The genetic and molecular basis of haploinsufficiency in flowering plants.

In diploid organisms, haploinsufficiency can be defined as the requirement for more than one fully functional copy of a gene. In contrast to most genes, whose loss-of-function alleles are recessive, loss-of-function alleles of haploinsufficient genes are dominant. However, forward and reverse genetic screens are biased toward obtaining recessive, loss-of-function mutations, and therefore, dominant mutations of all types are underrepresented in mutant collections. Despite this underrepresentation, haploinsufficient loci have intriguing implications for studies of genome evolution, gene dosage, stability of protein complexes, genetic redundancy, and gene expression. Here we review examples of haploinsufficiency in flowering plants and describe the underlying molecular mechanisms and evolutionary forces driving haploinsufficiency. Finally, we discuss the masking of haploinsufficiency by genetic redundancy, a widespread phenomenon among angiosperms.

Versatile mapping-by-sequencing with Easymap v.2.

Mapping-by-sequencing combines Next Generation Sequencing (NGS) with classical genetic mapping by linkage analysis to establish gene-to-phenotype relationships. Although numerous tools have been developed to analyze NGS datasets, only a few are available for mapping-by-sequencing. One such tool is Easymap, a versatile, easy-to-use package that performs automated mapping of point mutations and large DNA insertions. Here, we describe Easymap v.2, which also maps small insertion/deletions (InDels), and includes workflows to perform QTL-seq and variant density mapping analyses. Each mapping workflow can accommodate different experimental designs, including outcrossing and backcrossing, F2, M2, and M3 mapping populations, chemically induced mutation and natural variant mapping, input files containing single-end or paired-end reads of genomic or complementary DNA sequences, and alternative control sample files in FASTQ and VCF formats. Easymap v.2 can also be used as a variant analyzer in the absence of a mapping algorithm and includes a multi-threading option.

Mapping-by-Sequencing of Point and Insertional Mutations with Easymap.

Random mutagenesis followed by screening for phenotypes of interest is a widely used strategy for genetic dissection of biological pathways; however, identifying the causal gene traditionally required time-consuming mapping approaches based on iterative linkage analysis. Mapping-by-sequencing accelerates this process, efficiently linking the phenotype of a mutant to a narrow candidate genomic region, using next-generation sequencing (NGS) data from a mapping population segregating for the mutant phenotype. To enable researchers at any bioinformatics skill level to conduct mapping-by-sequencing, we developed the Easymap mapping software. In this protocol we break down the steps involved in mapping-by-sequencing. First, we describe different ways of obtaining a mapping population and the steps used to generate NGS data. Next, we show how to analyze the NGS data using Easymap and how to obtain a list of candidate mutations, along with comprehensive information for assessing the potential causality of each candidate. Thus, this protocol enables the user to conduct mapping-by-sequencing using Easymap, facilitating the identification of causal loci for a mutant phenotype of interest.

Easymap: A User-Friendly Software Package for Rapid Mapping-by-Sequencing of Point Mutations and Large Insertions.

Mapping-by-sequencing strategies combine next-generation sequencing (NGS) with classical linkage analysis, allowing rapid identification of the causal mutations of the phenotypes exhibited by mutants isolated in a genetic screen. Computer programs that analyze NGS data obtained from a mapping population of individuals derived from a mutant of interest to identify a causal mutation are available; however, the installation and usage of such programs requires bioinformatic skills, modifying or combining pieces of existing software, or purchasing licenses. To ease this process, we developed Easymap, an open-source program that simplifies the data analysis workflows from raw NGS reads to candidate mutations. Easymap can perform bulked segregant mapping of point mutations induced by ethyl methanesulfonate (EMS) with DNA-seq or RNA-seq datasets, as well as tagged-sequence mapping for large insertions, such as transposons or T-DNAs. The mapping analyses implemented in Easymap have been validated with experimental and simulated datasets from different plant and animal model species. Easymap was designed to be accessible to all users regardless of their bioinformatics skills by implementing a user-friendly graphical interface, a simple universal installation script, and detailed mapping reports, including informative images and complementary data for assessment of the mapping results. Easymap is available at http://genetics.edu.umh.es/resources/easymap; its Quickstart Installation Guide details the recommended procedure for installation.

Next-generation forward genetic screens: using simulated data to improve the design of mapping-by-sequencing experiments in Arabidopsis.

Forward genetic screens have successfully identified many genes and continue to be powerful tools for dissecting biological processes in Arabidopsis and other model species. Next-generation sequencing technologies have revolutionized the time-consuming process of identifying the mutations that cause a phenotype of interest. However, due to the cost of such mapping-by-sequencing experiments, special attention should be paid to experimental design and technical decisions so that the read data allows to map the desired mutation. Here, we simulated different mapping-by-sequencing scenarios. We first evaluated which short-read technology was best suited for analyzing gene-rich genomic regions in Arabidopsis and determined the minimum sequencing depth required to confidently call single nucleotide variants. We also designed ways to discriminate mutagenesis-induced mutations from background Single Nucleotide Polymorphisms in mutants isolated in Arabidopsis non-reference lines. In addition, we simulated bulked segregant mapping populations for identifying point mutations and monitored how the size of the mapping population and the sequencing depth affect mapping precision. Finally, we provide the computational basis of a protocol that we already used to map T-DNA insertions with paired-end Illumina-like reads, using very low sequencing depths and pooling several mutants together; this approach can also be used with single-end reads as well as to map any other insertional mutagen. All these simulations proved useful for designing experiments that allowed us to map several mutations in Arabidopsis.

Wound signaling of regenerative cell reprogramming.

Plants are sessile organisms that must deal with various threats resulting in tissue damage, such as herbivore feeding, and physical wounding by wind, snow or crushing by animals. During wound healing, phytohormone crosstalk orchestrates cellular regeneration through the establishment of tissue-specific asymmetries. In turn, hormone-regulated transcription factors and their downstream targets coordinate cellular responses, including dedifferentiation, cell cycle reactivation and vascular regeneration. By comparing different examples of wound-induced tissue regeneration in the model plant Arabidopsis thaliana, a number of key regulators of developmental plasticity of plant cells have been identified. We present the relevance of these findings and of the dynamic establishment of differential auxin gradients for cell reprogramming after wounding.

Antagonism at NMDA receptors, but not ß-adrenergic receptors, disrupts the reconsolidation of pavlovian conditioned approach and instrumental transfer for ethanol-associated conditioned stimuli.

RATIONALE: Reconsolidation is the process by which memories require restabilisation following destabilisation at retrieval. Since even old, well-established memories become susceptible to disruption following reactivation, treatments based upon disrupting reconsolidation could provide a novel form of therapy for neuropsychiatric disorders based upon maladaptive memories, such as drug addiction. Pavlovian cues are potent precipitators of relapse to drug-seeking behaviour and influence instrumental drug seeking through at least three psychologically and neurobiologically distinct processes: conditioned reinforcement, conditioned approach (autoshaping) and conditioned motivation (pavlovian-instrumental transfer or PIT). We have previously demonstrated that the reconsolidation of memories underlying the conditioned reinforcing properties of drug cues depends upon NMDA receptor (NMDAR)- and ß-adrenergic receptor (ßAR)-mediated signalling. However, it is unknown whether the drug cue memory representations underlying conditioned approach and PIT depend upon the same mechanisms. OBJECTIVES: Using orally self-administered ethanol as a reinforcer in two separate experiments, we investigated whether the reconsolidation of the memories underlying conditioned approach and PIT requires ßAR- and NMDAR-dependent neurotransmission. RESULTS: For ethanol self-administering but non-dependent rats, the memories underlying conditioned approach and PIT for a pavlovian drug cue were disrupted by the administration of the NMDAR antagonist MK-801, but not the administration of the ßAR antagonist propranolol, when given in conjunction with memory reactivation. CONCLUSIONS: As for natural reinforcers, NMDARs are required for the reconsolidation of all aspects of pavlovian drug memories, but ßARs are only required for the memory representation underlying conditioned reinforcement. These results indicate the potential utility of treatments based upon disrupting cue-drug memory reconsolidation in preventing relapse.

Toxicological assessment of beta-lapachone on organs from pregnant and non-pregnant rats.

Naphthoquinones have been studied extensively due to their activity as topoisomerase inhibitors. These enzymes are critical to DNA replication in cells. beta-Lapachone (beta-lap) is an o-naphthoquinone chemically obtained from lapachol. This work results in a toxicological evaluation of beta-lap in Wistar rats observing the following parameters: teratology, histology, hematology and serum biochemistry. The data demonstrate teratogenic action at the doses used, as well as hematological alterations in the total leukocytes, monocytes and segmented. The biochemical data demonstrated an increase in gamma glutamyl transferase, alkaline phosphatase and glutamate pyruvate transaminase levels. Histological study showed significant alterations in the spleen, however, the liver and kidney did not present significant alterations.

Determinaçäo de microhematócritos em centrífuga convencional / Microhematocrit determination in conventional centrifuge

A determinaçäo de microhematócritos é realizada em microcentrífugas, as quais nem sempre säo disponíveis em pequenos laboratórios de análises clínicas. Compara-se os valores de hematócritos realizados em tubos capilares utilizando-se centrífugas convencionais, com os valores de hematócritos obtidos segundo Wintrobe e empregando-se microcentrífugas. Os resultados obtidos indicam que a realizaçäo de microhematócritos em centrífuga convencional é perfeitamente viável