Deletion of the nuclear exosome component RRP6 leads to continued accumulation of the histone mRNA HTB1 in S-phase of the cell cycle in Saccharomyces cerevisiae

The nuclear exosome, a macromolecular complex of 3′ to 5′ exonucleases, is required for the post-transcriptional processing of a variety of RNAs including rRNAs and snoRNAs. Additionally, this complex forms part of a nuclear surveillance network where it acts to degrade any aberrantly processed mRNAs in the nucleus. The exosome complex has been implicated in the biogenesis pathway of general messenger RNAs through its interaction with the 3′-end processing machinery. During the cell cycle, yeast histone mRNAs accumulate in the S-phase and are rapidly degraded as cells enter the G2-phase. To determine if the exosome contributes to the cyclic turnover of yeast histone mRNAs, we examined the pattern of accumulation of ‘HTB1’ mRNA during the cell cycle in a deletion strain of ‘RRP6’, a component of the nuclear exosome. Our results show that cells lacking Rrp6p continue to accumulate HTB1 mRNA as the cell cycle proceeds. This continued accumulation appears to result from a delay in exit from S-phase in rrp6 cells. The accumulation of HTB1 mRNA in rrp6 cells is influenced by the interaction of the nuclear exosome with the 3′-end processing machinery although there is no evidence for differential regulation of histone mRNA 3′-end processing during the yeast cell cycle

Coordination Studies of Copper(II), Cobalt(II) and Iron(II) with Isomeric Pyridyl-Tetrazole Ligands

The reaction of 2-(2H-tetrazol-5-yl)pyridine (L1) with 1,6-dibromohexane results in formation of the isomers 2-(6′′-bromohexyl-(1-tetrazol-5-yl)pyridine (L2) and 2-(6′′-bromohexyl-(2-tetrazol-5-yl)pyridine (L3). Coordination reactions of L2 and L3 with CuCl2·2H2O, Co(SCN)2 and Fe(ClO4)2·H2O yielded the strongly coloured solids [Cu(II)(L2)Cl2]2 (1), [Cu(II)(L3)Cl2]2 (2), [Co(II)(L2)2(NCS)2] (3), [Co(II)(L3)2(NCS)2] (4), [Fe(II)(L2)2(H2O)2](ClO4)2 (5) and [Fe(II)(L3)2(H2O)2](ClO4)2 (6), containing high-spin metal centres for the Co(II) and Fe(II) compounds. X-ray crystal structures were obtained for complexes 1–5. In each complex, ligands L2 and L3 coordinate to the metal centre through the pyridyl N atom and the 1-N site of the tetrazole ring, and the pyridyl-tetrazole ligand remains planar in all cases except 3. Complexes 1 and 2 comprise a central Cu2Cl2 dimeric core with Cu(II) in an essentially square-pyramidal coordination environment. Complexes 3 and 4 contain Co(II) in a distorted octahedral coordination environment. In 3, the pyridyl and tetrazole rings of L2 are twisted with respect to each other and the complex adopts a puckered conformation in its equatorial plane. Complex 5 contains water molecules coordinated to Fe(II) in the axial sites, which form hydrogen bonds to the perchlorate counter anions

Coordination Studies of Copper(II), Cobalt(II) and Iron(II) with Isomeric Pyridyl-Tetrazole Ligands

The reaction of 2-(2H-tetrazol-5-yl)pyridine (L1) with 1,6-dibromohexane results in formation of the isomers 2-(6′′-bromohexyl-(1-tetrazol-5-yl)pyridine (L2) and 2-(6′′-bromohexyl-(2-tetrazol-5-yl)pyridine (L3). Coordination reactions of L2 and L3 with CuCl2·2H2O, Co(SCN)2 and Fe(ClO4)2·H2O yielded the strongly coloured solids [Cu(II)(L2)Cl2]2 (1), [Cu(II)(L3)Cl2]2 (2), [Co(II)(L2)2(NCS)2] (3), [Co(II)(L3)2(NCS)2] (4), [Fe(II)(L2)2(H2O)2](ClO4)2 (5) and [Fe(II)(L3)2(H2O)2](ClO4)2 (6), containing high-spin metal centres for the Co(II) and Fe(II) compounds. X-ray crystal structures were obtained for complexes 1–5. In each complex, ligands L2 and L3 coordinate to the metal centre through the pyridyl N atom and the 1-N site of the tetrazole ring, and the pyridyl-tetrazole ligand remains planar in all cases except 3. Complexes 1 and 2 comprise a central Cu2Cl2 dimeric core with Cu(II) in an essentially square-pyramidal coordination environment. Complexes 3 and 4 contain Co(II) in a distorted octahedral coordination environment. In 3, the pyridyl and tetrazole rings of L2 are twisted with respect to each other and the complex adopts a puckered conformation in its equatorial plane. Complex 5 contains water molecules coordinated to Fe(II) in the axial sites, which form hydrogen bonds to the perchlorate counter anions

A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens

The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-γ (IFN-γ) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours

Enhanced flavour profiles through radicicol induced genomic variation in the lager yeasts, Saccharomyces pastorianus

The yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and have acquired traits from the combined parental genomes such as ability to ferment a range of sugars at low temperatures and to produce aromatic flavour compounds, allowing for the production of lager beers with crisp, clean flavours. The polyploid strains are sterile and have reached an evolutionary bottleneck for genetic variation. Here we describe an accelerated evolution approach to obtain lager yeasts with enhanced flavour profiles. As the relative expression of orthologous alleles is a significant contributor to the transcriptome during fermentation, we aimed to induce genetic variation by altering the S. cerevisiae to S. eubayanus chromosome ratio. Aneuploidy was induced through the temporary inhibition of the cell's stress response and strains with increased production of aromatic amino acids via the Shikimate pathway were selected by resistance to amino acid analogues. Genomic changes such as gross chromosomal rearrangements, chromosome loss and chromosome gain were detected in the characterised mutants, as were single-nucleotide polymorphisms in ARO4, encoding for DAHP synthase, the catalytic enzyme in the first step of the Shikimate pathway. Transcriptome analysis confirmed the upregulation of genes encoding enzymes in the Ehrlich pathway and the concomitant increase in the production of higher alcohols and esters such as 2-phenylethanol, 2-phenylethyl acetate, tryptophol, and tyrosol. We propose that the polyploid nature of S. pastorianus genomes is an advantageous trait supporting opportunities for genetic alteration in otherwise sterile strain

Fermentation innovation through complex hybridization of wild and domesticated yeasts

The most common fermented beverage, lager beer, is produced by interspecies hybrids of the brewing yeast Saccharomyces cerevisiae and its wild relative S. eubayanus. Lager-brewing yeasts are not the only example of hybrid vigour or heterosis in yeasts, but the full breadth of interspecies hybrids associated with human fermentations has received less attention. Here we present a comprehensive genomic analysis of 122 Saccharomyces hybrids and introgressed strains. These strains arose from hybridization events between two to four species. Hybrids with S. cerevisiae contributions originated from three lineages of domesticated S. cerevisiae, including the major wine-making lineage and two distinct brewing lineages. In contrast, the undomesticated parents of these interspecies hybrids were all from wild Holarctic or European lineages. Most hybrids have inherited a mitochondrial genome from a parent other than S. cerevisiae, which recent functional studies suggest could confer adaptation to colder temperatures. A subset of hybrids associated with crisp flavour profiles, including both lineages of lager-brewing yeasts, have inherited inactivated S. cerevisiae alleles of critical phenolic off-flavour genes and/or lost functional copies from the wild parent through multiple genetic mechanisms. These complex hybrids shed light on the convergent and divergent evolutionary trajectories of interspecies hybrids and their impact on innovation in lager brewing and other diverse fermentation industries.Fil: Langdon, Quinn K.. University of Wisconsin; Estados UnidosFil: Peris, David. University of Wisconsin; Estados Unidos. Consejo Superior de Investigaciones Científicas; EspañaFil: Baker, Emily Clare. University of Wisconsin; Estados UnidosFil: Opulente, Dana A.. University of Wisconsin; Estados UnidosFil: Nguyen, Huu-Vang. Université Paris-Saclay; Francia. Institut National de la Recherche Agronomique; FranciaFil: Bond, Ursula. Trinity College; Estados UnidosFil: Gonçalves, Paula. Universidade Nova de Lisboa; PortugalFil: Sampaio, José Paulo. Universidade Nova de Lisboa; PortugalFil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; ArgentinaFil: Hittinger, Chris. University of Wisconsin; Estados Unido

Generacija novih genotipskih i fenotipskih svojstava prirodnih i umjetnih hibrida kvasaca

Evolution and genome stabilization have mostly been studied on the Saccharomyces hybrids isolated from natural and alcoholic fermentation environments. Genetic and phenotypic properties have usually been compared to the laboratory and reference strains, as the true ancestors of the natural hybrid yeasts are unknown. In this way the exact impact of different parental fractions on the genome organization or metabolic activity of the hybrid yeasts is difficult to resolve completely. In the present work the evolution of geno- and phenotypic properties is studied in the interspecies hybrids created by the cross-breeding of S. cerevisiae with S. uvarum or S. kudriavzevii auxotrophic mutants. We hypothesized that the extent of genomic alterations in S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii should affect the physiology of their F1 offspring in different ways. Our results, obtained by amplified fragment length polymorphism (AFLP) genotyping and karyotyping analyses, showed that both subgenomes of the S. cerevisiae x S. uvarum and of S. cerevisiae × S. kudriavzevii hybrids experienced various modifications. However, the S. cerevisiae × S. kudriavzevii F1 hybrids underwent more severe genomic alterations than the S. cerevisiae × S. uvarum ones. Generation of the new genotypes also influenced the physiological performances of the hybrids and the occurrence of novel phenotypes. Significant differences in carbohydrate utilization and distinct growth dynamics at increasing concentrations of sodium chloride, urea and miconazole were observed within and between the S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii hybrids. Parental strains also demonstrated different contributions to the final metabolic outcomes of the hybrid yeasts. A comparison of the genotypic properties of the artificial hybrids with several hybrid isolates from the wine-related environments and wastewater demonstrated a greater genetic variability of the S. cerevisiae × S. kudriavzevii hybrids. Saccharomyces cerevisiae × S. uvarum artificial and natural hybrids showed considerable differences in osmolyte tolerance and sensitivity to miconazole, whereas the S. cerevisiae × S. kudriavzevii hybrids exhibited differences also in maltotriose utilization. The results of this study suggest that chromosomal rearrangements and genomic reorganizations as post-hybridization processes may affect the phenotypic properties of the hybrid progeny substantially. Relative to their ancestors, the F1 segregants may generate different phenotypes, indicating novel routes of evolution in response to environmental growth conditions.Evolucija i stabilizacija genoma kvasca uglavnom se proučavaju s pomoću interspecijskih hibrida roda Saccharomyces, izoliranih iz prirodnih staništa ili tijekom alkoholnih fermentacija. Njihova genetska i fenotipska svojstva obično se uspoređuju sa svojstvima laboratorijskih i referentnih sojeva, budući da su izvorni roditeljski sojevi prirodnih hibrida kvasaca nepoznati. Na ovaj je način teško u potpunosti razumjeti utjecaj različitih roditeljskih frakcija na organizaciju genoma ili metaboličku aktivnost hibrida kvasaca. U ovom je radu proučena evolucija genotipskih i fenotipskih svojstava interspecijskih hibrida, nastalih križanjem kvasca S. cerevisiae s auksotrofnim mutantima kvasaca S. uvarum i S. kudriavzevii. Naša je hipoteza bila da bi genomske promjene nastale u hibridima S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii trebale na različite načine utjecati na fiziologiju njihovih F1 segreganata. Rezultati dobiveni genotipizacijom, tj. analizom polimorfizma duljine umnoženih fragmenata (engl. amplified fragment length polymorphism - AFLP) i kariotipizacijom pokazuju da su oba subgenoma hibrida S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii izmjenjena. Međutim, promjene genoma segreganata F1 hibrida S. cerevisiae × S. kudriavzevii bile su znatnije od onih segreganata hibrida S. cerevisiae × S. uvarum. Novi su genotipovi utjecali na fiziološke značajke hibrida te nastanak novih fenotipova. Bitna je razlika među hibridima S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii opažena u potrošnji šećera i različitoj dinamici rasta kod povećanih koncentracija natrijevog klorida, uree i mikonazola. Roditeljski su sojevi različito utjecali na konačnu metaboličku sliku hibrida kvasaca. Usporedbom genotipskih svojstava umjetnih hibrida s nekoliko hibrida izoliranih iz prirodnih staništa (vinograda i otpadnih voda) utvrđena je veća genetska raznolikost hibrida S. cerevisiae × S. kudriavzevii. Umjetni i prirodni hibridi Saccharomyces cerevisiae × S. uvarum bili su različito osjetljivi prema osmolitima i mikonazolu, dok su se hibridi S. cerevisiae × S. kudriavzevii razlikovali i u potrošnji maltotrioze. Iz dobivenih se rezultata može zaključiti da kromosomalna rekombinacija i genomska reorganizacija kao post-hibridizacijski procesi mogu značajno utjecati na fenotipska svojstva hibridnih potomaka. U usporedbi s roditeljskim sojevima, segreganti F1 mogu generirati različite fenotipove, što upućuje na zaključak da su specifični uvjeti rasta kvasaca uzrokovali nastanak novih evolucijskih tokova

Migration Patterns, Use of Stopover Areas, and Austral Summer Movements of Swainson\u27s Hawks

From 1995 to 1998, we tracked movements of adult Swainson’s Hawks (Buteo swainsoni), using satellite telemetry to characterize migration, important stopover areas, and movements in the austral summer. We tagged 46 hawks from July to September on their nesting grounds in seven U.S. states and two Canadian provinces. Swainson’s Hawks followed three basic routes south on a broad front, converged along the east coast of central Mexico, and followed a concentrated corridor to a communal area in central Argentina for the austral summer. North of 20°N, southward and northward tracks differed little for individuals from east of the continental divide but differed greatly (up to 1700 km) for individuals from west of the continental divide. Hawks left the breeding grounds mid-August to mid-October; departure dates did not differ by location, year, or sex. Southbound migration lasted 42 to 98 days, northbound migration 51 to 82 days. Southbound, 36% of the Swainson’s Hawks departed the nesting grounds nearly 3 weeks earlier than the other radio-marked hawks and made stopovers 9.0–26.0 days long in seven separate areas, mainly in the southern Great Plains, southern Arizona and New Mexico, and northcentral Mexico. The birds stayed in their nonbreeding range for 76 to 128 days. All used a core area in central Argentina within 23% of the 738 800-km2 austral summer range, where they frequently moved long distances (up to 1600 km). Conservation of Swainson’s Hawks must be an international effort that considers habitats used during nesting and non-nesting seasons, including migration stopovers