New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast

Copyright © 2015 John Wiley & Sons, Ltd. Funded by College of Life Science and Medicine, University of Aberdeen, UK This work was funded by a start-up grant from the College of Life Science and Medicine, University of Aberdeen, UK. I am grateful to J. Bähler, E. Hartsuiker, F. Klein, J. Kohli, K. Nasmyth, M. C. Whitby, the Leibniz Institute – German Collection of Microorganisms and Cell Cultures (DMSZ) and the National BioResource Project Japan (NBRP) for providing materials used in this study. I thank Alistair J. P. Brown and Takashi Kubota for critically reading this manuscript.

New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast

Copyright © 2015 John Wiley & Sons, Ltd. Funded by College of Life Science and Medicine, University of Aberdeen, UK This work was funded by a start-up grant from the College of Life Science and Medicine, University of Aberdeen, UK. I am grateful to J. Bähler, E. Hartsuiker, F. Klein, J. Kohli, K. Nasmyth, M. C. Whitby, the Leibniz Institute – German Collection of Microorganisms and Cell Cultures (DMSZ) and the National BioResource Project Japan (NBRP) for providing materials used in this study. I thank Alistair J. P. Brown and Takashi Kubota for critically reading this manuscript.

Development of a binary positive and negative protein fragment complementation assay using yeast cytosine deaminase

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Membrane protein production in the Yeast, S. cerevisiae

The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca2+-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.

Mechanisms of Cdc42 Polarization in Yeast

Polarization is important for the function and morphology of many different cell types. The keys regulators of polarity in eukaryotes are the Rho-family GTPases. In the budding yeast Saccharomyces cerevisiae, which must polarize in order to bud and to mate, the master regulator is the highly conserved Rho GTPase, Cdc42. During polarity establishment, active Cdc42 accumulates at a site on the plasma membrane characterizing the “front” of the cell where the bud will emerge. The orientation of polarization is guided by upstream cues that dictate the site of Cdc42 clustering. However, in the absence of upstream cues, yeast can still polarize in a random direction during symmetry breaking. Symmetry breaking suggests cells possess an autocatalytic polarization mechanism that can amplify stochastic fluctuations of polarity proteins through a positive feedback mechanism.

Two different positive feedback mechanisms have been proposed to polarize Cdc42 in budding yeast. One model posits that Cdc42 activation must be localized to a site at the plasma membrane. Another model posits that Cdc42 delivery must be localized to a particular site at the plasma membrane. Although both mechanisms could work in parallel to polarize Cdc42, it is unclear which mechanism is critical to polarity establishment. We directly tested the predictions of the two positive feedback models using genetics and live microscopy. We found that localized Cdc42 activation is necessary for polarity establishment.

While this explains how active Cdc42 localizes to a particular site at the plasma membrane, it does not address how Cdc42 concentrates at that site. Several different mechanisms have been proposed to concentrate Cdc42. The GDI can extract Cdc42 from membranes and selective mobilize GDP-Cdc42 in the cytoplasm. It was proposed that selectively mobilizing GDP-Cdc42 in combination with local activation could locally concentrate total Cdc42 at the polarity site. Although the GDI is important for rapid Cdc42 accumulation at the polarity site, it is not essential to Cdc42 concentration. It was proposed that delivery of Cdc42 by actin-mediated vesicle can act as a backup pathway to concentrate Cdc42. However, we found no evidence for an actin-dependent concentrating pathway. Live microscopy experiments reveal that prenylated proteins are not restricted to membranes, and can enter the cytoplasm. We found that the GDI-independent concentrating pathway still requires Cdc42 to exchange between the plasma membrane and the cytoplasm, which is supported by computational modeling. In the absence of the GDI, we found that Cdc42 GAP became essential for polarization. We propose that the GAP limits GTP-Cdc42 leak into the cytoplasm, which would be prohibitive to Cdc42 polarization.

Topoisomerase 1 (Top1)-associated Genome Instability in Yeast: Effects of Persistent Cleavage Complexes or Increased Top1 Levels

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.

Predicting Transcript Production Rates in Yeast With Sparse Linear Models

To provide biological insights into transcriptional regulation, a couple of groups have recently presented models relating the promoter DNA-bound transcription factors (TFs) to downstream gene’s mean transcript level or transcript production rates over time. However, transcript production is dynamic in response to changes of TF concentrations over time. Also, TFs are not the only factors binding to promoters; other DNA binding factors (DBFs) bind as well, especially nucleosomes, resulting in competition between DBFs for binding at same genomic location. Additionally, not only TFs, but also some other elements regulate transcription. Within core promoter, various regulatory elements influence RNAPII recruitment, PIC formation, RNAPII searching for TSS, and RNAPII initiating transcription. Moreover, it is proposed that downstream from TSS, nucleosomes resist RNAPII elongation.

Here, we provide a machine learning framework to predict transcript production rates from DNA sequences. We applied this framework in the S. cerevisiae yeast for two scenarios: a) to predict the dynamic transcript production rate during the cell cycle for native promoters; b) to predict the mean transcript production rate over time for synthetic promoters. As far as we know, our framework is the first successful attempt to have a model that can predict dynamic transcript production rates from DNA sequences only: with cell cycle data set, we got Pearson correlation coefficient Cp = 0.751 and coefficient of determination r2 = 0.564 on test set for predicting dynamic transcript production rate over time. Also, for DREAM6 Gene Promoter Expression Prediction challenge, our fitted model outperformed all participant teams, best of all teams, and a model combining best team’s k-mer based sequence features and another paper’s biologically mechanistic features, in terms of all scoring metrics.

Moreover, our framework shows its capability of identifying generalizable fea- tures by interpreting the highly predictive models, and thereby provide support for associated hypothesized mechanisms about transcriptional regulation. With the learned sparse linear models, we got results supporting the following biological insights: a) TFs govern the probability of RNAPII recruitment and initiation possibly through interactions with PIC components and transcription cofactors; b) the core promoter amplifies the transcript production probably by influencing PIC formation, RNAPII recruitment, DNA melting, RNAPII searching for and selecting TSS, releasing RNAPII from general transcription factors, and thereby initiation; c) there is strong transcriptional synergy between TFs and core promoter elements; d) the regulatory elements within core promoter region are more than TATA box and nucleosome free region, suggesting the existence of still unidentified TAF-dependent and cofactor-dependent core promoter elements in yeast S. cerevisiae; e) nucleosome occupancy is helpful for representing +1 and -1 nucleosomes’ regulatory roles on transcription.

Reduction of salt in yeasted wheat bread: impact on bread quality and solutions using sourdough fermented by functional lactic acid bacteria strains

The dietary intake of sodium chloride has increased considerably over the last few decades due to changes in the human diet. This higher intake has been linked to a number of diseases including hypertension and other cardiovascular diseases. Numerous international health agencies, as well as the food industry, have now recommended a salt intake level of 5-6 g daily, approximately half of the average current daily intake level. Cereal products, and in particular bread, are a major source of salt in the Western diet. Therefore, any reduction in the level of salt in bread could have a major impact on global health. However, salt is a critical ingredient in bread production, and its reduction can have a deleterious effect on the production process as well as on the final bread quality characteristics such as shelf-life, bread volume and sensory characteristics, all deviating from the bakers’ and consumers’ expectations. This work addresses the feasibility of NaCl reduction in wheat bread focusing on options to compensate NaCl with the use of functional sourdoughs. Three strains were used for the application of low-salt bread; L. amylovorus DSM19280, W. cibaria MG1 and L. reuteri FF2hh2. The multifunctional strain L. reuteri FF2hh2 was tested the first time and its application could be demonstrated successfully. The functionalities were based on the production of exopolysaccharides as well as the production of antifungal compounds. While the exopolysaccharides, mainly high molecular dextrans, positively influenced mainly bread loaf volume, crumb structure and staling rate, the strains producing antifungal compounds prolonged the microbial shelf life significantly and compensated the lack of salt. The impact on the sensory characteristics of bread were evaluated by descriptive sensory evaluation. The increase in surface area as well as the presence of organic acids impacted significantly on the flavour profile of the sourdough bread samples. The flavour attribute “salt” could be enhanced by sourdough addition and increased the salty perception. Furthermore, a trained sensory panel evaluated for the first time the impact of yeast activity, based on different salt and yeast concentrations, on the volatile aroma profile of bread crumb samples. The analytical measurements using high resolution gas chromatography and proton-transfer-reaction mass spectrometry (PTR-MS) resulted in significantly different results based on different yeast activities. Nevertheless, the extent of the result could not be recognised by the sensory panel analysing the odour profile of the bread crumb samples. Hence, the consumer cannot recognised low-salt bread by its odour. The use of sourdough is a natural option to overcome the broad range of technological issues caused by salt reduction and also a more popular alternative compared to existing chemical salt replacers.

Development of a binary positive and negative protein fragment complementation assay using yeast cytosine deaminase

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

Microtubule attachment and regulation mechanisms of the budding yeast kinetochore

Thesis (Master's)--University of Washington, 2016-08

Biofuels and high value added products from the yeast Rhodosporidium toruloides NCYC 921: Strategies towards a true cost-effective and environmentally sustainable integrated multiproduct driven biorefinery [Poster]

Single-cell oils (SCO) have been considered a promising source of 3rd generation biofuels mainly in the final form of biodiesel. However, its high production costs have been a barrier towards the commercialization of this commodity. The fast growing yeast Rhodosporidium toruloides NCYC 921 has been widely reported as a potential SCO producing yeast. In addition to its well-known high lipid content (that can be converted into biodiesel), is rich in high value added products such as carotenoids with commercial interest. The process design and integration may contribute to reduce the overall cost of biofuels and carotenoid production and is a mandatory step towards their commercialization. The present work addresses the biomass disruption, extraction, fractionation and recovery of products with special emphasis on high added valued carotenoids (beta-carotene, torulene, torularhodin) and fatty acids directed to biodiesel. The chemical structure of torularhodin with a terminal carboxylic group imposes an additional extra challenge in what concern its separation from fatty acids. The proposed feedstock is fresh biomass pellet obtained directly by centrifugation from a 5L fed-batch fermentation culture broth. The use of a wet instead of lyophilised biomass feedstock is a way to decrease processing energy costs and reduce downstream processing time. These results will contribute for a detailed process design. Gathered data will be of crucial importance for a further study on Life-Cycle Assessment (LCA).

Improvement of the yeast slide culture technique

Este trabalho tem como objetivo melhorar a técnica de cultura em lâmina para ser usada na avaliação da viabilidade de leveduras sob diferentes condições fisiológicas. Inicialmente, foram otimizadas as condições ideais para o cultivo em lâmina de uma estirpe laboratorial (BY4741) e de uma estirpe industrial (NCYC 1214) da levedura Saccharomyces cerevisiae. O melhor protocolo foi obtido utilizando: YEPD agar com uma espessura de cerca de 2 mm; 20 μL de uma suspensão de 1 x 105 células/mL para a estirpe BY4741 ou de 5 x 104 células/mL para a estirpe NCYC 1214; uma câmara de humedecimento com 100 μL de água desionizada e um tempo de incubação de 24 h, a 25 ° C. Com o objetivo de facilitar a contagem das microcolónias, foi adicionado um corante (calcofluor white, CFW) ao meio YEPD agar. Ensaios preliminares, em YEPD líquido, contendo diferentes concentrações de CFW, permitiram verificar que o corante, até 5,0 μg/L, não inibe o crescimento da levedura. Uma concentração de 2,5 μg/L de CFW permitiu a coloração da parede das leveduras, não se observando células com morfologia alterada, sendo esta a concentração de CFW selecionado nos estudos subsequentes. A técnica de cultura em lâmina, com ou sem CFW, foi aplicada para avaliar a viabilidade de células saudáveis (células em fase exponencial de crescimento), células submetidas a stress de etanol [células expostas a 20% (v/v) de etanol, a 25 ºC, durante 2 h] e células envelhecidas (células incubadas em água, a 25 ° C, durante 48 h), da estirpe laboratorial. A percentagem de células viáveis não foi significativamente diferente entre as duas técnicas (com ou sem CFW), após uma incubação de 24 horas. Finalmente, a técnica de cultura de lâmina, contendo CFW, foi comparada com duas técnicas habitualmente usadas na indústria cervejeira: fermentação de curta duração e determinação da percentagem de células gemuladas. Os resultados obtidos através da técnica de cultura de lâmina, desenvolvida, seguem um padrão similar aos obtidos nos ensaios de fermentação de curta duração e aos da determinação da percentagem de células gemuladas. Os resultados obtidos sugerem que a técnica de cultura em lâmina, combinada com CFW, parece ser uma alternativa, fácil, rápida (em 24 h) e reprodutível, relativamente ao método convencional (técnica de plaqueamento), para a avaliação da viabilidade de células de levedura. Deverá ser realizado trabalho adicional a fim de validar o método com estirpes industriais.