Patterns of cytotype distribution and genome size variation in the genus Sesleria Scop. (Poaceae)

Polyploidization has played an important role in the diversification of the genus Sesleria (Poaceae), which comprises c. 48 species and subspecies mostly distributed in Europe. The genus' centre of diversity clearly is the Balkan Peninsula, harbouring about 80% of the species, half of which are endemic to this area. We employed chromosome counts, measurements of absolute genome size and determination of relative DNA‐content for 460 populations belonging to 43 species of Sesleria. Our main aim was to provide essential baseline data for future molecular genetic reconstructions of the genus' evolutionary history. Relative genome size allowed for a mostly clear separation of four ploidy levels. The most frequent and widespread cytotypes are tetraploids followed by octoploids, while di‐ and dodecaploids were only found in a few species. We present first chromosome numbers for the tetraploid species S. doerfleri, S. phleoides, S. skipetarum and S. tuzsonii as well as for diploid S. ovata. Based on relative and partly also on absolute genome size measurements, ploidy level was determined in tetraploid S. rhodopaea and S. voronovii for the first time, and new cytotypes were identified in S. interrupta, S. kalnikensis and S. wettsteinii (tetraploids), S. caerulea, S. klasterskyi, S. latifolia, S. tenerrima, S. ujhelyii and S. vaginalis (octoploids), and S. albanica and S. vaginalis (dodecaploids). While most Sesleria species are ploidy‐uniform, several comprise two or even, in the case of S. vaginalis, three ploidy levels. Genome downsizing after polyploidization was confirmed by significant negative correlation between ploidy level and monoploid genome size. Finally, we found a significant increase in monoploid relative genome size towards the margin of the genus' distribution area, which may be triggered by increased activity of transposable element in populations exposed to environmental or genomic stress. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 126–143.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

Ctp1 and the MRN-Complex Are Required for Endonucleolytic Rec12 Removal with Release of a Single Class of Oligonucleotides in Fission Yeast

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5′ ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Δ and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal.     

Identification of a S-ribonuclease-binding protein in Petunia hybrida

To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBP1 may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.     

A conjugate of pyridine-4-aldoxime and atropine as a potential antidote against organophosphorus compounds poisoning

A conjugate of pyridine-4-aldoxime and atropine (ATR-4-OX) was synthesized and its antidotal efficiency was tested in vitro on tabun- or paraoxon-inhibited acetylcholinesterase (AChE) of human erythrocytes as well as in vivo using soman-, tabun- or paraoxon-poisoned mice. Its genotoxic profile was assessed on human lymphocytes in vitro and was found acceptable for further research. ATR-4-OX showed very weak antidotal activity, inadequate for soman or tabun poisoning. Conversely, it was effective against paraoxon poisoning both in vitro and in vivo. All animals treated with 5 % or 25 % LD(50) doses of the new oxime survived after administration of 10.0 or 16.0 LD(50) doses of paraoxon, respectively. Based on the persistence of toxicity symptoms in mice, the atropine moiety had questionable effects in attenuating such symptoms. It appears that ATR-4-OX has a therapeutic effect related to the reactivation of phosphylated AChE, but not to receptor antagonization.     

Prodigiosin from <Emphasis Type="BoldItalic">Vibrio</Emphasis> sp. DSM 14379; A New UV-Protective Pigment

Pigments such as melanin, scytonemin and carotenoids protect microbial cells against the harmful effects of ultraviolet (UV) radiation. The role in UV protection has never been assigned to the prodigiosin pigment. In this work, we demonstrate that prodigiosin provides a significant level of protection against UV stress in Vibrio sp. DSM 14379. In the absence of pigment production, Vibrio sp. was significantly more susceptible to UV stress, and there was no difference in UV survival between the wild-type strain and non-pigmented mutant. The pigment’s protective role was more important at higher doses of UV irradiation and correlated with pigment concentration in the cell. Pigmented cells survived high UV exposure (324 J/m²) around 1,000-fold more successfully compared to the non-pigmented mutant cells. Resistance to UV stress was conferred to the non-pigmented mutant by addition of exogenous pigment extract to the growth medium. A level of UV protection equivalent to that exhibited by the wild-type strain was attained by the non-pigmented mutant once the prodigiosin concentration had reached comparable levels to those found in the wild-type strain. In co-culture experiments, prodigiosin acted as a UV screen, protecting both the wild-type and non-pigmented mutants. Our results suggest a new ecophysiological role for prodigiosin.     

Ubiquitin ligases of the N-end rule pathway: assessment of mutations in UBR1 that cause the Johanson-Blizzard syndrome

Background

Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.

Methodology/Principal Findings

Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.

Conclusions/Significance

These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.