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Item 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 EHere, 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.Item 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 AJThe 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.Item 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 ALThe 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.Item 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 HPerceived 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.Item 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 ANParāroa 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.Item 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 ANThis 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.Item Identification of large ribosomal proteins required for the full activation of the protein kinase Gcn2 in Saccharomyces cerevisiae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Albany, New Zealand. EMBARGOED indefinitely.(Massey University, 2019) Anderson, Reuben AndrewProtein synthesis is a fundamental biological process that all organisms require for maintaining life, growth and development. The maintenance of amino acid levels, the building blocks of proteins, is essential for maintaining protein synthesis under all biological conditions. Hence, amino acid shortage can be deleterious to the cell. Therefore, cells harbour mechanisms to cope and overcome amino acid starvation. When eukaryotes are subjected to amino acid starvation, the resulting accumulation of uncharged tRNAs activates the protein kinase Gcn2, leading to phosphorylation of eIF2α and activation of the amino acid starvation response. Uncharged tRNAs are the signal of starvation, directly detected by Gcn2. Gcn2 must bind to the effector protein Gcn1 and both must contact ribosomes for Gcn2 activation. The current working model for how the starvation signal is delivered to Gcn2 postulates that these uncharged tRNAs bind in the A-site of the ribosome in a codon specific manner, which are subsequently transferred to Gcn2. Gcn1 is directly involved in this process but its exact involvement is unknown. To test the working model, it is paramount to investigate where Gcn1 and Gcn2 bind on the ribosome. Ribosomes consist of a large and small subunit, each containing multiple ribosomal proteins placed in unique locations. Identification of ribosomal proteins contacting Gcn1 or Gcn2 will allow for deduction of where Gcn1 and Gcn2 bind on the ribosome. This study aimed to determine Gcn1 and Gcn2 contact points on the large ribosomal subunit, usinga genetic approach. The hypothesis was that if an interaction of Gcn1 or Gcn2 with a particular large ribosomal protein (Rpl) is important for Gcn2 activation, then its overexpression would impair Gcn2 function. Overexpression of several large ribosomal proteins impaired cell growth on a medium triggering amino acid starvation, suggesting Gcn2 activation was impaired. Groups of two or more of these Rpls were found in several regions which contain ribosomal proteins shown or suggested to interact with Gcn1 or Gcn2 previously. This included a region containing the P-stalk proteins (part of the large ribosomal complex) known to contact Gcn2. A region close to the small ribosomal protein Rps10, known to contact Gcn1, was also identified. Another region with Rpls which contacts a protein eEF3, which is suggested to share similar ribosomal contacts as Gcn1, was identified.Item Investigating the evolutionary changes in Crabtree-negative yeasts during a long-term evolution experiment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand(Massey University, 2017) Morley, AnnabelThe Crabtree effect is a metabolic strategy that allows yeast to ferment in the presence of oxygen. This is of interest as not all yeasts display this strategy, and nearly 100 years after its discovery it is still unclear what the overall benefit is. Two key theories attempt to explain the emergence of this phenomenon, the make-accumulate-consume theory and the rate/yield trade-off theory. The aim of this thesis was to investigate whether a trade-off between rate and yield develops in Crabtree-negative yeasts over the course of 1500 generations in a high sugar environment. Chapter Two demonstrates that growth rate is more likely to increase than decrease while growth yield is more likely to decrease than increase in the isolate-derived populations of yeast. We find that species that started out relatively fast, changed little while the slower species had more significant gains in growth rate. With growth yield, the species with initially high yield lost more significantly than the already low yield species. This could suggest there is an overall optimum growth rate and growth yield, that the species are evolving towards. In Chapter Three, ethanol production was measured using colorimetric tests and no change was observed to support the development of the Crabtree effect in these populations after 1500 generations. In Chapter Four growth yield was investigated using flow cytometry and it was found that several yeast populations both increased in cell size and decreased in growth yield. This is an interesting observation that has been observed in several previous experimental evolution experiments. In Chapter Five, as cell size is often associated with ploidy changes, DNA content was measured using DAPI and SYTOX DNA stains, detected by flow cytometry. This did not provide any statistically significant conclusions but highlighted the importance of employing further techniques to analyse the DNA content of these populations. This thesis has illustrated the importance of studying the competitive behaviours of microorganisms in isolation, where selfish traits appear to thrive.Item Role of the ribosomal DNA repeats on chromosome segregation of Saccharomyces cerevisiae : a dissertation presented in fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand(Massey University, 2016) Quintana Rincon, Daniela MariaChromosome segregation is a highly conserved process that progresses with great accuracy. Failure of proper segregation can lead to genetic disorders, such as Down syndrome in humans. Interestingly, segregation errors found in human genetic disorders and associated with spontaneous abortions or stillbirths are frequent in the chromosomes containing the ribosomal RNA gene repeats (rDNA). The rDNA is essential for cell viability and growth as it encodes ribosomal RNA, a major component of ribosomes. In yeast, the rDNA locus has a unique cohesin-independent cohesion mechanism to hold sister chromatids together before separation, and behaves in unique ways with respect to replication, recombination and transcription. These rDNAspecific features may promote a chromosome segregation mechanism distinct from the rest of the genome. Therefore, the aim of this thesis was to test the hypothesis that the rDNA affects chromosome segregation. To test this hypothesis I focused on mitotic chromosome segregation, and used the model genetic organism, Saccharomyces cerevisiae. S. cerevisiae offers many advantages for testing this hypothesis, including its tolerance to aneuploidy and systems that have been developed to genetically manipulate the rDNA. I developed and optimized a chromosome loss assay (CLA) that measures the rate of chromosome loss during mitosis in S. cerevisiae. I modified a number of strains that had alterations associated with the rDNA, including strains deleted for the chromosomal rDNA repeats, with a reduction in rDNA copies, and with the rDNA translocated to a different chromosome, with specific phenotypic markers for detection of chromosome loss events. I then tested the chromosome loss rates of these strains using the CLA. My results demonstrate that the rDNA affects mitotic chromosome segregation fidelity at two levels. First, the rDNA increases the segregation fidelity of the rDNA-containing chromosome, defining a local chromosome segregation role for the rDNA. I found that this local effect is mediated by the rDNA binding protein Fob1, and I propose three potential mechanisms for how Fob1 mediates this role: (1) through establishment of rDNA recombination-intermediates that may help to stabilize the long rDNA locus; (2) through recruitment of condensin to establish intra-chromatid linkages that promote timely condensation of sister chromatids; or (3) through recruitment of a silencing complex to achieve an appropriate rDNA chromatin state for chromosome segregation. Second, I show that the rDNA has a global effect on chromosome segregation fidelity, with rDNA deletion or reduction in rDNA copies influencing the segregation of many or all chromosomes. Curiously, heterozygosity of rDNA state, regardless of what states are present, confers wild type missegregation rates. I rule out trivial explanations for this global effect, and instead propose that the rDNA affects segregation through changes in nucleolar structure and overall nuclear organization that impact spindle polarity and thus the fidelity of chromosome segregation. Together, these results define a new role for the rDNA in facilitating chromosome segregation, and one that acts at two different levels. This work provides insights into a novel beneficial role of the rDNA in chromosome segregation of S. cerevisiae, and the conserved mechanism of chromosome segregation across eukaryotes suggests the rDNA may play similar roles in more complex organisms. It will be interesting to determine if the rDNA also has beneficial role in meiosis, where the rDNA has been associated with missegregation.Item Genomes in space and time : insights into the functional three-dimensional organization of prokaryotic and eukaryotic genomes in response to environmental stimuli and cell cycle progression : a thesis presented in partial fulfilment of the requirements for the degree of Doctorate in Philosophy in Genetics at Massey University, Albany, New Zealand(Massey University, 2014) Grand, Ralph StefanThe specific three-dimensional organization of prokaryotic and eukaryotic genomes and its contribution to cellular functions is increasingly being recognized as critical. Bacterial chromosomes are highly condensed into a structure called the nucleoid. Despite the high degree of compaction in the nucleoid, the genome remains accessible to essential biological processes such as replication and transcription. Here I present the first high-resolution Chromosome Conformation Capture based molecular analysis of the spatial organization of the Escherichia coli nucleoid during rapid growth in rich medium and following an induced amino-acid starvation that promotes the stringent response. My analyses identified the presence of origin and terminus domains in exponentially growing cells. Moreover, I observe an increased number of interactions within the origin domain and significant clustering of SeqA binding sequences, suggesting a role for SeqA in clustering of newly replicated chromosomes. By contrast, “Histone-like” protein (i.e. Fis, IHF, H-NS) binding sites did not cluster suggesting that their role in global nucleoid organization does not manifest through the mediation of chromosomal contacts. Finally, genes that were down-regulated after induction of the stringent response were spatially clustered indicating that transcription in E. coli occurs at transcription foci. The successful progression of a cell through the cell cycle requires the temporal regulation of gene expression, the number and condensation levels of chromosomes and numerous other processes. Despite this, detailed investigations into how the genome structure changes through the cell cycle and how these changes correlate with functional changes have yet to be performed. Here I present the results of a high resolution study in which we used synchronized Fission yeast (Schizosaccharomyces pombe) cells to investigate changes in genome organization and transcription patterns during the cell cycle. The small size of the Fission yeast genome makes this organism particularly amenable to studies of the spatial organization of its chromosomes. I detected cell cycle dependent changes in connections within and between chromosomes. My results show that chromosomes are effectively circular throughout the cell cycle and that they remain connected even during the M phase, in part by the co-localization of repeat elements. Furthermore, I identified the formation and disruption of chromosomal interactions with specific groups of genes in a cell cycle dependent manner, linking genome organization and cell cycle stage specific transcription patterns. Determining the structure and transcript levels for matched synchronized cells revealed: 1) that telomeres of the same chromosome co-localization throughout the cell cycle, effectively circularizing the chromosomes; 2) that genes with high transcript levels are highly connected with other genomic loci and highly expressed genes at specific stages of the cell cycle; 3) that interactions have positive and negative effects on transcript levels depending on the gene in question; and 4) that metaphase chromosomes assume a ‘polymer melt’ like structure and remain interconnected with each other. I hypothesize that the observed correlations between transcript levels and the formation and disruption of cell cycle specific chromosomal interactions, implicate genome organization in epigenetic inheritance and bookmarking. Over the course of mitochondrial evolution, the majority of genes required for its function have been transferred and integrated into nuclear chromosomes of eukaryotic cells. The ongoing transfer of mitochondrial DNA to the nucleus has been detected, but its functional significance has not been fully elucidated. To determine whether the recently detected interactions between the mitochondrial and nuclear genomes (mt-nDNA interactions) in S. cerevisiae are part of a DNA-based communication system I investigated how the reduction in interaction frequency of two mt-nDNA interactions (COX1-MSY1 and Q0182-RSM7) affected the transcript level of the nuclear genes (MSY1 and RSM7). I found that the reduction in interaction frequency correlated with increases in MSY1 and RSM7 transcript levels. To further investigate whether mt-nDNA interaction could be detected in other organisms and characterize their possible functional roles, I performed Genome Conformation Capture (GCC) on Fission yeast cell cycle synchronized in the G1, G2 and M phases of the cell cycle. I detected mt-nDNA interactions that vary in strength and number between the G1, G2 and M phases of the Fission yeast cell cycle. Mt-nDNA interactions formed during metaphase were associated with nuclear genes required for the regulation of cell growth and energy availability. Furthermore, mt-nDNA interactions formed during the G1 phase involved high efficiency, early firing replicating origins of DNA replication. Collectively, these results implicate the ongoing transfer of regions of the mitochondrial genome to the nucleus in the regulation of nuclear gene transcription and cell cycle progression following exit from metaphase. I propose that these interactions represent an inter-organelle DNA-mediated communication mechanism.
