Epigenetics is an area of biology that studies heritable changes in gene ex- pression without any change in the DNA sequence. The most studied epige- netic mechanisms are DNA methylation, RNA interference and histone mod- ifications. There are over 130 different modifications that can be attached to histones, and the most commonly studied are methylation, acetylation, phos- phorylation, sumoylation and ubiquitination. The modifications, spread out through the genome, form the histone code, which recruits transcription fac- tors and modifies the accessibility of the DNA, which results in either active or silenced transcription. The silenced form of chromatin is known as heter- ochromatin and is usually found in regions of the chromosome that need to be highly regulated. To study epigenetics, the model organism Schizosac- charomyces pombe is used widely used. S. pombe is a single cell, rod shaped, fission yeast. The simplicity of S. pombe and its similarities to high- er eukaryotes makes it a good model organism for studying epigenetics. We find that, when mutating evolutionary conserved amino acids in the Clr2 protein, which is involved in heterochromatin formation in S. pombe, there is a change in silencing in different heterochromatic regions. When constructs of Clr2 with the BAH domain deleted are overexpressed, there is an increase in silencing in the central core centromere of chromosome II of S. pombe.

Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild type Arabidopsis plants regarding their sensitivity to low temperature, and the size and height of the plants.

MSCAGR (Animal Science) / Department of Animal Science / The overall objective of this study was to investigate the effects of level of dietary concentrate and live yeast (LY) on milk yield, milk composition, rumen fermentation and nitrogen metabolism in lactating dairy cows. Four primiparous Holstein dairy cows in early lactation (average weight 500 ±9 kg and 20 days in milk (DIM)) were used in a 4 x 4 Latin Square design for a period of 44 days. The animals had seven days of adaptation to the treatments and four days for measurements. The treatments were, Low concentrate to forage (C:F) diet (40:60) with no additive, High C:F diet (60:40) with no additive, High C:F diet with LY and Low C:F diet with LY.Cows weremilked at 06h00 and 16h00 daily and milk samples were analysed for fat, protein, lactose milk urea nitrogen (MUN) and somatic cell count (SCC). Proximate analysis of feed samples was done and daily feed intake was recorded. Weight and body condition score were determined at beginning and end of every experimental period. Faecal and urinary nitrogen (N)were determined. In vitro batch fermentation was conducted to determine ruminal fermentation kinetics. Data generated from the feeding trial was analysed for a 4 x 4 Latin square design (LSD) using the PROC MIXED procedure of SAS (2009) and data for the invitro trialwassubjected to ANOVA using PROC GLM (SAS Institute, 2009) for a complete randomized design. Addition of LY affected only dry matter intake (DMI) (P<0.05), which effect was pronounce when cows were fed low (40:60) C:F diet resulting in better feed efficiency(FE). Cows fed high C:F diet consumed more feed, produced more milk with high fat and protein content (P<0.05). Both LY and C:F reduced (P<0.05) N intake as result of low DMI, but reduced (P<0.05) N excretion in manure. Addition of LY decreased ruminal ammonia and increased total VFA’s (P<0.05). The effects on ammonia suggest a better utilisation of diet proteins and probably more incorporation of products of CP degradation into microbial proteins, which support the observed reduced manure N excretion. The opposite was observed with high C:F diet, which increased ammonia and decreased total VFA’s. Propionate and butyrate were increased and decreased, respectively by high C:F diet.Addition of LY reduced SCC and MUN compared to control.The effects of LY were better pronounced on most parameters at low C:F diet. It is therefore recommended that the effects of LY be tested at low C:F on a larger scale of animals over longer periods to observe its effect of the rest of the parameters.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / Production of bioethanol from lignocellulosic biomass has gained significant attention worldwide as an alternative fuel source for the transportation sector without affecting food supply. Efficient conversion of pentose sugars (L-arabinose and D-xylose) produced during hydrolysis of hemicellulose to ethanol can enhance the economic viability. In this study, a total of 390 yeasts isolated from Marula wine, the gut of dung beetles, herbal concoctions and banana residues were screened for the ability to ferment L-arabinose and D-xylose. Fourteen yeasts were able to ferment both pentose sugars and ten strains were subjected to an adaptation process in the presence of acetic acid using L-arabinose as carbon source. Four adapted strains of Meyerozyma caribbica were able to ferment L-arabinose to ethanol and arabitol in the presence of 3 g/L acetic acid at 35 °C. Meyerozyma caribbica Mu 2.2f fermented D-xylose, L-arabinose and a mixture of D-xylose and L-arabinose to produce 1.7, 3.0 and 1.9 g/L ethanol, respectively, compared to the parental strain with 1.5, 1.0 and 1.8 g/L ethanol, respectively, in the absence of acetic acid. The adapted strain of M. caribbica Mu 2.2f produced 3.6 and 0.8 g/L ethanol from L-arabinose and D-xylose, respectively in the presence of acetic acid while the parental strain failed to grow. In the bioreactor, the adapted strain of M. caribbica Mu 2.2f produced 5.7 g/L ethanol in the presence of 3 g/L acetic acid with an ethanol yield and productivity of 0.338 g/g and 0.158 g/L/h, respectively at a KLa value of 3.3 h-1. The adapted strain produced 26.7 g/L arabitol with a yield of 0.900 g/g at a KLa value of 4.9 h-1. Meyerozyma caribbica Mu 2.2f could potentially be used to produce ethanol and arabitol under stressed conditions. / National Research Foundation (NRF)

Yeast probiotics and paraprobiotics, live and inactivated yeast cells, respectively, improve health and performance of livestock by stabilizing the intestinal microbial community. They have also been used for infection prevention and treatment. Despite much research already conducted, the mechanism of direct antagonism, or adhesion of bacteria to the probiotic/paraprobiotic, is under characterized. Additionally, it is unknown which probiotic/paraprobiotic is optimal to use for specific infections. The interactions between the yeast and certain pathogens were analyzed qualitatively with scanning electron microscopy (SEM) and quantitatively with membrane filtration assays. Gram-positive bacteria were found to exhibit specificity under SEM. Through membrane filtration, Listeria monocytogenes exhibited binding to all samples (P<0.05), while Salmonella Typhimurium exhibited binding (P<0.001) with all samples except with 2338. Escherichia coli O157:H7 only bound to the probiotics (P<0.001). With a better understanding of how specific yeast probiotics and paraprobiotics interact with bacteria, specific therapies can be administered to combat infections.

No description available.

As the name suggests, synthetic biology designs new biology using human power, knowledge, and creativity. Biology is vast, complicated, and all-inclusive, and so is synthetic biology. I believe synthetic biology is the Utopia of biologists, chemists, physicists, material scientists, engineers,and computer scientists. It is a newly emerged and vastly growing field that can impact and improve our lives in many aspects. I dare to say that anything you see that is done by biology can, in the future, be done better by synthetic biology since, on top of having biology as a teacher and as a template, synthetic biology has the benefit of creative and rational design provided by the human brain. In a way, it is the next step in evolution. In this thesis, we worked on some yeast synthetic biology applications. We used engineered yeasts to create bandages to enhance and accelerate the healing of diabetic wounds, make biosensors for pathogenic bacteria and a small molecule metabolite (glucose) important in diabetic patients, and design a community of cells that could contain artificial intelligence. Chapter 1 gives a short introduction and background information regarding diabetes, wound healing, and advanced healing therapies. Chapter 2 is focused on engineering yeasts to secrete wound-healing proteins and in vitro and cell-based studies on the engineered yeasts and secreted recombinant proteins. Chapter 3 presents two wound dressings that contain engineered live yeasts as active ingredients. This chapter includes further in vitro and cell-based studies to assess the functionality of the designed dressings. Chapter 4 focuses on in vivo experiments to study the wound-healing properties of the designed live yeast dressings. Finally, Chapter 5 presents two other projects: one on live yeast biosensors and one on designing modular smart yeast communities that can do computation based on neural network algorithms.

No description available.

The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield. / Ph. D. / Trees are unique among plants since they have extremely long life spans and the ability to generate large quantities of woody biomass. The woody biomass derived from forest trees can function to provide renewable energy in the form of biofuels. The process of wood formation is complex and requires coordinated activation of genes involved in multiple metabolic pathways. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA. These protein-DNA interactions regulate gene expression during plant growth and development. In this study, new regulators of genes known to be involved in wood formation were identified using the Yeast One-Hybrid (Y1H) assay. One of the proteins identified, PtrRAD1 had not been previously linked to wood formation and was a candidate for further characterization. Further testing of PtrRAD1 by the Yeast Two-Hybrid (Y2H) assay resulted in identification of a protein-protein interaction with Populus trichocarpa DIVARICATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay to identify numerous interacting proteins, in addition to those reported previously in other species. Further characterization of PtrDRIF1, identified an N-terminal region capable of forming interactions with MYB/SANT domain proteins, and C-terminal region that interacted with homeodomain proteins. PtrRAD1, PtrDRIF1, and the homeodomain containing PtrWOX13c were able to form a complex in an Y2H-bridge assay. Combined, these findings support a potential role for PtrDRIF1 in regulating wood polarity, wood formation, and stem cell proliferation.

Department of Food Science and Technology / MSCPNT