A near-deterministic mutational hotspot in Pseudomonas fluorescens is constructed by multiple interacting genomic features.

Mutation - whilst stochastic - is frequently biased toward certain loci. When combined with selection this results in highly repeatable and predictable evolutionary outcomes. Immotile variants of the bacterium Pseudomonas fluorescens (SBW25) possess a 'mutational hotspot' that facilitates repeated occurrences of an identical de novo single nucleotide polymorphism when re-evolving motility, where ≥95% independent lines fix the mutation ntrB A289C. Identifying hotspots of similar potency in other genes and genomic backgrounds would prove valuable for predictive evolutionary models, but to do so we must understand the genomic features that enable such a hotspot to form. Here we reveal that genomic location, local nucleotide sequence, gene strandedness and presence of mismatch repair proteins operate in combination to facilitate the formation of this mutational hotspot. Our study therefore provides a framework for utilising genomic features to predict and identify hotspot positions capable of enforcing near-deterministic evolution.

Relaxin augments the inflammatory IL6 response in the choriodecidua.

UNLABELLED: Intrauterine infection frequently leads to preterm birth (PTB), with the pathophysiology involving activation of the innate immune system and its associated inflammatory response. The choriodecidua produces relaxin (RLN) and elevated levels are associated with preterm premature rupture of the fetal membranes. However, it is not increased in bacterially-mediated PTB, but may act as an endogenous sterile inflammatory mediator. Elevated systemic RLN levels from the corpus luteum are also associated with PTB, but the mechanism is unknown. In clinical obstetrics, intrauterine inflammation or infection can coexist with elevated RLN. Therefore, in this study, we further characterized the effects of RLN alone or together with an inflammatory mediator on the production of IL1B, CSF2 (GM-CSF), IL6, IL8 and TNF, from chorionic cytotrophoblasts (CyT), decidual fibroblasts (DF) and stromal cells (DSC), using interleukin-1 beta (IL1B) to mimic sterile inflammation or lipopolysaccharide (LPS) for bacterial infection. Endogenous differences between the cells showed that the CyT expressed more RLN, its receptor RXFP1 and the RXFP1 splice variant D. CyT also showed the most robust cAMP response to RLN with increased IL6 secreted after 4 h, preceded by increased transcription at 1 h, likely due to activation of RXFP1 and cAMP. When all cell types were treated with IL1B and RLN, RLN augmented secretion of IL6 and IL8 from CyT and DF, but not DSC. Similarly, RLN augmented LPS-induced IL6 secretion from CyT and DF. Despite the structural similarity between TLR4 and RXFP1, blocking TLR4 in CyT had no effect on RLN-induced IL6 secretion, suggesting specific activation of RXFP1. Thus, we have shown that in the presence of a low level of intrauterine inflammation/infection, elevated RLN could act on the CyT and DF to augment the inflammatory response, contributing to the pathophysiology of PTB. SUMMARY: RLN augments the inflammatory responses induced by IL1B or LPS in chorionic cytotrophoblasts and decidual fibroblasts.

Relaxin modulates proinflammatory cytokine secretion from human decidual macrophages.

Relaxin (RLN) is a systemic hormone from the corpus luteum, and its levels remain low during normal human gestation. Indeed, elevation of circulating RLN has long been associated with preterm birth, for which there has been no physiological explanation. Recent studies have shown that RLN suppresses endotoxin-induced cytokine secretion from THP-1 monocytic cells by acting on the glucocorticoid receptor (GR), but its effects on primary macrophages are unknown. Therefore, in the present study, we examined the effects of RLN on cytokine secretion from primary decidual macrophages (DMs) obtained at term before labor. Unlike THP-1 cells, RLN had no effects on the cytokine responses induced by either lipopolysaccharide (LPS) or interleukin (IL) 1B, mimicking infection-induced or sterile inflammation, respectively. However, RLN alone for 4 h significantly decreased (P < 0.05) colony-stimulating factor 2 (CSF2; also known as granulocyte-macrophage colony-stimulating factor) and IL8 but for 24 h significantly increased IL6 (P < 0.01). We show that DMs express both the RLN receptor (RXFP1) and the GR. RLN suppression of CSF2 and IL8 was sensitive to the GR-antagonist mifepristone (RU-486). However, RLN activation of RXFP1 induced a dose-dependent cAMP response, which when mimicked by forskolin also caused significantly increased (P < 0.05) secretion of IL6. Thus, RLN may be anti-inflammatory in DMs via activation of the GR but proinflammatory via activation of RXFP1 and cAMP. In summary, we have shown that RLN targeting DMs may modulate proinflammatory cytokine secretion at the maternal-fetal interface and contribute to the localized inflammatory response associated with parturition in women.

Regulation of dikaryon-expressed genes by FRT1 in the basidiomycete Schizophyllum commune.

The gene FRT1 has previously been shown to induce homokaryotic fruiting in transformation recipients of the basidiomycete Schizophyllum commune. In this paper, we demonstrate by gene disruption experiments that FRT1 is dispensable for dikaryotic fruiting. Nonfruiting homokaryotic FRT1 disruptant strains exhibited enhanced aerial growth of mycelia compared to wild type. Introduction of a functional FRT1 allele into the disruptant restored the wild-type colony morphology. Transcript abundance of the dikaryon-expressed SC1 and SC4 hydrophobin genes and the SC7 gene were greatly elevated in homokaryotic FRT1 disruptant strains. Growth of the disruptant strains under continuous light was found to inhibit the elevation of SC1 and SC4 transcript levels, but not of SC7 mRNA. These data suggest that the role of FRT1 in vegetatively growing homokaryons is to act as a negative regulator of dikaryon-expressed genes.

The mushroom-inducing gene Frt1 of Schizophyllum commune encodes a putative nucleotide-binding protein.

Fruiting bodies (mushrooms) can be induced to form in unmated, normally non-fruiting strains of the basidiomycete fungus Schizophyllum commune by the ectopic genomic integration of a cloned gene called Frt1. Thus, the normal requirement of mating for mushroom formation is bypassed. Sequence analysis of genomic and cDNA clones revealed that the Frt1 gene encodes a predicted polypeptide of 192 amino acids, interrupted by three short introns. The FRT1 protein is predicted to be of M(r) 21,625 and does not have significant overall similarity to any known proteins. Analysis of the predicted amino acid sequence revealed the presence of a P-loop motif, a conserved sequence found in nucleotide-binding proteins. A potential site for Mg2+ binding is predicted to reside next to the P-loop at Thr24. The possible functional significance of these and other residues within FRT1 was examined using site-directed mutagenesis, followed by transformation of these mutant alleles of Frt1 back into S. commune. Mutation of the middle glycine of the P-loop completely abolished the fruit-inducing activity of cloned Frt1. Substitution of an alanine residue for Thr24 also resulted in mutant clones with no fruit-inducing activity. The possibility of an interaction between two closely spaced threonine residues within FRT1 was suggested by transformation experiments utilizing mutant Frt1 alleles with specific combinations of mutations at these sites. Taken together, the results of our mutagenesis experiments suggest the possibility that activity of the predicted FRT1 protein could be altered by nucleotide binding and coordination of Mg2+. Northern blot hybridization experiments indicate that Frt1 activity is probably not controlled at the transcriptional level.

A mushroom-inducing DNA sequence isolated from the Basidiomycete, Schizophyllum commune.

A DNA sequence capable of inducing the de novo development of fruiting bodies (mushrooms) when integrated into the genome of unmated, nonfruiting strains of the Basidiomycete Schizophyllum commune has been isolated and partially characterized. This sequence, designated FRT1, overrides the normal requirement of a mating interaction for fruiting in this organism. It has been shown to integrate stably in different chromosome locations and appears to be trans-acting. It also enhances the normal process of fruiting that occurs after mating. Additional DNA sequences with similarity to FRT1 were detected within the genome of the strain of origin by hybridization of labeled FRT1 DNA to blots of digested genomic DNAs. FRT1 and the genomic sequences similar to it were shown to be genetically linked. Southern hybridization experiments suggested sequence divergence at the FRT1 locus between different strains of S. commune. A testable model for how FRT1 may act as a key element in the pathway for the differentiation of fruiting bodies is presented as a working hypothesis for further investigation.

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA.

Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35-36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.