Browsing by Subject "yeast"

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  • Yang, Yingying (Helsingfors universitet, 2013)
    The literature review presented the effects of the polyglutamate chain on the biological and nutritional properties of folates and the main methods used for folate assays, with a special emphasis on the approaches to studying intact polyglutamates. A brief introduction regarding safety aspects of folate fortification was also given. The aim of this study was to develop a UPLC-FLR/PDA method for simultaneous determination of polyglutamyl folate vitamers. Chromatographic conditions were optimised for the resolution of polyglutamyl 5-methyltetrahydrofolates and major naturally-occurring monoglutamates. Method validation was conducted for both the UPLC method and affinity chromatography. Applicability of the validated method was evaluated on lupin flour, faba bean flour, and dry yeast, which were subjected to preparatory treatments with and without deconjugation. In addition, the effects of the sequential modification of preparatory treatments on the folate content and composition were investigated by using both the UPLC method and Lactobacillus rhamnosus assay. A desirable separation of target polyglutamates and monoglutamates was successfully achieved on the BEH C18 UPLC column within 11 minutes. The optimised UPLC method showed satisfactory selectivity, linearity, and sensitivity for the determination of methylated polyglutamates in the femtomole range and monoglutamates in the picogram range. Affinity chromatography showed satisfactory recoveries for polyglutamyl 5-methyltetrahydrofolates, but not for 5-formyl polyglutamates. In all three selected foods, 5-methyltetrahydrofolate was the dominant folate vitamer. Meanwhile, the analysis of undeconjugated samples showed that in the intact methylated folate pools, pentaglutamate predominated in legume flours and heptaglutamate in dry yeast. In addition, different sequences of enzyme and purification pretreatments were found to significantly affect both the total measurable folates and the folate profiles. Our standard preparatory procedures comprising simultaneous treatments with amylase and conjugase, then protease and affinity purification resulted in the greatest yield of total folates, but UPLC analysis indicated incomplete deconjugation. However, a modification in which deconjugation was conducted as the last step enhanced hydrolysis efficiency.
  • Lohva, Henri (Helsingfors universitet, 2016)
    Saccharomyces cerevisiae is a popular organism in the production of biofuels, chemicals and pharmaceuticals. This is thanks to a good understanding of its metabolism, GRAS status and the ease of modification. Traditionally its genetic modification has been based on the use of selectable markers. Modifying multi gene pathways has required a sequential process consisting of multiple single gene disruptions together with marker recycling. Additionally, many industrial S. cerevisiae strains are polyploid and lack the same tools for their modification as laboratory strains. In this study we sought to develop CRISPR/Cas9 based genetic engineering method for the modification of industrial S. cerevisiae strains. The CRISPR/Cas9 system is based on the adaptive immunity system of bacteria. It makes use of the Cas9 endonuclease which produces double stranded DNA brake to any location determined by a gRNA molecule. This causes the activation of DNA repair mechanisms which can be utilized to for the genomic integration of a template DNA. This makes transformation events much more likely and thus enables producing multiple modifications at once and removes the need for the of use selectable markers. In our approach Cas9 and gRNA were transformed into the cell in a plasmid together with a separate template DNA molecule. We used this method to remove lyp1, ura3 and can1 genes from diploid and polyploid industrial S. cerevisiae strains multiple genes at a time. Simultaneously we evaluated the effect of the NHEJ repair mechanism on CRISPR/Cas9 by repeating the tests with a deletion strain missing the ku70 gene required by NHEJ. Finally the method was used for the metabolic engineering by integrating the five gene violacein metabolic pathway into two loci in a single transformation event. This study demonstrated the CRISPR/Cas9 method is well suited for the modification of industrial S. cerevisiae strains and is capable of modifying up to three loci at a time in a polyploid yeast strain.
  • Verni, Michela; Rizzello, Carlo Giuseppe; Coda, Rossana (2019)
    Cereals are one of the major food sources in human diet and a large quantity of by-products is generated throughout their processing chain. These by-products mostly consist of the germ and outer layers (bran), deriving from dry and wet milling of grains, brewers’ spent grain originating from brewing industry, or others originating during bread-making and starch production. Cereal industry by-products are rich in nutrients, but still they end up as feed, fuel, substrates for biorefinery, or waste. The above uses, however, only provide a partial recycle. Although cereal processing industry side streams can potentially provide essential compounds for the diet, their use in food production is limited by their challenging technological properties. For this reason, the development of innovative biotechnologies is essential to upgrade these by-products, potentially leading to the design of novel and commercially competitive functional foods. Fermentation has been proven as a very feasible option to enhance the technological, sensory, and especially nutritional and functional features of the cereal industry by-products. Through the increase of minerals, phenolics and vitamins bioavailability, proteins digestibility, and the degradation of antinutritional compounds as phytic acid, fermentation can lead to improved nutritional quality of the matrix. In some cases, more compelling benefits have been discovered, such as the synthesis of bioactive compounds acting as antimicrobial, antitumoral, antioxidant agents. When used for baked-goods manufacturing, fermented cereal by-products have enhanced their nutritional profile. The key factor of a successful use of cereal by-products in food applications is the use of a proper bioprocessing technology, including fermentation with selected starters. In the journey toward a more efficient food chain, biotechnological approaches for the valorization of agricultural side streams can be considered a very valuable help.
  • Zhang, Yuetuan (Helsingin yliopisto, 2015)
    The literature review described the importance of folate enhancement to human health especially to coeliac patients with an introduction to folate analysis, pseudocereals and possible fortification methods. The aim of this study was to study the natural folate enhancement methods in pseudocereal matrix. Pseudocereal materials consisted of buckwheat, amaranth and quinoa, each of which was subjected to three different treatments: germination, fermentation and combined treatment. Total folate determination was based on an official microbiological assay method (Lactobacillus rhamnosus ATCC 7469). Germination of pseudocereals lasted for 4–5 days. Fermentation was conducted using either baking yeast Saccharomyces cerevisiae ALKO743 or LAB Streptococcus thermophilus ABM5097. All germinated whole grain pseudocereals indeed showed a significant increase in total folate content. Specifically, the increase was 5.4-fold in buckwheat, 5-fold in amaranth and 2.6-fold in quinoa. Fermentation of native pseudocereals also enhanced total folate level. As for the combined treatment, the total folate level of germinated seeds did not further significantly increase or decrease in later fermentation period. Although more studies are needed for processing real pseudocereal foods, our study showed great potential of folate enhancement using germination or fermentation.
  • Isakova, Elena P.; Matushkina, Irina N.; Popova, Tatyana N.; Dergacheva, Darya; Gessler, Natalya N.; Klein, Olga; Semenikhina, Anastasya; Deryabina, Yulia; La Porta, Nicola; Saris, Nils-Eric L. (2020)
    In this study, we evaluated the metabolic profile of the aerobic microorganism of Endomyces magnusii with a complete respiration chain and well-developed mitochondria system during long-lasting cultivation. The yeast was grown in batches using glycerol and glucose as the sole carbon source for a week. The profile included the cellular biological and chemical parameters, which determined the redox status of the yeast cells. We studied the activities of the antioxidant systems (catalases and superoxide dismutases), glutathione system enzymes (glutathione peroxidase and reductase), aconitase, as well as the main enzymes maintaining NADPH levels in the cells (glucose-6-phosphate dehydrogenase and NADP(+)-isocitrate dehydrogenase) during aging of Endomyces magnusii on two kinds of substrates. We also investigated the dynamics of change in oxidized and reduced glutathione, conjugated dienes, and reactive oxidative species in the cells at different growth stages, including the deep stationary stages. Our results revealed a similar trend in the changes in the activity of all the enzymes tested, which increased 2-4-fold upon aging. The yeast cytosol had a very high reduced glutathione content, 22 times than that of Saccharomyces cerevisiae, and remained unchanged during growth, whereas there was a 7.5-fold increase in the reduced glutathione-to-oxidized glutathione ratio. The much higher level of reactive oxidative species was observed in the cells in the late and deep stationary phases, especially in the cells using glycerol. Cell aging of the culture grown on glycerol, which promotes active oxidative phosphorylation in the mitochondria, facilitated the functioning of powerful antioxidant systems (catalases, superoxide dismutases, and glutathione system enzymes) induced by reactive oxidative species. Moreover, it stimulated NADPH synthesis, regulating the cytosolic reduced glutathione level, which in turn determines the redox potential of the yeast cell during the early aging process.
  • Azizkhani, Mohammad (Helsingin yliopisto, 2020)
    Brewer's yeasts metabolize sugars and produce ethanol and CO2. This study aimed to investigate the relation between the assimilation of sugars in all-malt wort and isotopic signature of carbon and oxygen in the evolved CO2 from brewery fermentations. The isotopic composition of CO2 was measured by a tunable diode laser absorption spectrometer. The isotopic data obtained with automatic sampling, on-line, and in real-time. Wort samples were collected with 3 h intervals to quantify the residual sugars by high-performance liquid chromatography. Patterns of changes in δ13C and δ18O values were unique to experiments with each yeast type. The common overall ascending trend in δ13C and δ18O values in all experiments can be described by kinetic fractionation of isotopes, which explains that in a bioreaction the lighter isotopes participate in the reaction more readily than the heavier ones. Therefore, the early emergence of light isotopologues of CO2 may be a consequence of the fermentation of light isotopologues of sugars. A sudden decrease and then increase in delta values were observed in all experiments before the residual concentrations of glucose and fructose reach their lowest levels. This can be an indicator of the complete assimilation of monosaccharides by yeast. In the fermentations that yeast was able to consume maltose, δ13C and δ18O values raised considerably in a short period. Concurrently, maltose approached its eventual residual concentrations indicating an endpoint for its utilization by yeast. Our results confirm the hypothesis of a connection between the assimilation of sugars and the isotopic signature of evolved CO2 during brewery fermentations. The findings support the potential of off-gas isotopic analysis to monitor sugar assimilation in brewery fermentations.
  • Chandrasekar Rajendran, Suresh Chander (Helsingfors universitet, 2014)
    Idli is a popular cereal-legume fermented food of Indian origin. It is steam cooked from fermented (lactic acid-yeast) batter of rice (cereal) and black gram (legume). Idli preparation process includes three major steps – soaking of rice and black gram, grounding and fermentation. The idli preparation process is laborious, as the whole procedure takes about 20 hours. Further, the fermented batter has a shelf life of 4-5 days at 4 ºC. Literature studies reveal less efforts has been taken to improve shelf life and nutritional quality of idli. The overall aim of this thesis was to improve the quality of idli batter by mild heat treatment (Objective 1) and through microbial applications (Objective 2-4). First, the fermented idli batter was mild heat (MH) treated (57, 60, 63, 66 and 70 ºC ) to reduce the high (10.5 log cfu/g) lactic acid bacteria and yeast counts for enhancing the shelf stability at refrigerated storage. MH treatment (at 70 ºC) induced the highest reduction (3.6 log cfu/g) without affecting the pasting profile of idli batter. During storage study (upto 10 days at 4 ºC) the microbial counts further decreased without change in pH.The second objective was to monitor the changes in physicochemical properties and B-vitamin (riboflavin, folate and vitamin B12) levels in idli batter fermentation on addition of starters - Lactococcus lactis N8 (SAA1) and Saccharomyces boulardii (YEA1). Fermentation profiles were recorded individually and in combination of starters. SAA1 and YEA1 were able to enhance or retain riboflavin and folate levels, but no change in vitamin B12 levels were observed during fermentation. Further, YEA1 individually and in combination with SAA1 significantly improved the idli batter volume, implying high gas production. The third objective was to produce nisin in idli batter by addition of SAA1 (nisin producer). The results highlighted SAA1 was capable of producing nisin in idli. However, the produced nisin was degraded by the activity of indigenous LAB and yeast in idli batter. The final objective of this thesis was to determine the viability of probiotic Bacillus coagulans (BAC1) spores after cooking (steaming and microwaving) and during storage (at 4 ºC) of idli batter. Microwave cooking resulted in higher reduction of BAC1 than steam cooking. However, 5.4 log cfu/g of BAC1 spores were still viable in steamed idli from the initial added amount (8.2 log cfu/g). The BAC1 spores were not stable in idli batter suggesting spore outgrowth during storage. In summary, these results present different strategies and information for future process and product developments in idli.
  • Back, Anu (Helsingfors universitet, 2010)
    Selvitin työssäni yhdeksän vapaankaupan pötsilääkkeen sisällön koostumusta ja vaikutusta pötsissä. Vertasin valmisteiden ohjeannosten mukaisia määriä ainesosakohtaisesti niiden tarpeellisuuteen, kirjallisuudessa suositeltuihin hoitoannoksiin ja lehmän päivittäiseen saantiin rehuista. Ruokintatasoksi valittiin yksinkertaisuuden vuoksi 20 kg KA/pv, joista 12 kg KA tulee säilörehusta, 8 kg KA ohrasta ja 2 kg KA rypsirouheesta. Voimakas väkirehuruokinta tai väkirehun osuuden liian nopea lisääminen ruokinnassa altistavat pötsin happamoitumiselle. Luontaisen puskuroinnin ollessa riittämätöntä, pötsin happamuutta voidaan alentaa puskuroivilla tai alkaloivilla aineilla. Yhdeksästä valmisteesta viisi sisältää happamuutta alentavia aineita. Näistä viidestä valmisteesta kahdessa (ReCovin pötsin pH pasta, Correct pH Kombi) aineiden määrä on riittävä. Natriumbikarbonaatti (ruokasooda) on puskuri ja magnesiumoksidi on alkaloiva aine, molemmat ovat tehokkaita happamuuden alentajia. Myös hiivat alentavat pötsinesteen happamuutta. Ne vähentävät maitohapon kertymistä ja lisäävät kuitua sulattavien bakteerien määrää pötsissä. Kolmessa valmisteessa yhdeksästä on riittävästi hiivaa (Super Vetrumin -jauhe, Rumelan, Correct Pötsi-Potku). Propyleeniglykoli on tehokkaana märehtijän verensokeria kohottavana aineena tarpeellinen herumiskaudella olevan syömättömän lehmän tukihoidossa. Propyleeniglykolia on kahdessa valmisteessa (Rumex pH-pasta, Correct Pötsi-Potku). Hivenaineista seleenin on todettu lisäävän alkueläinten määrää pötsissä, ja mikrobit käyttävät sitä proteiinisynteesissään. Kobolttia tarvitaan B12-vitamiinin synteesiin. B12-vitamiinia tarvitaan märehtijän energia-aineenvaihdunnalle välttämättömässä glukoneogeneesissä. Kobolttia on riittävästi viidessä valmisteessa (ReCovin Pötsin pH-pasta, Super Vetrumin -jauhe, Rumelan, Rumex, Rumevit), seleeniä vain yhdessä (ReCovin Pötsin pH-pasta). Mikrobitoiminnan häiriössä B-vitamiinien synteesi pötsissä saattaa vähentyä. Varsinkin B1-vitamiinin eli tiamiinin puute tiaminaasin tuotannon takia happamassa pötsissä ja sen yhteys kerebrokortikaali nekroosiin on hyvin tunnettu. B3-vitamiinin eli niasiinin on todettu tehostavan pötsimikrobien proteiinisynteesiä. B-vitamiineja on lisätty riittävästi neljään valmisteeseen (Biorumin, Super Vetrumin -jauhe, Rumex, Rumevit). Tutkielmassani pohdin myös millainen olisi hyvä pötsilääke. Pötsilääkkeisiin valitsin 8 edellä mainittua hyödyllistä ainetta. Happaman pötsin hoitoon suosittelen natriumbikarbonaattia ja/tai magnesiumoksidia ja hiivaa, niiden happamuutta alentavan vaikutuksen takia. Tiamiinia (B1-vitamiini), koska siitä on happamassa pötsissä todennäköisesti puutetta, ja propyleeniglykolia tukihoidoksi energiavajeeseen. Yksinkertaisen pötsihäiriön hoitoon suosittelen hiivaa, kobolttia, seleenimetioniinia (orgaaninen seleeni) ja niasiinia (B3-vitamiini) niiden pötsimikrobistoa elvyttävän vaikutuksen takia, ja propyleeniglykolia energiavajeeseen. Yhtä tärkeänä, kuin pötsihäiriöiden lääkitsemistä, pidän niiden ennaltaehkäisyä, jossa tärkeimpiä asioita ovat nopeiden ruokinnanmuutosten välttäminen ja rehujen hyvä laatu. Lisäksi on hyvä muistaa kuivan heinän edulliset vaikutukset märehtimistä, syljen erittymistä ja pötsin liikkeitä ylläpitävänä rehuna. Riittävä syljen erittyminen on tärkeä pötsinesteen happamuutta alentava tekijä.
  • EFSA Panel Nutr Novel Foods Food A; Turck, Dominique; Heinonen, Marina (2020)
    Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on chromium-enriched biomass of Yarrowia lipolytica as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is the dried and heat-killed chromium-enriched biomass of Y. lipolytica. This yeast species is widespread in nature, can be found in the environment and in foods, and was attributed the qualified presumption of safety (QPS) status for production purposes in 2018, including food and feed products based on biomass of the yeast. The production process, fermentation in the presence of chromium chloride, includes a heat-killing step of the yeast, resulting in the absence of viable Y. lipolytica in the NF. The maximum total chromium content of the NF is 23 mu g Cr/g, with the chromium present as Cr(III). The applicant proposed to use the NF as a food supplement. The target population proposed by the applicant is the general population from 3 years of age onwards, with maximum proposed use levels of 2 g/day for children from 3 to 9 years of age and 4 g/day thereafter. At the proposed use levels of the NF, the combined intake of chromium provided by the NF, in addition to a background diet high in chromium, would result in total chromium intakes well below the tolerable daily intake (TDI) for chromium(III) for all target population groups. The Panel concludes that the NF, chromium-enriched biomass of Y. lipolytica, is safe under the proposed conditions of use. (C) 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.
  • EFSA Panel Nutr Novel Foods Food A; Turck, Dominique; Heinonen, Marina (2020)
    Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on selenium-enriched biomass of Yarrowia lipolytica as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is the dried and heat-killed selenium-enriched biomass of Y. lipolytica. This yeast species is widespread in nature, can be found in the environment and in foods, and was attributed the qualified presumption of safety (QPS) status for production purposes in 2018, including food and feed products based on biomass of the yeast. The production process, fermentation in the presence of sodium selenite, includes a heat-killing step of the yeast, resulting in the absence of viable Y. lipolytica in the NF. The maximum total selenium content in the NF is 200 mu g Se/g, mainly present as organic selenium compounds. The applicant proposed to use the NF as a food supplement. The target population proposed by the applicant is the general population from 3 years of age onwards, with maximum proposed use levels of 0.2 g/day for children from 3 to 9 years of age and 1 g/day thereafter. The Panel considers that the yeast biomass is not of safety concern at the proposed use levels. The Panel also considers that the selenium provided by the NF is as safe as selenium from other dietary sources. However, the Panel notes that, at the use levels proposed by the applicant, the intake of the NF could, in combination with a background diet high in selenium, lead to total selenium intakes exceeding the UL for selenium in all target population groups, except for children from 7 to 9 years. Furthermore, the Panel considers that newly emerging data warrant a reassessment of the UL for selenium. (C) 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.
  • Turck, Dominique; Castenmiller, Jacqueline; de Henauw, Stefaan; Hirsch-Ernst, Karen-Ildico; Kearney, John; Maciuk, Alexandre; Mangelsdorf, Inge; McArdle, Harry J.; Naska, Androniki; Pelaez, Carmen; Pentieva, Kristina; Siani, Alfonso; Thies, Frank; Tsabouri, Sophia; Vinceti, Marco; Cubadda, Francesco; Engel, Karl-Heinz; Frenzel, Thomas; Heinonen, Marina; Marchelli, Rosangela; Neuhauser-Berthold, Monika; Poting, Annette; Poulsen, Morten; Sanz, Yolanda; Schlatter, Josef Rudolf; van Loveren, Henk; Ackerl, Reinhard; Knutsen, Helle Katrine (2019)
    Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on Yarrowia lipolytica yeast biomass as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is the dried and heat-killed biomass of Yarrowia lipolytica, a yeast species that is widespread in nature and which can be found in the environment as well as in foods. The production process is sufficiently described and does not raise safety concerns. The information provided on the composition of the NF is sufficient and does not raise safety concerns. The applicant proposed to use the NF as a food supplement in the form of capsules, tablets or powder. The target population for the NF is the general population above 3 years of age. The maximum proposed daily use levels are 3 g/day for children from 3 years to less than 10 years of age and 6 g/day thereafter. Taking into account the composition of the NF and the proposed use levels, the consumption of the NF is not nutritionally disadvantageous. No relevant toxicological information was provided. The Panel considers that given the qualified presumption of safety (QPS) status for production purposes of Yarrowia lipolytica and the fact that the production process of the NF does not raise safety concerns, no toxicological studies are needed for the safety assessment of the NF. The Panel concludes that the NF, Yarrowia lipolytica yeast biomass, is safe under the proposed conditions of use. (C) 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.
  • EFSA Panel Nutr Novel Foods Food (2019)
    Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on Yarrowia lipolytica yeast biomass as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is the dried and heat-killed biomass of Yarrowia lipolytica, a yeast species that is widespread in nature and which can be found in the environment as well as in foods. The production process is sufficiently described and does not raise safety concerns. The information provided on the composition of the NF is sufficient and does not raise safety concerns. The applicant proposed to use the NF as a food supplement in the form of capsules, tablets or powder. The target population for the NF is the general population above 3 years of age. The maximum proposed daily use levels are 3 g/day for children from 3 years to less than 10 years of age and 6 g/day thereafter. Taking into account the composition of the NF and the proposed use levels, the consumption of the NF is not nutritionally disadvantageous. No relevant toxicological information was provided. The Panel considers that given the qualified presumption of safety (QPS) status for production purposes of Yarrowia lipolytica and the fact that the production process of the NF does not raise safety concerns, no toxicological studies are needed for the safety assessment of the NF. The Panel concludes that the NF, Yarrowia lipolytica yeast biomass, is safe under the proposed conditions of use. (C) 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.
  • Karjalainen, Eveliina (Helsingin yliopisto, 2019)
    Yeasts are significant plant pathogens, yet plant-yeast interactions are poorly understood. Mammals have immune receptors for yeast-specific MAMPs (microbe-associated molecular patterns), suggesting that plants might have similar receptors as well. Detection of MAMPs by PRRs (pattern recognition receptors) triggers the plant immune system, leading to the first phase of PTI (pattern-triggered immunity). The yeast cell wall is layered, so only some of the potential MAMP molecules, like mannans, are easily available for the plant receptors. In order to utilize yeasts and PRR-based yeast resistance in agriculture and forestry, it is crucial to identify these receptors and gain a better understanding of their functions in plant-yeast interactions. PRRs can be divided into two groups, RLPs (receptor-like proteins) and RLKs (receptor-like kinases). Some of the L-type lectin receptor kinases have already been shown to participate in the interactions between plants and fungi or fungal like pathogens, making them promising candidates for yeast receptors. G-type lectins remain less studied but have promising genes as well. In this thesis, we use forward and reverse genetics methods to show that lectin receptor kinases might include putative yeast pattern recognition receptors. We also establish the first protocol and genetic screen for the identification of plant PRRs participating in the recognition of yeast cell wall MAMPs. Only genes present in both silver birch (Betula pendula) and arabidopsis (Arabidopsis thaliana) were examined to enable following studies with an Arabidopsis model system of the interactions between silver birch and the dimorphic birch pathogen Taphrina betulina. This information can later be used in plant production with other plants and their yeasts as well.
  • Wei, Ting; Najmi, Saman M.; Liu, Hester; Peltonen, Karita; Kucerova, Alena; Schneider, David A.; Laiho, Marikki (2018)
    Summary Inhibition of RNA polymerase I (Pol I) is a promising strategy for modern cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme, but how this exceptional response is enforced is not known. Here, we define key elements requisite for the response. We show that Pol I preinitiation factors and polymerase subunits (e.g., RPA135) are required for BMH-21-mediated degradation of RPA194. We further find that Pol I inhibition and induced degradation by BMH-21 are conserved in yeast. Genetic analyses demonstrate that mutations that induce transcription elongation defects in Pol I result in hypersensitivity to BMH-21. Using a fully reconstituted Pol I transcription assay, we show that BMH-21 directly impairs transcription elongation by Pol I, resulting in long-lived polymerase pausing. These studies define a conserved regulatory checkpoint that monitors Pol I transcription and is activated by therapeutic intervention.