additional exposure(s). Herein, we report the development of a rapid mass spectrometric (MS) method to identify OPNA metabolites directly from urine with no sample preparation. Synthetic urine spiked with multiple OPNA metabolites was analyzed using an atmospheric solids analysis probe (ASAP) attached to a high resolution mass spectrometer. The alkyl methylphosphonic acid metabolites resulting from hydrolysis of sarin, cyclosarin, soman, and Russian VX were clearly detectable down to a level of 1.0 ng/ml. The ability to rapidly detect OPNA metabolites in unprepared urine allows for the design of a field-deployable device that could afford field personnel the ability to rapidly screen individuals for specific OPNA exposure. In addition, this provides proof-of-concept evidence that a fieldable ASAP-MS device could afford personnel the ability to rapidly detect OPNAs on skin, equipment, and other porous surfaces. Published 2012. This article is a US Government work and is in the public domain in the USA.

It is known that cyanide is converted to thiocyanate in the presence of the enzyme rhodanese. The enzyme is activated by sulfur transfer from an appropriate sulfur donor. The activated enzyme then binds cyanide and transfers the sulfur atom to cyanide to form thiocyanate. This project began as an exploration of the ability of disulfides to act as sulfur donors in the rhodanese-mediated detoxification of cyanide. To our surprise, and contrary to expectations based on efficacy studies in vivo, our in vitro results showed that disulfides are rather poor sulfur donors. The transfer of a sulfur atom from a disulfide to the enzyme must occur via cleavage of a carbon-sulfur bond either of the original disulfide or in a mixed disulfide arising from the reaction of rhodanese with the original disulfide. Extending the reaction time and addition of chloride anion (a nucleophile) did not significantly change the results of the experiment. Using ultrasound as a means of accelerating bond cleavage also had a minimal effect. Those results ruled out cleavage of the carbon-sulfur bond in the original disulfide but did not preclude formation of a mixed disulfide. S-Methyl methylthiosulfonate (MTSO) was used to determine whether a mixed disulfide, if formed, would result in transfer of a sulfur atom to rhodanese. While no thiocyanate was formed in the reaction between cyanide and rhodanese exposed to MTSO, NMR analysis revealed that MTSO reacted directly with cyanide anion to form methyl thiocyanate. This result reveals the body's possible use of oxidized disulfides as a first line of defense against cyanide intoxication. The oxidation of disulfides to the corresponding thiosulfinate or thiosulfonate will result in facilitating their reaction with other nucleophiles. The reaction of an oxidized disulfide with a sulfur nucleophile from glutathione could be a plausible origin for the cyanide metabolite 2-aminothiazoline-4-carboxylic acid.

Eukaryotic genomes can be organized into distinct domains of heterochromatin or euchromatin. In the fission yeast Schizosaccharomyces pombe , assembly of heterochromatin at the silent mating-type region is critical for cell fate determination in the form of mating-type switching. Here, we report that the ubiquitin ligase, Msc1, is a critical factor required for proper cell fate determination in S. pombe . In the absence of Msc1, the in vivo mobility of Swi6 at heterochromatic foci is compromised, and centromere heterochromatin becomes hyperenriched with the heterochromatin binding protein Swi6/HP1. However, at the mating-type locus, Swi6 recruitment is defective in the absence of Msc1. Therefore, Msc1 links maintaining dynamic heterochromatin with proper heterochromatin assembly and cell fate determination. These findings have implications for understanding mechanisms of differentiation in other organisms.

In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.

An unidentified white powder collected as evidence in an intelligence investigation was characterized exclusively by nuclear magnetic resonance (NMR) analysis. A small fraction of the powder dissolved in D 2 O was subjected to a series of one- and two-dimensional techniques which were used to elucidate the molecular structure of the powder's major component and positively identify it as the scopolamine biotoxin. Quantitative one-dimensional experiments identified individual proton and carbon atom sites, and conventional 14 N spectroscopy detected a single nitrogen atom site. Heteronuclear single quantum coherence data correlated all protons to their directly bonded carbon atom, and together with the quantitative spectra, were used to determine the number of protons directly bonded to each carbon atom. The presence of a methyl, carboxyl, and a benzyl group was also identified from these data. Correlation spectroscopy detected a three proton and a nine proton J H,H network, representing a CH 2 CH moiety and seven carbon atom ring, respectively. These five elements were assembled into an almost complete molecular structure by using long-range, J-coupled, 'H- 13 C pairs detected by heteronuclear multiple bond correlation (HMBC) spectroscopy and 1 H- 1 H dipolar-coupled pairs found from nuclear Overhauser effect spectroscopy (NOESY) data. Additional oxygen atom sites were inferred from 1 H- 13 C correlation intensities in the HMBC spectra along with 'H and 13 C chemical shift values, or directly from NOESY correlations. Only a single oxygen atom site could not be inferred from NMR data, but its presence was inferred from comparisons to target analyte structures to complete the structure of the scopolamine molecule. To confirm these results, an ethanol/H 2 O solution of the powder was analyzed by direct infusion into an ion trap mass spectrometer. A prominent base signal was observed at m/z 304.1 amu, corresponding to the protonated molecular ion of scopolamine. Subsequently, the ion was selected and subjected to collision-induced dissociation, producing characteristic major MS/MS fragments at m/z 138.1 and 156.1. Comparisons of 1 H and 13 C chemical shift values and J H,H values measured from our NMR data were found to agree very favorably with previously reported values for scopolamine in D 2 O.

An analytical method for determining exposure to 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD) has been enhanced. The method is based on the cleavage of adducted HD (protein-hydroxyethylthioethyl esters) to produce thiodiglycol. Following cleavage, a deuterated internal standard is added, and the analytes are extracted, derivatized, and analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry. Inclusion of a concentration step, addition of solid sodium bicarbonate to neutralize excess derivatization reagent, and optimization of method and instrument conditions provided dramatic increases in signal-to-noise ratio. A five-day precision and accuracy study was conducted, including interday and intraday unknown analysis. Linearity was verified by a R(2)0.9995 for all five curves evaluated. The precision and accuracy of the assay were demonstrated to be excellent by evaluation of the interday and intraday unknown samples (10% relative standard deviation and relative error in most cases). Statistical treatment of the method blanks and calibration results demonstrated a reduction in the limit of quantitation from 25 nM (HD, human plasma, in vitro) to 1.56 nM. Sample and calibration stability through the analytical sequence was established by the inclusion of continuing calibration verification standards (5% error). Short-term sample stability was verified by reinjection of a calibration set after 18 days (R(2) = 0.9997). Quantitative agreement with the previous method was supported by the analysis of a 50 nM standard protein sample (HD, rat plasma) with both methodologies (1% error).

Sulfur mustard (HD) is an alkylating agent that reacts rapidly with macromolecular targets resulting in the formation of stable adducts providing depots for markers of exposure. The purpose of this study was to validate an analytical procedure for detection of HD-plasma protein adducts and to establish the utility of the method in an HD rat inhalation study. Calibration curves were prepared in human and rat plasma at six levels of HD (12.5 to 400 nM). Correlation coefficients for the mean data were 0.9987 for human and 0.9992 for rat plasma. The percent coefficient of variation (%CV) derived from the mean concentration data ranged from 0.53 to 14.1% in human (n = 5) and 0.57 to 10.63% in rat (n = 6) plasma. Intraday and interday precision and accuracy studies were conducted at three concentration levels (25, 150, 300 nM) to represent low, medium, and high concentrations of HD relative to those employed in the calibration curve. Precision and accuracy were assessed by determining %CV and % error, respectively. For intra- and interday studies, the %CVs and absolute % errors were less than 15%. The limits of quantitation were 20.88 nM for human and 16.73 nM for rat plasma. In animal studies, rats received nebulized HD at six doses. The data indicate a dose-dependent relationship between maximal plasma concentrations and dose administered (R(2) = 0.9728). Results from this study indicate an accurate, precise, and sensitive method. The method was useful in determining plasma protein adduct formation in a rat inhalation model.

Eukaryotes have hundreds (sometimes thousands) of ribosomal RNA (rRNA) genes whose transcription by RNA polymerase I helps establish the proliferative ability of cells by dictating the pace of ribosome production and protein synthesis. Interestingly, only a subset of the total rRNA gene pool is active at any one time, making rRNA genes attractive for understanding the dynamic balance between gene silencing and activation. However, the fact that rRNA genes are essentially identical in sequence in a pure species has been an obstacle to telling apart the active and inactive genes. Nature has provided one solution to this conundrum in the form of the epigenetic phenomenon, nucleolar dominance: the transcriptional silencing of one parental set of rRNA genes in a genetic hybrid. Parental genes in hybrids typically differ in sequence as well as expression, allowing a definition of the chromatin modifications of rRNA genes in the on and off states in vivo. By exploiting nucleolar dominance in plants, we recently showed that concerted changes in DNA methylation and histone methylation comprise an epigenetic switch that turns rRNA genes on and off. Independent studies using mouse and human cells have led to similar conclusions, implicating chromatin modifications as important components of the regulatory networks that control the effective dosage of active rRNA genes.

Vitamin K1 in infant formulas and milk products is determined by reversed-phase liquid chromatography (LC) with UV detection. The sample is hydrolyzed enzymatically, and the vitamin is extracted with hexane. Fractionation by normal-phase semipreparative LC is followed by analytical LC, with quantitation by the internal standard technique. Recovery of the analyte was 97.4 ± 2.8%. Linearity was established between 0.05 and 4.0 fig/mL. The limit of quantitation is 0.5 μg/100 g for milk powder, which allows the method to quantitate endogenous levels of vitamin K1.

Exposures to organophosphorus nerve agents (OPNA) remain a threat to both civilian and military populations. Verification of exposures typically involves determinations of urinary metabolites or adducted proteins in blood. Urinary alkyl methylphosphonic acid metabolites resulting from hydrolysis of OPNAs provide a convenient marker for OPNA exposure. In a military setting, urine is a relatively easy sample to obtain, and a rapid turnaround for analyses for the identification of metabolites is critical for field commanders. Timely information on use and identity of OPNAs facilitates decisions regarding employment of personal protective equipment and additional strategies to mitigate additional exposure(s). Herein, we report the development of a rapid mass spectrometric (MS) method to identify OPNA metabolites directly from urine with no sample preparation. Synthetic urine spiked with multiple OPNA metabolites was analyzed using an atmospheric solids analysis probe (ASAP) attached to a high resolution mass spectrometer. The alkyl methylphosphonic acid metabolites resulting from hydrolysis of sarin, cyclosarin, soman, and Russian VX were clearly detectable down to a level of 1.0 ng/ml. The ability to rapidly detect OPNA metabolites in unprepared urine allows for the design of a field-deployable device that could afford field personnel the ability to rapidly screen individuals for specific OPNA exposure. In addition, this provides proof-of-concept evidence that a fieldable ASAP-MS device could afford personnel the ability to rapidly detect OPNAs on skin, equipment, and other porous surfaces. Published 2012. This article is a US Government work and is in the public domain in the USA.

Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor's rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana –derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generation.

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.

Three assays useful for detecting specific RNA transcripts are primer extension, S1 nuclease protection, and reverse-transcription-cleaved amplified polymorphic sequence (RT-CAPS) analysis. All three of these techniques are used routinely for gene expression analyses and allow insights not possible by RNA blot (northern blot) hybridization. In this chapter, we describe how the primer extension, S1 nuclease protection, and RT-CAPS methods can be used to discriminate one or more parental or progenitor alleles in hybrids or allopolyploids. We discuss the rationale for using the different techniques and provide examples of the data generated.

Three assays useful for detecting specific RNA transcripts are primer extension, S1 nuclease protection, and reverse-transcription-cleaved amplified polymorphic sequence (RT-CAPS) analysis. All three of these techniques are used routinely for gene expression analyses and allow insights not possible by RNA blot (northern blot) hybridization. In this chapter, we describe how the primer extension, S1 nuclease protection, and RT-CAPS methods can be used to discriminate one or more parental or progenitor alleles in hybrids or allopolyploids. We discuss the rationale for using the different techniques and provide examples of the data generated.

Eukaryotes have hundreds (sometimes thousands) of ribosomal RNA (rRNA) genes whose transcription by RNA polymerase I helps establish the proliferative ability of cells by dictating the pace of ribosome production and protein synthesis. Interestingly, only a subset of the total rRNA gene pool is active at any one time, making rRNA genes attractive for understanding the dynamic balance between gene silencing and activation. However, the fact that rRNA genes are essentially identical in sequence in a pure species has been an obstacle to telling apart the active and inactive genes. Nature has provided one solution to this conundrum in the form of the epigenetic phenomenon, nucleolar dominance: the transcriptional silencing of one parental set of rRNA genes in a genetic hybrid. Parental genes in hybrids typically differ in sequence as well as expression, allowing a definition of the chromatin modifications of rRNA genes in the on and off states in vivo. By exploiting nucleolar dominance in plants, we recently showed that concerted changes in DNA methylation and histone methylation comprise an epigenetic switch that turns rRNA genes on and off. Independent studies using mouse and human cells have led to similar conclusions, implicating chromatin modifications as important components of the regulatory networks that control the effective dosage of active rRNA genes.

Histone acetylation and deacetylation are connected with transcriptional activation and silencing in many eukaryotic organisms. Gene families for enzymes that accomplish these modifications show a surprising multiplicity in sequence and expression levels, suggesting a high specificity for different targets. We show that mutations in Arabidopsis (Arabidopsis thaliana) HDA6, a putative class I histone deacetylase gene, result in loss of transcriptional silencing from several repetitive transgenic and endogenous templates. Surprisingly, total levels of histone H4 acetylation are only slightly affected, whereas significant hyperacetylation is restricted to the nucleolus organizer regions that contain the rDNA repeats. This switch coincides with an increase of histone 3 methylation at Lys residue 4, a modified DNA methylation pattern, and a concomitant decondensation of the chromatin. These results indicate that HDA6 might play a role in regulating activity of rRNA genes, and this control might be functionally linked to silencing of other repetitive templates and to its previously assigned role in RNA-directed DNA methylation.

Eukaryotes regulate the effective dosage of their ribosomal RNA (rRNA) genes, expressing fewer than half of the genes at any one time. Likewise, genetic hybrids displaying nucleolar dominance transcribe rRNA genes inherited from one parent but silence the other parental set. We show that rRNA gene dosage control and nucleolar dominance utilize a common mechanism. Central to the mechanism is an epigenetic switch in which concerted changes in promoter cytosine methylation density and specific histone modifications dictate the on and off states of the rRNA genes. A key component of the off switch is HDT1, a plant-specific histone deacetylase that localizes to the nucleolus and is required for H3 lysine 9 deacetylation and subsequent H3 lysine 9 methylation. Collectively, the data support a model in which cytosine methylation and histone deacetylation are each upstream of one another in a self-reinforcing repression cycle.

Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.

A new study of ribosomal RNA gene silencing in the mouse provides clues as to how repressive chromatin states become established. At center stage is a chromatin remodeling complex that recruits DNA methyltransferase and histone deacetylase activities to the gene promoter, initiating a process that includes de novo DNA methylation, methylation of histone H3 on Lys9 and heterochromatin protein binding.