Characterization of Novel Di-, Tri-, and Tetranucleotide Microsatellite Primers Suitable for Genotyping Various Plant Pathogenic Fungi with Special Emphasis on Fusaria and Mycospherella graminicola

The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence of Mycospherlla graminicola and Fusarium graminearum. A total of 20 new microsatellite primers as easy-to-score markers were developed. Microsatellite primer PCR (MP-PCR) yielded highly reproducible and complex genomic fingerprints, with several bands ranging in size from 200 to 3000 bp. Of the 20 primers tested, only (TAGG)4, (TCC)5 and (CA)7T produced a high number of polymorphic bands from either F. graminearum or F. culmorum. (ATG)5 led to successful amplifications in M. graminicola isolates collected from Germany. Percentage of polymorphic bands among Fusarium species ranged from 9 to 100%. Cluster analysis of banding patterns of the isolates corresponded well to the established species delineations based on morphology and other methods of phylogenetic analysis. The current research demonstrates that the newly designed microsatellite primers are reliable, sensitive and technically simple tools for assaying genetic variability in plant pathogenic fungi

FONZIE: An optimized pipeline for minisatellite marker discovery and primer design from large sequence data sets

<p>Abstract</p> <p>Background</p> <p>Micro-and minisatellites are among the most powerful genetic markers known to date. They have been used as tools for a large number of applications ranging from gene mapping to phylogenetic studies and isolate typing. However, identifying micro-and minisatellite markers on large sequence data sets is often a laborious process.</p> <p>Results</p> <p>FONZIE was designed to successively 1) perform a search for markers via the external software Tandem Repeat Finder, 2) exclude user-defined specific genomic regions, 3) screen for the size and the percent matches of each relevant marker found by Tandem Repeat Finder, 4) evaluate marker specificity (i.e., occurrence of the marker as a single copy in the genome) using BLAST2.0, 5) design minisatellite primer pairs via the external software Primer3, and 6) check the specificity of each final PCR product by BLAST. A final file returns to users all the results required to amplify markers. A biological validation of the approach was performed using the whole genome sequence of the phytopathogenic fungus <it>Leptosphaeria maculans</it>, showing that more than 90% of the minisatellite primer pairs generated by the pipeline amplified a PCR product, 44.8% of which showed agarose-gel resolvable polymorphism between isolates. Segregation analyses confirmed that the polymorphic minisatellites corresponded to single-locus markers.</p> <p>Conclusion</p> <p>FONZIE is a stand-alone and user-friendly application developed to minimize tedious manual operations, reduce errors, and speed up the search for efficient minisatellite and microsatellite markers departing from whole-genome sequence data. This pipeline facilitates the integration of data and provides a set of specific primer sequences for PCR amplification of single-locus markers. FONZIE is freely downloadable at: <url>http://www.versailles-grignon.inra.fr/bioger/equipes/leptosphaeria_maculans/outils_d_analyses/fonzie</url></p

Subtle Alterations in PCNA-Partner Interactions Severely Impair DNA Replication and Repair

Dynamic switching of PCNA-partner interactions is essential for normal DNA replication and repair in yeast

Friedreich's Ataxia (GAA)n•(TTC)n Repeats Strongly Stimulate Mitotic Crossovers in Saccharomyces cerevisae

Expansions of trinucleotide GAA•TTC tracts are associated with the human disease Friedreich's ataxia, and long GAA•TTC tracts elevate genome instability in yeast. We show that tracts of (GAA)230•(TTC)230 stimulate mitotic crossovers in yeast about 10,000-fold relative to a “normal” DNA sequence; (GAA)n•(TTC)n tracts, however, do not significantly elevate meiotic recombination. Most of the mitotic crossovers are associated with a region of non-reciprocal transfer of information (gene conversion). The major class of recombination events stimulated by (GAA)n•(TTC)n tracts is a tract-associated double-strand break (DSB) that occurs in unreplicated chromosomes, likely in G1 of the cell cycle. These findings indicate that (GAA)n•(TTC)n tracts can be a potent source of loss of heterozygosity in yeast

Cdk1 Targets Srs2 to Complete Synthesis-Dependent Strand Annealing and to Promote Recombinational Repair

Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA–independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair

Water mass distributions in the Southern Ocean derived from a parametric analysis of mixing water masses

An empirical method to reconstruct distributions of mixing water masses is developed and applied to two Southern Ocean ( Australian sector) data sets. The method is a parametric extension of the well-known Optimum Multiparameter (OMP) analysis, therefore called POMP ( parametric OMP). The main development consists of parameterizing the mixing fractions as a function of position ( e. g. latitude and depth) and estimating the parameters of the resulting function, instead of estimating the mixing fractions for every sampling point individually. Because this enables to reduce the total number of unknowns to be estimated, the proposed adjustments result in more robust estimates of the mixing fractions, as illustrated on the Southern Ocean CLIVAR-SR3 data set ( early spring 2001). An additional consequence of the new working scheme is that the mixing distributions are smoothed. It is emphasized that the degree of smoothing should be chosen with care in order not to neglect any significant features. Statistical rules to do this are applied. Application of the POMP method to the second Southern Ocean data set (SAZ' 98, summer 1998) revealed the importance of a careful selection of the mixing source water types. This study was limited to the subantarctic region and upper 1000 m, where variations in water mass characteristics and distributions are most important. Indeed, it seems necessary to redefine the source water characteristics for each data set. The direction of these changes suggests that interannual variability or long term changes of water masses overwhelm the seasonal variability

Comment on: Paleoclimatic inference from stable isotope profiles of accretionary biogenic hardparts - a quantitative approach to the evaluation of incomplete data, by Wilkinson, B.H., Ivany, L.C., 2002. Palaeogeogr. Palaeocl. Palaeoecol. 185, 95-114

The time base reconstruction method proposed by Wilkinson and Ivany [Paleoclimatic inference from stable isotope profiles of accretionary biogenic hardparts - a quantitative approach to the evaluation of incomplete data. Palaeogeogr. Palaeocl. Palaeoecol. 185 (2002), 95-114] can be further refined. They have shown that variations in accretion rate can be reconstructed from variations in the period of the measured signal. Here, it is shown that such variations influence not only the period, but also the phase of the signal. So, a refined estimator for the time base is constructed, which takes both period and phase variations into account

Non-culprit lesions detected during primary PCI: treat invasively or follow the guidelines?

Item does not contain fulltextAIMS: Evidence regarding the optimal treatment of non-culprit lesions detected during primary PCI is lacking. Our aim was to investigate whether early invasive treatment improves left ventricular ejection fraction (EF) and prevents major adverse cardiac events (MACE). METHODS AND RESULTS: Of 121 patients with at least one non-culprit lesion, 80 were randomised to early FFRguided PCI (invasive group), and 41 to medical treatment (conservative group). Primary endpoint was EF at six months, secondary endpoints included MACE. In the invasive group, early angiography was performed 7.5 days (5-20) after primary PCI. Forty percent of the non-culprit lesions did not show haemodynamic significance (FFR > 0.75). Subsequent PCI of at least one non-culprit lesion was performed in 52%, PCI without preceding FFR was performed in 8% and elective CABG was done in 4%. No in-hospital events occurred in the conservative group. After six months, EF was comparable (59+/-9% vs. 57+/-9%, p=0.362), and there was no difference in MACE between invasively and conservatively treated patients (21 vs. 22%, p=0.929). CONCLUSIONS: An invasive strategy towards non-culprit lesions does not lead to an increase in EF or a reduction in MACE. The functional stenosis severity of non-culprit lesions is frequently overestimated.1 april 201

Experiences and needs of partners as informal caregivers of patients with major low anterior resection syndrome : a qualitative study

Purpose: After treatment, many rectal cancer survivors are confronted with ongoing bowel problems, called low anterior resection syndrome. The cancer diagnosis and treatment poses a burden on informal caregivers and results in higher levels of psychological distress and loneliness which is persistent after treatment as well. Our study aimed to investigate the experiences and needs of partners as informal caregivers of patients with major low anterior resection syndrome. Methods: A ground theory study was conducted. Semi-structured interviews with partners as informal caregivers of patients with major low anterior syndrome were performed in three hospitals between 2017 and 2019. In the first phase, maximum variation sampling was used and later theoretical sampling. Data analysis was done using the constant comparative method and investigators triangulation. Results: Twenty partners as informal caregivers were interviewed until data saturation. Low anterior resection syndrome of their partner was overwhelming and they failed to live a normal life. They had the feeling that they stood at the side-line and partly because of that felt lonely. Partners experienced three levels of loneliness: because of their changed own life, the changed life of their partner and the changed life in the environment. Conclusion: Low anterior resection syndrome has a large impact on the lives of partners as informal caregivers and induces loneliness at three levels. It is key that the HCPs of the interdisciplinary team understand this impact. A clinical nurse specialist/oncology nurse navigator could help in alleviate that burden by addressing both patients' and partners' needs