Development of single nucleotide polymorphisms in Phaseolus vulgaris and related Phaseolus spp

In this study, new single nucleotide polymorphism (SNP) markers were developed for common bean (Phaseolus vulgaris L.) and related Phaseolus species. The applied strategy presents new and interesting aspects, such as the choice of accessions used, which was aimed at capturing a large portion of the genetic diversity present in the common bean, with particular focus on wild and domesticated materials from Mesoamerica and the identification of loci for sequencing. Indeed, the primer pairs for 34 loci were designed with the main strategy being to search for single-copy orthologous genes among the legumes (for use in other legume species and comparative analyses). The 10 remaining loci were selected as being near to domestication quantitative trait loci or detected as putatively under selection during domestication in previous studies. To provide an efficient and inexpensive genotyping platform for geneticists and breeders, we used sequence data to develop 60 new SNP markers for KASPar assay genotyping. The same sample was also genotyped with SNP markers developed for common bean in other studies for the same assay. This allowed testing for systematic bias according to the criteria chosen to select the genotypes in which the genetic diversity is surveyed during SNP discovery. Finally, we show that most of the SNP markers worked well in a set of accessions of other species belonging to the Phaseolus genus. The genetic resources developed will be very useful not only for breeding, but also for biodiversity conservation management and evolutionary studies on legumes.     

Balanced polymorphisms and their divergence in a Heliconius butterfly

The evolution of mimicry in similarly defended prey is well described by the Müllerian mimicry theory, which predicts the convergence of warning patterns in order to gain the most protection from predators. However, despite this prediction, we can find great diversity of color patterns among Müllerian mimics such as Heliconius butterflies in the neotropics. Furthermore, some species have evolved the ability to maintain multiple distinct warning patterns in single populations, a phenomenon known as polymorphic mimicry. The adaptive benefit of these polymorphisms is questionable since variation from the most common warning patterns is expected to be disadvantageous as novel signals are punished by predators naive to them. In this study, we use artificial butterfly models throughout Central and South America to characterize the selective pressures maintaining polymorphic mimicry in Heliconius doris. Our results highlight the complexity of positive frequency‐dependent selection, the principal selective pressure driving convergence among Müllerian mimics, and its impacts on interspecific variation of mimetic warning coloration. We further show how this selection regime can both limit and facilitate the diversification of mimetic traits.     

Reproducibility and complications in gene searches: linkage on chromosome 6, heterogeneity, association, and maternal inheritance in juvenile myoclonic epilepsy

Evidence for genetic influences in epilepsy is strong, but reports identifying specific chromosomal origins of those influences conflict. One early study reported that human leukocyte antigen (HLA) markers were genetically linked to juvenile myoclonic epilepsy (JME); this was confirmed in a later study. Other reports did not find linkage to HLA markers. One found evidence of linkage to markers on chromosome 15, another to markers on chromosome 6, centromeric to HLA. We identified families through a patient with JME and genotyped markers throughout chromosome 6. Linkage analysis assuming equal male-female recombination probabilities showed evidence for linkage (LOD score 2.5), but at a high recombination fraction (theta), suggesting heterogeneity. When linkage analysis was redone to allow independent male-female thetas, the LOD score was significantly higher (4.2) at a male-female theta of.5,.01. Although the overall pattern of LOD scores with respect to male-female theta could not be explained solely by heterogeneity, the presence of heterogeneity and predominantly maternal inheritance of JME might explain it. By analyzing loci between HLA-DP and HLA-DR and stratifying the families on the basis of evidence for or against linkage, we were able to show evidence of heterogeneity within JME and to propose a marker associated with the linked form. These data also suggest that JME may be predominantly maternally inherited and that the HLA-linked form is more likely to occur in families of European origin.     

DNA Methylation Signatures Triggered by Prenatal Maternal Stress Exposure to a Natural Disaster: Project Ice Storm

Background

Prenatal maternal stress (PNMS) predicts a wide variety of behavioral and physical outcomes in the offspring. Although epigenetic processes may be responsible for PNMS effects, human research is hampered by the lack of experimental methods that parallel controlled animal studies. Disasters, however, provide natural experiments that can provide models of prenatal stress.

Methods

Five months after the 1998 Quebec ice storm we recruited women who had been pregnant during the disaster and assessed their degrees of objective hardship and subjective distress. Thirteen years later, we investigated DNA methylation profiling in T cells obtained from 36 of the children, and compared selected results with those from saliva samples obtained from the same children at age 8.

Results

Prenatal maternal objective hardship was correlated with DNA methylation levels in 1675 CGs affiliated with 957 genes predominantly related to immune function; maternal subjective distress was uncorrelated. DNA methylation changes in SCG5 and LTA, both highly correlated with maternal objective stress, were comparable in T cells, peripheral blood mononuclear cells (PBMCs) and saliva cells.

Conclusions

These data provide first evidence in humans supporting the conclusion that PNMS results in a lasting, broad, and functionally organized DNA methylation signature in several tissues in offspring. By using a natural disaster model, we can infer that the epigenetic effects found in Project Ice Storm are due to objective levels of hardship experienced by the pregnant woman rather than to her level of sustained distress.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.     

Proteomic identification of a two-component regulatory system in Pseudoalteromonas haloplanktis TAC125

The capability of microorganisms to utilize different carbohydrates as energy source reflects the availability of these substrates in their habitat. Investigation of the proteins involved in carbohydrate usage, in parallel with analysis of their expression, is then likely to provide information on the interaction between microorganisms and their ecosystem. We analysed the growth behaviour of the marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in the presence and in the absence of different carbon source. A marked increase in the optical density was detected when l-malate was added to the growth medium. Bacterial proteins differently expressed in the presence of l-malate were identified by proteomic profiling experiments. On the basis of their relative increase, six proteins were selected for further analyses. Among these, the expression of a putative outer membrane porin was demonstrated to be heavily induced by l-malate. The presence of a functionally active two-component regulatory system very likely controlled by l-malate was found in the upstream region of the porin gene. A non functional genomic porin mutant was then constructed showing a direct involvement of the protein in the uptake of l-malate. To the best of our knowledge, the occurrence of such a regulatory system has never been reported in Pseudoalteromonads so far and might constitute a key step in the development of an effective inducible cold expression system.     

Design and synthesis of N6-substituted-4′-thioadenosine-5′-uronamides as potent and selective human A3 adenosine receptor agonists

On the basis of a bioisosteric rationale, 4′-thionucleoside analogues of IB-MECA (N⁶-(3-Iodo-benzyl)-9-(5′-methylaminocarbonyl-β-d-ribofuranosyl)adenine), which is a potent and selective A₃ adenosine receptor (AR) agonist, were synthesized from d-gulonic acid γ-lactone. The 4′-thio analogue (5h) of IB-MECA showed extremely high binding affinity (K_i = 0.25 nM) at the human A₃AR and was more potent than IB-MECA (K_i = 1.4 nM). Bulky substituents at the 5′-uronamide position, such as cyclohexyl and 2-methylbenzyl, in this series of 2-H nucleoside derivatives were tolerated in A₃AR binding, although small alkyl analogues were more potent.