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Elucidating the mechanism of action of an antifungal or cytotoxic compound is a time-consuming process. Yeast chemogenomic profiling provides a compelling solution to the problem but is experimentally complex. Here, we demonstrate the use of a highly simplified yeast chemical genetic assay comprising just 89 yeast deletion strains, each diagnostic for a specific mechanism of action. We use the assay to investigate the mechanism of action of two antifungal chalcone compounds, trans-chalcone and 4'-hydroxychalcone, and narrow down the mechanism to transcriptional stress. Crucially, the assay eliminates mechanisms of action such as topoisomerase I inhibition and membrane disruption that have been suggested for related chalcone compounds. We propose this simplified assay as a useful tool to rapidly identify common off-target mechanisms.

The ribosomal DNA (rDNA) array of Saccharomyces cerevisiae has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct presentations that arise as spatial adaptations to changes in the nuclear morphology triggered during mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of nuclear components yet colocalizes with the vacuole. We show that the rDNA-associated nuclear envelope (NE) often reshapes into a ladle to accommodate the vacuole in the SUL, with the nucleus becoming bilobed and doughnut-shaped. Finally, we demonstrate that the formation of the rDNA loop and the SUL require TORC1, membrane synthesis and functional vacuoles, yet is independent of nucleus–vacuole junctions and rDNA-NE tethering.

The ribosomal DNA array (rDNA) of Saccharomyces cerevisiae has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct morphologies that arise as spatial adaptations to changes in the nuclear morphology triggered during the mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of any nuclear component yet colocalizes with the vacuole. We finally show that the formation and maintenance of the rDNA loop and the SUL require TORC1 and membrane synthesis. We propose that the rDNA-associated nuclear envelope (NE) reshapes into a loop to accommodate the vacuole, with the nucleus becoming bilobed.

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and even anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses and GFP-tagged DNA damage markers suggest that non-recombinational modifications of late replication intermediates may account for the shift in the PFGE behavior. The fact that this shift does not trigger G2/M checkpoints further supports this statement since checkpoints are active for other replicative stresses in the absence of Top2. We propose that the prolonged absence of Top2 activity leads to a general chromosome structural change. This change might interlock chromatids together with catenations and thus contribute to the formation of anaphase bridges in top2 mutants.

It has been recently demonstrated that yeast cells are able to partially regress chromosome segregation in telophase as a response to DNA double-strand breaks (DSBs), likely to find a donor sequence for homology-directed repair (HDR). This regression challenges the traditional concept that establishes anaphase events as irreversible, hence opening a new field of research in cell biology. Here, the nature of this new behavior in yeast is summarized and the underlying mechanisms are speculated about. It is also discussed whether it can be reproduced in other eukaryotes. Overall, this work brings forwards the need of understanding how cells attempt to repair DSBs when transiting the latest stages of mitosis, i.e., anaphase and telophase.

Naphthoquinones are among the most active natural products obtained from plants and microorganisms. Naphthoquinones exert their biological activities through pleiotropic mechanisms that include reactivity against cell nucleophiles, generation of reactive oxygen species (ROS), and inhibition of proteins. Here, we report a mechanistic antiproliferative study performed in the yeast Saccharomyces cerevisiae for several derivatives of three important natural naphthoquinones: lawsone, juglone, and β-lapachone. We have found that (i) the free hydroxyl group of lawsone and juglone modulates toxicity; (ii) lawsone and juglone derivatives differ in their mechanisms of action, with ROS generation being more important for the former; and (iii) a subset of derivatives possess the capability to disrupt mitochondrial function, with β-lapachones being the most potent compounds in this respect. In addition, we have cross-compared yeast results with antibacterial and antitumor activities. We discuss the relationship between the mechanistic findings, the antiproliferative activities, and the physicochemical properties of the naphthoquinones.

A series of symmetric polyoxygenated dibenzofurans with 2-methylbutyril moieties at C-4 and C-6 were obtained from commercial phloroglucinol through a sequence of reactions that include monoacylation, iodination, Suzuki-Miyaura coupling, oxidative dimerization and cyclization. Some of the compounds obtained were active against Gram-positive bacteria, including multiresistant Staphylococcus aureus clinical isolates. The dibenzofuran 28 with propyl chains at C-2 and C-8 exhibited the best antibacterial activity with values comparable to those of the natural dibenzofuran achyrofuran. From the obtained results some structure-activity relationships were outlined.

Based on previous Topoisomerase II docking studies of naphthoquinone derivatives, a series of naphthoquinone-coumarin conjugates was synthesized through a multicomponent reaction from aromatic aldehydes, 4-hydroxycoumarin and 2-hydroxynaphthoquinone. The hybrid structures were evaluated against the α isoform of human topoisomerase II (hTopoIIα), Escherichia coli DNA Gyrase and E. coli Topoisomerase I. All tested compounds inhibited the hTopoIIα-mediated relaxation of negatively supercoiled circular DNA in the low micromolar range. This inhibition was specific since neither DNA Gyrase nor Topoisomerase I were affected. Cleavage assays pointed out that naphthoquinone-coumarins act by catalytically inhibiting hTopoIIα. ATPase assays and molecular docking studies further pointed out that the mode of action is related to the hTopoIIα ATP-binding site.

The interplay between regulated cell death (RCD) and mitotic catastrophe (MC) determines much of the success of many anticancer treatments. Down-regulation of Topoisomerase II (Top2) is the more direct and devastating form of MC since it happens concomitantly with the formation of anaphase bridges in cells proficient for G2/M checkpoints. Herein, we have characterized in budding yeast the consequences for the cell progeny of a top2 MC. Clonogenic and microcolony experiments, in combination with vital stains, showed that 75% of daughter cells become senescent immediately after the top2 MC; they are unable to divide but remain alive. Decline in cell vitality occurred slowly, not even reaching 50% of cells 24 h after the MC, and uncoordinatedly when comparing pairs of daughters. Genetic experiments showed that the RCD mediator Mca1/Yca1 does not modify the senescence/death outcome. We thus conclude that MC mediated by Top2 deficiency leads to short-term senescence that eventually ends up in non-regulated cell death. Furthermore, we showed that the ability to divide in the short term can be modulated by the chromosome ploidy, suggesting that gross chromosome imbalances during segregation may account for senescence in the top2 progeny. Indeed, we found that diploid long-term survivors of the top2 MC are prone to genomic imbalances such as trisomies, uniparental disomies and terminal loss of heterozygosity (LOH), the latter affecting the longest chromosome arms.

Joint molecules (JMs) are intermediates of homologous recombination (HR). JMs rejoin sister or homolog chromosomes and must be removed timely to allow segregation in anaphase. Current models pinpoint Holliday junctions (HJs) as a central JM. The canonical HJ (cHJ) is a four-way DNA that needs specialized nucleases, a.k.a. resolvases, to resolve into two DNA molecules. Alternatively, a helicase–topoisomerase complex can deal with pairs of cHJs in the dissolution pathway. Aside from cHJs, HJs with a nick at the junction (nicked HJ; nHJ) can be found in vivo and are extremely good substrates for resolvases in vitro. Despite these findings, nHJs have been neglected as intermediates in HR models. Here, I present a conceptual study on the implications of nicks and nHJs in the final steps of HR. I address this from a biophysical, biochemical, topological, and genetic point of view. My conclusion is that they ease the elimination of JMs while giving genetic directionality to the final products. Additionally, I present an alternative view of the dissolution pathway since the nHJ that results from the second end capture predicts a cross-join isomerization. Finally, I propose that this isomerization nicely explains the strict crossover preference observed in synaptonemal-stabilized JMs in meiosis.

Homologous recombination (HR) is a preferred mechanism to deal with DNA replication impairments. However, HR synapsis gives rise to joint molecules (JMs) between the nascent sister chromatids, challenging chromosome segregation in anaphase. Joint molecules are resolved by the actions of several structure-selective endonucleases (SSEs), helicases and topoisomerases. Previously, we showed that yeast double mutants for the Mus81-Mms4 and Yen1 SSEs lead to anaphase bridges (ABs) after replication stress. Here, we have studied the role of the Mph1 helicase in preventing these anaphase aberrations. Mph1, the yeast ortholog of Fanconi anaemia protein M (FANCM), is involved in the removal of the D-loop, the first JM to arise in canonical HR. Surprisingly, the absence of Mph1 alone did not increase ABs, rather, it blocked cells in G2. Interestingly, in the search for genetic interactions with functionally related helicases and translocases, we found additive effects on the G2 block and post-G2 aberrations between mph1&Delta, and knockout mutants for Srs2, Rad54 and Rad5. Based on these interactions, we suggest that Mph1 acts coordinately with these helicases in the non-canonical HR-driven fork regression mechanism to bypass stalled replication forks.

Cycling events in nature start and end to restart again and again. In the cell cycle, whose purpose is to become two where there was only one, cyclin-dependent kinases (CDKs) are the beginning and, therefore, phosphatases must play a role in the ending. Since CDKs are drivers of the cell cycle and cancer cells uncontrollably divide, much attention has been put into knocking down CDK activity. However, much less is known on the consequences of interfering with the phosphatases that put an end to the cell cycle. We have addressed in recent years the consequences of transiently inactivating the only master cell cycle phosphatase in the model yeast Saccharomyces cerevisiae, Cdc14. Transient inactivation is expected to better mimic the pharmacological action of drugs. Interestingly, we have found that yeast cells tolerate badly a relatively brief inactivation of Cdc14 when cells are already committed into anaphase, the first cell cycle stage where this phosphatase plays important roles. First, we noticed that the segregation of distal regions in the chromosome arm that carries the ribosomal DNA array was irreversibly impaired, leading to an anaphase bridge (AB). Next, we found that this AB could eventually be severed by cytokinesis and led to two different types of genetically compromised daughter cells. All these previous studies were done in haploid cells. We have now recently expanded this analysis to diploid cells and used the advantage of making hybrid diploids to study chromosome rearrangements and changes in the ploidy of the surviving progeny. We have found that the consequences for the genome integrity were far more dramatic than originally envisioned.

Chromosome morphology in Saccharomyces cerevisiae is only visible at the microscopic level in the ribosomal DNA array (rDNA). The rDNA has been thus used as a model to characterize condensation and segregation of sister chromatids in mitosis. It has been established that the metaphase structure (“loop”) depends, among others, on the condensin complex; whereas its segregation also depends on that complex, the Polo-like kinase Cdc5 and the cell cycle master phosphatase Cdc14. In addition, Cdc14 also drives rDNA hypercondensation in telophase. Remarkably, since all these components are essential for cell survival, their role on rDNA condensation and segregation was established by temperature-sensitive (ts) alleles. Here, we show that the heat stress (HS) used to inactivate ts alleles (25 ºC to 37 ºC shift) causes rDNA loop condensation in metaphase-arrested wild type cells, a result that can also be mimicked by other stresses that inhibit the TORC1 pathway. Because this condensation might challenge previous findings with ts alleles, we have repeated classical experiments of rDNA condensation and segregation, yet using instead auxin-driven degradation alleles (aid alleles). We have undertaken the protein degradation at lower temperatures (25 ºC) and concluded that the classical roles for condensin, Cdc5, Cdc14 and Cdc15 still prevailed. Thus, condensin degradation disrupts rDNA higher organization, Cdc14 and Cdc5 degradation precludes rDNA segregation and Cdc15 degradation still allows rDNA hypercompaction in telophase. Finally, we provide direct genetic evidence that this HS-mediated rDNA condensation is dependent on TORC1 but, unlike the one observed in anaphase, is independent of Cdc14.

β-Lapachone (β-lap) is a promising antitumour drug currently undergoing clinical trials. Although it is known that β-lap generates reactive oxygen species (ROS), its actual mechanism of action is still controversial. Especially important is to determine whether concomitant DNA or microtubule damage is the key target of its antitumour properties and whether DNA damage is mediated by topoisomerases as previously suggested. Here, we have searched for determinants of β-lap cytotoxicity in the model organism Saccharomyces cerevisiae through a mechanism-driven approach whereby several pathways of the DNA and microtubule integrity responses, as well as the anti-oxidant response, were downregulated and the outcome of β-lap treatment examined. We also included in the analysis several β-lap derivatives expected to modify drug bioavailability and activity. We found that neither topoisomerase II nor microtubules contributed to yeast sensitivity to β-lap and its equitoxic derivative 3-bromo-β-lapachone. Instead, we found that oxidative and related environmental stresses were primarily responsible for toxicity. Accordingly, Yap1, the central transcription factor in the antioxidant response in yeast, together with several components involved in stress tolerance (i.e., Snf1 and Hog1) and chromatin remodelling (i.e., the SWR1 and RSC complexes), played major roles in protection against β-lapachone. Critically, we show that dioxygen enhanced toxicity and that ROS scavengers protected cells from it. Furthermore, we show that both quinones resulted in cell death in a manner which cytologically resembled apoptosis/necrosis. We thus conclude that β-lap is toxic to yeast through massive ROS production that either directly kills the cells or else triggers programmed cell death.

We describe a new, efficient, sensitive, and fast single-tube multiple-PCR protocol for the identification of the most clinically significant Staphylococcus spp. and the simultaneous detection of the methicillin and mupirocin resistance loci. The protocol identifies at the species level isolates belonging to S. aureus , S. epidermidis , S. haemolyticus , S. hominis , S. lugdunensis , and S. saprophyticus .

A set of 5-ethynylarylnaphthalimides was synthesized by Sonogashira cross-coupling reactions and evaluated for antiproliferative and antitopoisomerase II in vitro activities. Furthermore docking studies of these molecules as DNA-intercalators were carried out and the in vivo DNA-damaging activity was also determined with the model organism Saccharomyces cerevisiae. From the obtained results three naphthalimides 6, 13 and 14 showed strong topoisomerase II inhibitory activity. These three molecules also presented good docking scores as DNA-intercalators using a self-complementary oligodeoxynucleotide d(ATGCAT)2 as a model, and compounds 13 and 14 were among the most cytotoxic in the in vivo DNA-damaging activity.

Currently, there is a pressing need for novel antibacterial agents against drug-resistant bacteria, especially those which have been common in our communities and hospitals, such as methicillin-resistant Staphylococcus aureus (MRSA). The South American plant Achyrocline satureioides ("Marcela") has been widely used in traditional medicine for a number of diseases, including infections. Several crude extracts from this plant have shown good antimicrobial activities in vitro. In the search for the active principle(s) that confers these antimicrobial activities, we have processed the dichloromethane extract from the aerial parts of the plant. One of the isolated compounds showed extraordinary antibacterial activities against a set of clinically relevant Gram-positive strains that widely differ in their antibiogram profiles. This compound was identified as achyrofuran on the basis of its spectroscopic and physical data. We determined the MIC to be around 0.1 μM (0.07 μg/ml) for the reference methicillin-resistant and vancomycin-intermediate S. aureus strain NRS402. Moreover, nanomolar concentrations of achyrofuran killed 10(6) bacteria within 12 h. Based on the presence of the 2,2'-biphenol core, we further studied whether achyrofuran killed bacteria through a mechanism of action similar to that reported for the naturally occurring antibiotic MC21-A. Indeed, we found that achyrofuran was not bacteriolytic by itself although it greatly compromised membrane impermeability as determined by increased SYTOX Green uptake.

A series of dihydropyran embelin derivatives was synthesized through a direct and highly efficient approach based on a domino Knoevenagel intramolecular hetero-Diels–Alder reaction from natural embelin (1), using unsaturated aldehydes in the presence of organocatalysts such as ethylendiamine diacetate or l-proline. The aliphatic aldehydes yielded exclusively trans adducts, while mixtures of trans and cis isomers were found in reactions with aromatic aldehydes, with the cis form always predominating. Some of the compounds obtained were active and selective against Gram-positive bacteria, including multiresistant Staphylococcus aureus clinical isolates. Fil: Peña, Rosalyn. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica ; España Fil: Martín, Pedro. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica ; España Fil: Feresin, Gabriela Egly. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Universidad Nacional de San Juan. Facultad de Filosofía, Humanidades y Artes. Instituto de Ciencias Básicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tapia, Aníbal Alejandro. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Universidad Nacional de San Juan. Facultad de Filosofía, Humanidades y Artes. Instituto de Ciencias Básicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Machín, Félix. Hospital Universitario Nuestra Señora de Candelaria; España Fil: Estevez Braun, Ana. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica ; España

In the yeast Hansenula polymorpha, the YNT1 gene encodes the high affinity nitrate transporter, which is repressed by reduced nitrogen sources such as ammonium or glutamine. Ynt1 protein is degraded in response to glutamine in the growth medium. Ynt1 disappears independently of YNT1 glutamine repression as shown in strains where YNT1 repression is abolished. Ynt1-green fluorescent protein chimera and a mutant defective in vacuolar proteinase A (deltapep4) showed that Ynt1 is degraded in the vacuole in response to glutamine. The central hydrophilic domain of Ynt1 contains PEST-like sequences whose deletion blocked Ynt1 down-regulation. Site-directed mutagenesis showed that Lys-253 and Lys-270, located in this sequence, were involved in internalization and subsequent vacuolar degradation of Ynt1. Ynt1-ubiquitin conjugates were induced by glutamine and not nitrate. We conclude that glutamine triggers Ynt1 down-regulation via ubiquitinylation of lysines in the central hydrophilic domain, and proteolysis in the vacuole.

Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5-Smc6 complexes. The Smc5-Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5-Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5-Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.

Ynt1 is the only high-affinity nitrate uptake system in Hansenula polymorpha. Nitrate uptake was directly correlated with the Ynt1 levels and shown to be independent of nitrate reductase (NR) activity levels. Ynt1 failed to transport chlorate and, as a result, strains lacking YNT1 were sensitive to chlorate, as is the wild-type. Nitrite uptake in a wild-type strain was partially inhibited by nitrate to levels shown by a YNT1-disrupted strain in which, in turn, nitrite transport was not inhibited by nitrate. It is concluded that nitrite uptake takes place by two different transport systems: Ynt1 and a nitrite-specific transporter(s). The nitrite-specific transport system was induced by nitrate; consistently, no induction was observed in strains lacking the transcription factor YNA1, which is involved in nitrate and nitrite induction of the nitrate assimilatory structural genes. Ynt1 presents its optimal rate for nitrite uptake at pH 6, while pH 4 was optimal for the specific nitrite uptake system(s). At pH 5.5, the contribution of Ynt1 to high-affinity nitrate and nitrite uptake was around 95% and 60%, respectively. The apparent Km of Ynt1 for nitrate and nitrite is in the microM range, as is the specific nitrite uptake system for nitrite. The analysis of the effect of the reduced nitrogen sources on nitrate assimilation revealed that glutamine inactivates nitrate and nitrite transport, dependent on Ynt1, but not the nitrite-specific system.

The role of nitrate reductase (NR) in the regulation of the nitrate assimilation pathway was evaluated in the yeast Hansenula polymorpha. Posttranscriptional regulation of NR in response to reduced nitrogen sources and the effect of a heterologous NR on the transcriptional regulation of nitrate-assimilatory gene expression was examined. The strain bearing YNR1 (nitrate reductase gene) under the control of the methanol-induced MOX (methanol oxidase) promoter showed that NR is active in the presence of reduced nitrogen sources. In cells incubated with glutamine plus nitrate, rapamycin abolished nitrogen catabolite repression, NR activity being very similar to that in cells induced by nitrate alone. This reveals the involvement of the Tor-signalling pathway in the transcriptional regulation of H. polymorpha nitrate assimilation genes. To assess the role of NR in nitrate-assimilatory gene expression, different strains lacking YNR1, or both YNR1 and YNT1 (high-affinity nitrate transporter) genes, or expressing the tobacco NR under the YNR1 promoter, were used. Tobacco NR abolished the constitutive nitrate-assimilatory gene induction shown by an NR gene disruptant strain. Moreover, in strains lacking the high-affinity nitrate transporter and NR this deregulation disappeared. These facts discard the role of NR protein in the transcriptional induction of the nitrate-assimilatory genes and point out the involvement of the high-affinity nitrate transporter as a part of the nitrate-signalling pathway.

Genomic instability is a common feature found in cancer cells . Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanisms that explain cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient loss yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes, and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity, are detected mainly on the ribosomal DNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins, and controlling spindle dynamics in a well-defined window within anaphase, we speculate that the transient loss of Cdc14 activity causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny.

Nitrate assimilation genes encoding a nitrate transporter (YNT1), nitrite reductase (YNI1), a Zn(II)(2)Cys(6) transcriptional factor involved in nitrate induction (YNA1) and the nitrate reductase (YNR1) are clustered in the yeast Hansenula polymorpha. A second gene, termed YNA2 (yeast nitrate assimilation), was located seven nucleotides away from the 3' region of YNR1 gene. The cluster is flanked by an ORF encoding a protein with similarity to glutathione-S-transferase on the YNT1 side and an ORF with similarity to Saccharomyces cerevisiae Rad3p on the YNA2 side. The disruption of YNA2 confers the resulting null mutant strain with inability to grow in nitrate. The YNA2 gene encodes a putative protein of 618 residues bearing in the N-terminus the consensus sequence Cys-X(2)-Cys-X(6)-Cys-X(5-16)-Cys-X(2)-Cys-X(6-8)-Cys characteristic of the Zn(II)(2)Cys(6) transcriptional factors. YNA2 is therefore a member of the H. polymorpha nitrate assimilation gene cluster which is transcribed in the opposite direction to the rest of the members. Yna2p shares about 27% similarity with the H. polymorpha Yna1p Zn(II)(2)Cys(6) transcriptional factor involved in nitrate induction. Unlike the wild-type, the yna2::URA3 strain showed no expression of the nitrate assimilation genes when incubated in nitrate for 2 h. With regard to YNA2 expression, similar YNA2 transcript levels were observed in ammonium and in ammonium plus nitrate, but about a four-fold higher expression was observed in nitrate. However, this induction by nitrate of the YNA2 gene was not observed in the Deltayna1::URA3 strain. On the contrary, the pattern of YNA1 expression was the same in the wild-type as in the yna2::URA3 strain, indicating that YNA2 does not affect YNA1 expression.

An integrative expression vector based on promoter and terminator transcriptional sequences from the Hansenula polymorpha nitrate reductase gene (YNR1) has been developed to express nitrate assimilation plant genes in the nitrate assimilatory yeast H. polymorpha. Using this vector a plant nitrate reductase cDNA (tobacco Nia2) was expressed for the first time in a nitrate assimilatory yeast. The heterologous nitrate reductase produced retained its biochemical and physiological properties such as its NADH-dependent nitrate reductase activity, and allowed growth in nitrate containing media in a strain lacking endogenous nitrate reductase activity. In the transgenic strain, maximum tobacco nitrate reductase activity was about 70% of that presented in the wild-type. On the other hand, the disappearance of nitrate reductase activity correlated with that of the enzyme protein in response to the addition of ammonium to the medium and took place more rapidly in the transgenic strain than in the wild-type. Nitrate reductase activity of the recombinant strain assayed in the presence of Mg2+ was about 30% of that observed when assayed with EDTA. This result, together with a decreased growth rate in nitrate, suggests that tobacco nitrate reductase could be partially inactivated in H. polymorpha by phosphorylation and binding of 14-3-3-like proteins. These results show that H. polymorpha is a useful yeast heterologous expression system for studying plant proteins involved in nitrate assimilation.

The new prenylated phloroglucinol α-pyrones 1–3 and the new dibenzofuran 4, together with the known 23-methyl-6-O-demethylauricepyrone (5), achyrofuran (6), and 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphaliin A), were isolated from the aerial parts of Achyrocline satureioides. Their structures were determined by 1D and 2D NMR spectroscopic studies, while the absolute configuration of the sole stereogenic center of 1 was established by vibrational circular dichroism measurements in comparison to density functional theory calculated data. The same (S) absolute configuration of the α-methylbutyryl chain attached to the phloroglucinol nucleus was assumed for compounds 2–6 based on biogenetic considerations. Derivatives 7–16 were prepared from 1 and 5, and the antimicrobial activities of the isolated metabolites and some of the semisynthetic derivatives against a selected panel of Gram-positive and Gram-negative bacteria, as well as a set of yeast molds, were determined.

Downregulation of separase, condensin, Smc5/6, topoisomerase II and Cdc14 in Saccharomyces cerevisiae yields anaphase bridges formed by unresolved sister chromatids (SCBs). Here we report that the overlapping actions of the structure-selective endonucleases (SSEs) Mus81-Mms4/EME1 and Yen1/GEN1, but not Slx1-Slx4, are also essential to prevent the formation of spontaneous SCBs that depend on the homologous recombination pathway. We further show that the frequency of SCBs is boosted after mild replication stress and that they contain joint molecules enriched in non-canonical forms of the Holliday junction (HJ), including nicked-HJ (nHJ). We show that SCBs are mostly reversible upon activation of either Mus81-Mms4 or Yen1 in late anaphase, which is concomitant with the disappearance of non-canonical HJs and restoration of viable progeny. On the basis of these findings, we propose a model where unresolved recombination intermediates are a source of mitotic SCBs, and Mus81-Mms4 and Yen1 play a central role in their resolution in vivo.

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake., This work was supported by grants from the Dirección General de Investigación Científica y Técnica (P97-1484) and Gobierno de Canarias (PI1998) to J.M.S. G.P. and F.M. had fellowships from the Gobierno de Canarias.

The only canonical Holliday junction (HJ) resolvase identified in eukaryotes thus far is Yen1/GEN1. Nevertheless, Yen1/GEN1 appears to have a minor role in HJ resolution, and, instead, other structure-specific endonucleases (SSE) that recognize branched DNA play the leading roles, Mus81-Mms4/EME1 being the most important in budding yeast. Interestingly, cells tightly regulate the activity of each HJ resolvase during the yeast cell cycle. Thus, Mus81-Mms4 is activated in G2/M, while Yen1 gets activated shortly afterwards. Nevertheless, cytological studies have shown that Yen1 is sequestered out of the nucleus when cyclin-dependent kinase activity is high, i.e., all of the cell cycle but G1. We here show that the mitotic master phosphatase Cdc14 targets Yen1 to the nucleus in early anaphase through the FEAR network. We will further show that this FEAR-mediated Cdc14-driven event is sufficient to back-up Mus81-Mms4 in removing branched DNA structures, which are especially found in the long chromosome arms upon replication stress. Finally, we found that MEN-driven Cdc14 re-activation in late anaphase is essential to keep Yen1 in the nucleus until the next G1. Our results highlight the essential role that early-activated Cdc14, i.e., through the FEAR network, has in removing all kind of non-proteinaceous linkages that preclude faithful sister chromatid segregation in anaphase. In addition, our results support the general idea of Yen1 acting as a last resource endonuclease to deal with any remaining HJ that might compromise genetic stability during chromosome segregation. © 2014 Landes Bioscience., This work was supported by Instituto de Salud Carlos III (PS09/00106 and PS12/00280), Fundación Canaria de Investigación y Salud (08/42 to J.G.L.) and Spanish Ministry of Education (FPU fellowship AP2009-2511 to J.G.L.). All these programs were co-financed with the European Commission's ERDF structural funds.

Coupling of embelin with aldehydes and the primary enaminones (III) provides a new, straightforward one-pot approach to dihydropyridine derivatives of type (IV).

A series of dihydropyran and dihydropyridin embelin derivatives were synthesized through a novel and straightforward, one pot protocol based on a three component reaction with embelin, aldehydes and cyclic enaminones as synthetic imputs. The type of substituent on the nitrogen atom of the β-enaminone is key to obtain nitrogenated or oxygenated rings. The obtained compounds were active against Gram-positive bacteria, including multiresistant Staphylococcus aureus clinical isolates Fil: Peña, Rosalyn. Instituto Canario de Investigaciones del Cáncer; España. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica "Antonio González"; España Fil: Jiménez Alonso, Sandra. Instituto Canario de Investigaciones del Cáncer; España. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica "Antonio González"; España Fil: Feresin, Gabriela Egly. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Universidad Nacional de San Juan. Facultad de Filosofía, Humanidades y Artes. Instituto de Ciencias Básicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tapia, Aníbal Alejandro. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Universidad Nacional de San Juan. Facultad de Filosofía, Humanidades y Artes. Instituto de Ciencias Básicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Méndez Alvarez, Sebastián. Hospital Universitario Nuestra Señora de la Candelaria; España. Universidad de la Laguna; España Fil: Machín, Félix. Universidad de la Laguna; España. Hospital Universitario Nuestra Señora de la Candelaria; España Fil: Ravelo, Ángel G.. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica "Antonio González"; España. Instituto Canario de Investigaciones del Cáncer; España Fil: Estevez Braun, Ana. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica "Antonio González"; España. Instituto Canario de Investigaciones del Cáncer; España

β-Lapachone (β-lap) is a promising antitumoral agent. DNA base oxidation and alkylation are among the expected damages by β-lap. Herein, we have explored the role that the homologous recombination pathway (HR), a critical DNA repair process in Saccharomyces cerevisiae, has in the cytotoxic profile of β-lap. We have further compared β-lap to the closely related compound menadione and the well-known alkylating agent methyl methanesulfonate (MMS). Surprisingly, we found that β-lap does not trigger HR, as seen for (i) the mutant sensitivity profiles, (ii) concentration-dependent arrest profiles, (iii) absence of nuclear DNA repair factories, and (iv) frequency of recombination between direct repeats.

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction., Author Summary When cells divide they must segregate copies of their chromosomes to each of their daughters. A particular harmful situation arises when those copies are glued to each other (i.e., nondisjunction) at the moment of division. Previously, it has been possible to genetically favour this scenario, yet it has been difficult to limit the extent of nondisjunction to a single chromosome. We have developed and studied a yeast model where we control nondisjunction of one of its sixteen chromosomes. We show that dividing cells manage to complete nuclear and cell fission and therefore break that chromosome. We further show that new daughter cells then trigger a DNA damage response, yet only after they initiate a new round of replication. Remarkably, an uncommon repair strategy seems to be used to deal with this damage, which involves part of the homologous recombination machinery (i.e., RPA complex and Rad52) but lacks its primary sensor Mre11. Importantly though, both daughter cells arrest their cell cycle in G2 to prevent further damage from occurring. After a while, the cell that still carries an entire copy of the chromosome often survives, leading to aberrant forms of the chromosome in the progeny.

Special issue Models and observations of Marine Systems.--16 pages, 20 figures, 2 tables, We test the skill of a polynomial fit to reproduce the upper ocean (down to 750 m) salinity in the eastern North Atlantic (from the Canary Islands to the Iberian Peninsula, approximately 12° × 12°) as a function of temperature and depth. A historical database, constructed by merging several regional datasets, is used. An ANOVA test is performed to determine the optimum degree of temperature and depth in the polynomial fit. The polynomial coefficients are estimated by solving an inverse model where we control the size of both coefficients and residuals. We divide the basin in 21 zones (2° × 2°) and four regions (each comprising several zones), and run the inversion for the whole basin, as well as for each individual region and zone. This allows us to assess the sensitivity of the model to changes in the spatial domain, and to investigate the spatial variability of the polynomial coefficients. Regions are defined by applying a cluster analysis to objectively group those zones with similar oceanographic properties. The seasonality of the coefficients is addressed with data from the whole basin and individual regions. We find that, for either the whole basin or individual regions, seasonal coefficients predict salinity more accurately than annual ones, but annual coefficients per zone yet provide the best results. The depth-averaged error estimating salinity is less than 0.086 psu, This work has been supported by the Spanish Ministerio de Educación y Ciencia through projects CANOA (CMT2005-00444/MAR) and MIDAS4 (ESP2005-06823-C05), and by the European Union through project OASIS (EVK3-CT-2002-00073-OASIS). The first author has been partly supported by a contract funded by the Spanish Ministerio de Educación y Ciencia through the Juan de la Cierva Programme

A series of pyranonaphthoquinone derivatives related to the known topoisomerase II inhibitor eleutherin 1 have been shown to act as specific topoisomerase II catalytic inhibitors, with several analogues displaying greater potency than the natural product itself. Amongst the compounds tested were the natural products ventiloquinone L 4 and thysanone 8 with a diverse range of topoisomerase II inhibition properties being observed. Interestingly, the natural products are generally weaker inhibitors than their synthetic counterparts, emphasising that subtle changes in the basic molecular structure of a natural product led to significant changes in the inhibition profile. It has also been demonstrated for the first time that analogues related to nanaomycin A and cardinalin-type dimeric pyranonaphthoquinones exhibit potent topoisomerase II inhibitory properties. With respect to structural features, it appears that the nature of the substituents at C1 on the pyran ring and oxygenated substituents on the aryl ring are critical for anti-topoII activity. Importantly, the topoisomerase II inhibition strength does not correlate well with the measured cytotoxicity against yeast, indicating that other molecular features in the pyranonaphthoquinone family must be considered for the design and use of this structural class as highly specific topoisomerase II inhibitors.