Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Calcium ions trigger the exposure of phosphatidylserine on the surface of necrotic cells

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an “eat-me” signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a “two-step” pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.

The Classic Lobe Eye Phenotype of Drosophila Is Caused by Transposon Insertion-Induced Misexpression of a Zinc-Finger Transcription Factor

Drosophila Lobe (L) alleles were first discovered ∼100 years ago as spontaneous dominant mutants with characteristic developmental eye defects. However, the molecular basis for L dominant eye phenotypes has not been clearly understood. A previous work reported identification of CG10109/PRAS40 as the L gene, but subsequent analyses suggested that PRAS40 may not be related to L. Here, we revisited the L gene to clarify this discrepancy and understand the basis for the dominance of L mutations. Genetic analysis localized the L gene to Oaz, which encodes a homolog of the vertebrate zinc finger protein 423 (Zfp423) family transcriptional regulators. We demonstrate that RNAi knockdown of Oaz almost completely restores all L dominant alleles tested. Lrev6-3, a revertant allele of the L2 dominant eye phenotype, has an inframe deletion in the Oaz coding sequence. Molecular analysis of L dominant mutants identified allele-specific insertions of natural transposons (roo[ ]L1, hopper[ ]L5, and roo[ ]Lr) or alterations of a preexisting transposon (L2-specific mutations in roo[ ]Mohr) in the Oaz region. In addition, we generated additional L2-reversion alleles by CRISPR targeting at Oaz. These new loss-of-function Oaz mutations suppress the dominant L eye phenotype. Oaz protein is not expressed in wild-type eye disc but is expressed ectopically in L2/+ mutant eye disc. We induced male recombination between Oaz-GAL4 insertions and the L2 mutation through homologous recombination. By using the L2-recombined GAL4 reporters, we show that Oaz-GAL4 is expressed ectopically in L2 eye imaginal disc. Taken together, our data suggest that neomorphic L eye phenotypes are likely due to misregulation of Oaz by spontaneous transposon insertions.

A deregulated stress response underlies distinct INF2 associated disease profiles

Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Here we systematically characterize a large panel (>50) of autosomal dominant mutants of the actin regulator inverted formin 2 (INF2) that have been reported to cause the podocytic kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot Marie-Tooth disease (CMT). We found that INF2 mutations lead to deregulated activation of the formin and a constitutive stress response in cultured cells, primary patient cells and Drosophila nephrocytes. Using quantitative live-cell imaging we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. Furthermore, we could clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and CMT. Our results indicate that cellular profiling of disease-associated mutations can contribute substantially to sequence-based phenotype predictions.

Hepatitis A Virus Mutant Spectra under the Selective Pressure of Monoclonal Antibodies: Codon Usage Constraints Limit Capsid Variability

ABSTRACT Severe structural constraints in the hepatitis A virus (HAV) capsid have been suggested as the reason for the lack of emergence of new serotypes in spite of the occurrence of complex distributions of mutants or quasispecies. Analysis of the HAV mutant spectra under immune pressure by the monoclonal antibodies (MAbs) K34C8 (immunodominant site) and H7C27 (glycophorin binding site) has revealed different evolutionary dynamics. Populations composed of complex ensembles of mutants with very low fitness or single dominant mutants with high fitness permit the acquisition of resistance to each of the MAbs, respectively. Deletion mutants were detected as components of the mutant spectra: up to 61 residues, with an average of 19, and up to 83 residues, with an average of 45, in VP3 and VP1 proteins, respectively. A clear negative selection of those replacements affecting the residues encoded by rare codons of the capsid surface has been detected through the present quasispecies analysis, confirming a certain beneficial role of such clusters. Since these clusters are located near or at the epitope regions, the need to maintain such clusters might prevent the emergence of new serotypes.

Dominant and Pharmacologically Sensitized ENU Mutagenesis Screens Uncover Novel Regulators of Hematopoiesis and Model Hematopoietic Disease.

To generate new mouse models of human hematopoietic disease and increase our knowledge of the genetic networks that control hematopoiesis, we are performing dominant (generation 1 or G1) and pharmacologically-sensitized forward ethylnitrososurea (ENU) mutagenesis screens. Mice are phenotyped by saphenous vein peripheral blood analysis using an automated hematological analyzer. ENU is ideally suited to generating models of human disease and annotating gene function because the spectrum of mutations (point mutations generating leading to subtle amino acid substitutions, splicing errors, or premature termination) are similar to those often found in human disease. Furthermore, null mutations often do not represent the full extent of a gene’s function, requiring multiple alleles to fully define gene function. While dominant mutations can unequivocally cause some human diseases, often mutations in multiple genes interact and contribute to disease progression. Thus, we have developed sensitized screens that induce transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea) and analyzing the recovery in peripheral blood levels of red blood cells, white blood cells and platelets. This strategy enables identification of hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. The induced cytopenia recovery assay is also being used as a secondary phenotyping assay for some of our G1 dominant mutants. The combined dominant and sensitized screens have yielded 14 heritable dominant mutants to date plus four additional mutants in hereditary testing. The array of mutations that we are analyzing are models for the following diseases: polycythemia, thrombocythemia, leukocytosis, anemia, and thrombocytopenia. I will discuss the progress of the mutagenesis screen and several ENU mutants, including a novel mutation in the protein tyrosine kinase Jak2, leading to thrombocythemia. This point mutation in the protein kinase domain will help us to dissect the recently discovered role of Jak2 in Myeloproliferative Diseases including Essential Thrombocythemia.