Journal Articles

Permanent URI for this collectionhttps://mro.massey.ac.nz/handle/10179/7915

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Has files: YesSubject: search.filters.subject.Saccharomyces cerevisiae

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    Rapid yeast-based screen for Functionally Relevant Amino Acids (RS-FRAA) in a protein
    (Elsevier Inc, 2023-03-17) Ghuge AA; Anderson RA; Gottfried S; Daube C; Koloamatangi SMBMJ; Schiemann AH; Sattlegger E
    Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces cerevisiae in-yeast recombination, and semi-quantitative growth assays. We also describe a mating step to reduce the occurrence of false positive findings due to ectopic mutations. The only requirement is that the protein elicits a phenotype in Saccharomyces cerevisiae.
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    A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation (research article)
    (PLOS, 2022-11-28) Gottfried S; Koloamatangi SMBMJ; Daube C; Schiemann AH; Sattlegger E; Lustig AJ
    The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.
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    Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals-Illustrated with Four Actin Cytoskeleton Proteins
    (MDPI (Basel, Switzerland), 2020-03-10) Akram Z; Ahmed I; Mack H; Kaur R; Silva RC; Castilho BA; Friant S; Sattlegger E; Munn AL
    The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: 1) yeast Hof1p/mammalian PSTPIP1, 2) yeast Rvs167p/mammalian BIN1, 3) yeast eEF1A/eEF1A1 and eEF1A2 and 4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.
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    Saccharomyces cerevisiae Yeast-Based Supplementation as a Galactagogue in Breastfeeding Women? A Review of Evidence from Animal and Human Studies
    (MDPI (Basel, Switzerland), 2021-03) Jia LL; Brough L; Weber JL; Demmelmair H
    Perceived insufficient milk production (PIM) adversely affects breastfeeding duration. Women sometimes use galactagogues with the intent to increase breast milk production and support lactation. Saccharomyces cerevisiae yeast-based supplement (SCYS) is an inactive form of Saccharomyces cerevisiae yeast (SCY) either obtained from the fermentation process or grown on molasses. Anecdotal evidence suggests SCYS is a galactagogue. SCYS is promoted on the internet as a galactagogue in various forms and doses. Dietary supplementation with SCYS during gestation and lactation significantly increases milk yield in ruminants. No human study has evaluated efficacy of SCYS as a galactagogue. SCYS is rich in B vitamins, beta-glucan, mannan oligosaccharides and bioavailable chromium; these may impact breast milk production or composition, thus may alleviate PIM. The safety of taking SCYS during lactation is not well studied. Studies have reported contamination of SCYS with ochratoxin A (OTA) as well as minor side effects from SCYS. Studies are needed to evaluate the efficacy of SCYS on breast milk production and composition and to assess the safety of taking SCYS during lactation in humans.
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    Phenotypic and genotypic characterisation of Lactobacillus and yeast isolates from a traditional New Zealand Māori potato starter culture
    (Elsevier BV, 2022-08-26) Sun J; Silander O; Rutherfurd-Markwick K; Wen D; Davy TP-P; Mutukumira AN
    Parāroa Rēwena is a traditional Māori sourdough produced by fermentation using a potato starter culture. The microbial composition of the starter culture is not well characterised, despite the long history of this product. The morphological, physiological, biochemical and genetic tests were conducted to characterise 26 lactic acid bacteria (LAB) and 15 yeast isolates from a Parāroa Rēwena potato starter culture. The results of sugar fermentation tests, API 50 CHL tests, and API ID 32 C tests suggest the presence of four different LAB phenotypes and five different yeast phenotypes. 16S rRNA and 26S rRNA sequencing identified the LAB as Lacticaseibacillus paracasei and the yeast isolates as Saccharomyces cerevisiae, respectively. Multilocus sequence typing (MLST) of the L. paracasei isolates indicated that they had identical genotypes at the MLST loci, to L. paracasei subsp. paracasei IBB 3423 or L. paracasei subsp. paracasei F19. This study provides new insights into the microbial composition of the traditional sourdough Parāroa Rēwena starter culture.
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    Identification of dominant lactic acid bacteria and yeast in rice sourdough produced in New Zealand
    (Elsevier BV, 2021-10-21) Yang Q; Rutherfurd-Markwick K; Mutukumira AN
    This study characterised a commercial New Zealand gluten free (GF) rice sourdough and its starter culture composition. Acidity of the mother sourdough, dough before proofing and dough after proofing was determined during the production of rice sourdough bread, and colour was measured for the baked bread. Yeast and lactic acid bacteria (LAB) were enumerated in the rice sourdough samples and representative colonies characterised using API kits and sequenced by the Internal Transcribed Spacer and 16 S rRNA region. Sourdough LAB isolates were identified as Lactobacillus (L.) papraplantarum DSM 10667 and L. fermentarum CIP 102980 and the yeast isolates as Saccharomyces (S.) cerevisiae CBS 1171. Dough acidity increased significantly (p < 0.05) during fermentation due to the metabolic activities of the sourdough cultures. After baking, the colour of the rice sourdough bread crust was similar to that of unleavened wheat bread (golden brown). The improved colour of the rice sourdough bread crust may be a result of combined use of sourdough technique and optimal baking conditions. The results of this study may allow bakers to improve the overall quality of GF rice sourdough baked bread by selecting suitable fermentation and baking parameters.