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The transformation of wine yeasts with glucanase, xylanase and pectinase genes for improved clarification and filterability of wineStrauss, Marlene 03 1900 (has links)
Thesis (MScAgric) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Cellulose is by far the most abundant carbohydrate available from plant biomass.
These biopolymers are therefore an important renewable source of food, fuels and
chemicals. Cellulose is embedded in a matrix of hemicellulose, lignin and pectin and
is composed of repeating glucose units linked by p-1,4-glycosidic bonds. The
individual molecules are held together by hydrogen bonds, forming largely crystalline
fibres. The hemicellulose, which is a low molecular weight heteropolysaccharide,
coats and binds the cellulose microfibrils, preventing the cellulose from becoming too
crystalline. Three predominant types of hemicelluloses are recognised, namely 1,3-
and 1,4-p-D-galactans, 1,4-p-D-mannans and 1,4-p-D-xylans, which are named
according to the sugar type that forms the polymer backbone. Pectic substances
contain rhamnogalacturonan backbones in which 1,4-linked a-D-galacturonan chains
are interrupted at intervals with a-L-rhamnopyranosyl residues carrying neutral side
chains. Two groups of enzymes, cellulases and pectinases, are required for the
microbial utilisation of crystalline cellulose and pectin. Cellulases are
multicomponent complexes that are often composed of endoglucanases,
exoglucanases and cellobiases. Cellobiose is the major end product of concerted
endoglucanase and exoglucanase activity. Cellobiose is then hydrolysed to glucose
by p-glucosidases. The enzymatic breakdown of pectic polymers occurs by the deesterifying
action of the saponifying enzymes, pectinesterase, releasing the methyl
groups of the pectin molecule, and by hydrolase or lyase action of the
depolymerases (pectin lyase, pectate lyase and polygalacturonase), splitting the a-
1.4-glycosidic linkages in the polygalacturonate chain.
The yeast Saccharomyces cerevisiae has been used extensively in the alcoholic
beverage industry for fermentations of wine, beer and other alcoholic beverages for
many years. However, it is unable to produce extracellular depolymerising enzymes
that can efficiently degrade polysaccharides, which are the main cause of
clarification and filtration problems. Enzyme preparations have been used in the
alcoholic beverage industries to degrade haze-forming polysaccharides, thereby
improving the filterability and quality of products such as beer and wine. An
alternative would be to develop S. cerevisiae strains that produce extracellular
polysaccharidases, enabling the yeast to degrade polysaccharides without the
addition of commercial enzyme preparations. These strains can also be very useful
in improving the quality of wine, as well as cutting the costs of the winemaking
process. The objective of this study was to investigate the effects of two transformed
S. cerevisiae strains on different wine grape varieties.
The following genes have been cloned and characterised previously: the
Aspergillus niger endo-p-xylanase gene (xynC), the Butyrivibrio fibrisolvens endo-|3-
1.4-glucanase gene (endl), the Erwinia chrysanthemi pectate lyase gene (pelE) and
the Erwinia carotovora polygalacturonase gene (p e h l). The yeast alcohol dehydrogenase I gene promoter (ADH1p), the alcohol dehydrogenase II gene
terminator (ADH2j), the tryptophan synthase gene terminator (TRP5r) and the yeast
mating-type pheromone a-factor secretion signal sequence (MFcrfs) were used to
compile the following gene constructs: ADH1 p-MFa1 s-end1-TRP5r (designated
END1), A DH1 p-xyn C-A DH2T (designated XYN4), ADH1 p-MFa1 s-peh1 -TRP5t
(designated PEH1) and ADH1 p-MFa1 s-pelE-TRP5r (designated PELE).
Two yeast integrating plasmids were constructed, one containing the END1 and
XYN4 gene cassettes and the other containing the PEH1-PELE cassette. These two
plasmids were then integrated into the URA3 locus of two separate industrial wine
yeast strains of S. cerevisiae. To facilitate selection of the industrial yeast
transformants in the absence of auxotrophic markers, the integrating plasmid
containing the END1 and XYN4 gene cassettes was issued with the dominant
selectable Geneticin G418-resistance {G f) marker. The integrating plasmid
harbouring the PEH1-PELE gene cassette was issued with the dominant selectable
sulphumetronmethyl resistance (SMR1) marker. The introduction of these plasmids
into commercial wine yeast strains directed the synthesis of END1, XYN4, PELE and
PEFI1 transcripts and the production of extracellular biologically active endo-P-1,4-
glucanase, endo-(3-xylanase, pectate lyase and polygalacturonase.
These recombinant yeasts were capable of extracting more colour from grape
skins of certain varieties, as well as leading to more freeflow wine as a result of the
more effective degradation of glucans, xylans and pectins in the skins. They also led
to decreased turbidity in the wine, making it more filterable.
Future work will entail further investigation of the effects of these recombinant
yeasts on different white and red wine grape varieties.
Another objective of this study was to screen non-Saccharomyces wine yeasts for
the production of extracellular hydrolytic enzymes. The reason for this part of the
thesis was to determine the types of extracellular hydrolytic enzymes that are
produced and to determine which genera produce which kinds of extracellular
enzymes. A total of 237 yeast isolates, belonging to the genera Kloeckera, Candida,
Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and
Kluyveromyces, were screened for the production of extracellular pectinases,
proteases, (3-glucanases, lichenases, p-glucosidases, cellulases, xylanases,
amylases and sulphite reductase activity. These yeasts were all isolated from
grapes and clarified grape juice to ensure that they were yeasts found in must during
the initial stages of fermentation. This information can be used to pave the way to
pinpoint the specific effects in wine of these enzymes produced by the so-called wild
yeasts associated with grape must. This information can also be used to transform
Saccharomyces wine yeasts with some of the genes from these non-Saccharomyces
yeasts for the production of extracellular hydrolytic enzymes.
However, future research will have to be done to determine the extent of the
activity of these enzymes in wine fermentations and to obtain better knowledge of the
physiological and metabolical features of non-Saccharomyces yeasts. / AFRIKAANSE OPSOMMING: Sellulose is verreweg die volopste koolhidraat in plantbiomassa. Hierdie biopolimere
is dus ‘n baie belangrike hernubare bron van voedsel, brandstof en chemikaliee.
Sellulose is in 'n matriks van hemisellulose, lignien en pektien gebed en is uit
herhaalde glukose eenhede, wat deur middel van (3-1,4-glukosidiese bindings geheg
is, saamgestel. Die individuele molekules word deur waterstofbindings aan mekaar
geheg, wat aanleiding gee tot die vorming van kristallyne vesels. Die hemisellulose,
wat 'n lae molekulere gewig heteropolisakkaried is, bedek en bind die sellulose
vesels en verhoed daarmee die vorming van vesels wat te kristallyn is. Drie
predominante tipes hemisellulose word herken en sluit 1,3- en 1,4-p-D-galaktane,
1,4-p-D-mannane en 1,4-p-D-xylane in, wat vernoem word volgens die
suikereenhede wat die polimeerruggraat vorm. Pektiene bestaan uit 'n
rhamnogalakturonaanruggraat waarin 1,4-gekoppelde a-D-galakturonaankettings
periodiek met a-L-rhamnopiranosiel residue, bevattende neutrale sykettings,
onderbreek word. Twee groepe ensieme, nl. pektinase en sellulase, word deur
mikrobes vir die benutting van kristallyne pektinase en sellulase vereis. Sellulase is
multikomponent komplekse wat dikwels uit endoglukanase, ekso-glukanase en
sellobiase saamgestel is. Sellobiose is die hoof eindproduk van die saamgestelde
aktiwiteit tussen endoglukanase en ekso-glukanase en word verder gehidroliseer tot
glukose deur |3-glukosidases. Die ensimatiese afbraak van pektien polimere vind
deur die de-esterifiserings aksie van die versepings ensiem, pektienesterase, plaas.
Dit lei tot die vrystelling van die metielgroepe van die pektienmolekuul. Deur die
hidrolase of liase aksie van die depolimerase (pektien liase, pektaatliase en
poligalakturonase), split die a-1,4-glukosidiese verbindings in die
poligalakturonaatketting.
Die gis Saccharomyces cerevisiae word al vir jare ekstensief in die alkoholbedryf
vir die fermentasie van verskeie produkte, veral druiwe, gebruik. S. cerevisiae besit
egter nie die vermoe om ekstrasellulere depolimiserende ensieme wat vir die
effektiewe degradasie van polisakkariede verantwoordelik is, te produseer nie, wat
die hoof oorsaak van die verhelderings- en filtreringsprobleme in onder andere wyn
en bier is. Dit veroorsaak ook dat S. cerevisiae nie oor die vermoe beskik om
waasvormende polisakkariede in wyn te degradeer nie. Tans word ensiempreparate
in die alkoholiese bedryf vir die degradasie van die probleem
polisakkariede gebruik. Sodoende word die filtreerbaarheid en kwaliteit van wyn en
bier verbeter. ‘n Goeie alternatief is die ontwikkeling van S. cerevisiae-rasse wat oor
die vermoe beskik om ekstrasellulere polisakkarase te produseer en dus
polisakkariede self sonder die byvoeging van eksterne kommersiele
ensiempreparate te degradeer. Hierdie rasse sal baie voordelig wees vir die
verbetering van wynkwaliteit, sowel as vir die vermindering van die kostes verbonde
aan die wynmaakproses. Die objektief van hierdie studie is dus om die uitwerking van twee getransformeerde S. cerevisiae rasse, wat ekstrasellulere polisakkarases
produseer, op verskillende wyndruifvarieteite na te vors.
Die volgende gene is reeds voorheen gekloneer en gekarakteriseer: die endo-pxylanase-
geen (xynC) van Aspergillus niger, die endo-p-1,4-glukanase-geen (endl)
van Butyrivibrio fibrisolvens, die pektaatliase-geen (pe/E) van Erwinia chrysanthemi
en die poligalakturonase-geen (p e h l) van Erwinia carotovora. Die
alkoholdehidrogenase-geenpromotor (ADH1P), die alkoholdehidrogenase IIgeentermineerder
(ADH2T), die gistriptofaansintase geen se termineerder (TRP5t)
en die sekresiesein van die gisferomoon a-faktor (MFa1s) is gebruik om die
volgende geenkonstrukte saam te stel: ADH1 p-MFa1 s-end1 -TRP5t (toekend as
END1), ADH1 p-xynC-ADH2T (bekend as XYN4), ADH1 p-MFa1 s-peh1-TRP5T
fbekend as PEH1), and ADH1 p-MFa1 s-pelE-TRP5T (bekend as PELE).
Twee gisintegrerings plasmiede is gekonstrueer, een wat die END1- en XYN4-
geenkassette bevat en die ander wat die PEH1-PELE-kasset besit. Hierdie twee
plasmiede is daarna in twee aparte industriele wyngisrasse van S. cerevisiae by die
URA3 lokus geintegreer. Vir die seleksie van die industriele wyngistransformante in
die afwesigheid van ouksotrofiese merkers, is die dominante selekteerbare Geneticin
G418 weerstandbiedende (G f) merker in die END1- en XYA/4-geenkassetbevattende
plasmied geintegreer. Die dominante selekteerbare sulfumetronmetielweerstandbiedende
(SMR1) merker is in die integreringsplasmied, wat die PEH1-
PELE-geenkasset bevat, geintegreer vir seleksie. Transformasie van hierdie
plasmiede in kommersiele wyngisrasse het tot die direkte sintese van die END1-,
XYN4-, PELE- en PEH1-transkripte aanleiding gegee, sowel as tot die produksie van
die biologies aktiewe ekstrasellulere endo-P-1,4-glukanase, endo-P-xylanase,
pektaatliase en poligalaturonase.
Tydens die wynmaakproses het bogenoemde rekombinante giste aanleiding
gegee tot verhoogde kleurekstraksie uit die druifdoppe van sekere varieteite, asook
tot verhoogde vryvloei wyn. Dit is verkry deur die effektiewe degradasie van die
glukane, xilane en pektiene in die doppe. Die rekombinante giste het ook verlaagde
turbiditeit in die wyn tot gevolg gehad, wat die wyne makliker filtreerbaar maak.
Hierdie werk was net die eerste stap. In die toekoms sal verdere navorsing
gedoen moet word om die presiese effekte van hierdie rekombinante giste op
verskillende rooi en wit druifvarieteite te bepaal.
‘n Ander fokus van hierdie tesis was om nie-Saccharomyces wyngiste vir die
produksie van ekstrasellulere hidrolitiese ensieme te selekteer. Die rede hiervoor is
om te bepaal watter tipes ekstrasellulere hidrolitiese ensieme geproduseer word,
asook watter ensieme deur watter genera geproduseer word, ‘n Totaal van 237 gisisolate
wat tot die generas Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia,
Zygosaccharomyces, Hanseniaspora en Kluyveromyces behoort, is vir die produksie
van ekstrasellulere pektinase, protease, p-glukanase, lichenase, p-glukosidase,
sellulase, xilanase, amilase en sulfiet reduktase-aktiwiteit getoets. Hierdie giste is
almal vanaf druiwe en druiwesap geVsoleer om te verseker dat dit wel giste is wat gedurende die beginfases van fermentasie in die mos teenwoordig is. Hierdie
inligting kan nou verder gebruik word om die spesifieke effekte wat hierdie ensieme,
wat deur die sogenaamde wilde giste geproduseer word, tydens die beginfases van
fermentasies op die mos het, te bepaal. Hierdie inligting kan ook in die toekoms
gebruik word om Saccharomyces-wyngiste met gene van die ri\e-Saccharomycesgiste
te transformeer om ekstrasellulere hidrolitiese ensieme vir die degradasie van
die problematiese polisakkariede in wyn te produseer.
Daar sal egter in die toekoms baie navorsing gedoen moet word om die omvang
van hierdie ensiemaktiwiteite in wynfermentasies te bepaal, asook om meer kennis
te bekom oor die fisiologiese en metaboliese samestelling van nie-Saccfraromyces
wyngiste.
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Comparative 'omic' profiling of industrial wine yeast strainsRossouw, Debra 12 1900 (has links)
Thesis (PhD(Agric) Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / The main goal of this project was to elucidate the underlying genetic factors responsible for the
different fermentation phenotypes and physiological adaptations of industrial wine yeast strains. To
address this problem an ‘omic’ approach was pursued: Five industrial wine yeast strains, namely
VIN13, EC1118, BM45, 285 and DV10, were subjected to transcriptional, proteomic and exometabolomic
profiling during alcoholic fermentation in simulated wine-making conditions. The aim
was to evaluate and integrate the various layers of data in order to obtain a clearer picture of the
genetic regulation and metabolism of wine yeast strains under anaerobic fermentative conditions.
The five strains were also characterized in terms of their adhesion/flocculation phenotypes,
tolerance to various stresses and survival under conditions of nutrient starvation.
Transcriptional profiles for the entire yeast genome were obtained for three crucial stages during
fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late
stationary phase (day 14). Analysis of changes in gene expression profiles during the course of
fermentation provided valuable insights into the genetic changes that occur as the yeast adapt to
changing conditions during fermentation. Comparison of differentially expressed transcripts
between strains also enabled the identification of genetic factors responsible for differences in the
metabolism of these strains, and paved the way for genetic engineering of strains with directed
modifications in key areas. In particular, the integration of exo-metabolite profiles and gene
expression data for the strains enabled the construction of statistical models with a strong predictive
capability which was validated experimentally.
Proteomic analysis enabled correlations to be made between relative transcript abundance and
protein levels for approximately 450 gene and protein pairs per analysis. The alignment of
transcriptome and proteome data was very accurate for interstrain comparisons. For intrastrain
comparisons, there was almost no correlation between trends in protein and transcript levels, except
in certain functional categories such as metabolism. The data also provide interesting insights into
molecular evolutionary mechanisms that underlie the phenotypic diversity of wine yeast strains.
Overall, the systems biology approach to the study of yeast metabolism during alcoholic
fermentation opened up new avenues for hypothesis-driven research and targeted engineering
strategies for the genetic enhancement/ modification of wine yeast for commercial applications.
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Putative promoter sequences for differential expression during wine fermentations / by Renata Martina Polotnianka.Polotnianka, Renata Martina January 1996 (has links)
Includes bibliographies. / 104, [64] leaves, [18] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the isolation of putative promoter sequences that can produce differential expression of a gene during anaerobic wine fermentations, the use of these sequences in the development of expression vectors and the application of this work to the production of genetically engineered wine yeasts for commercial purposes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997?
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Evolution of the Vacuolar H+-ATPase Enzyme ComplexFinnigan, Gregory Charles, 1983- 06 1900 (has links)
xvii, 167 p. : ill. (some col.) / The vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit enzyme complex responsible for acidification of cellular organelles. The V-ATPase hydrolyzes ATP to pump protons across membranes to create an electrochemical gradient. Acidification of vesicular compartments is critical in numerous biological processes including protein trafficking, endocytosis, and ion homeostasis; defects in V-ATPase function can also lead to human diseases. While the function of the V-ATPase enzyme is highly conserved across eukaryotes, the molecular architecture of this protein complex has undergone unique structural changes through evolutionary time. The goal of this work is to investigate the assembly, transport, and evolution of this critical molecular machine in the model organism <italic>Saccharomyces cerevisiae</italic>. A series of genetic screens was performed in budding yeast to identify factors and pathways that are involved in promoting full V-ATPase function. I utilized several "assembly factor" alleles to serve as sensitized genetic backgrounds to partially reduce enzyme function; this work implicated sphingolipid composition in promoting full vacuolar ATPase enzyme function. I also used ancestral gene reconstruction to analyze the two isoforms of subunit a of the V<sub>0</sub> subdomain (Vph1p and Stv1p) by recreating the most recent common ancestral subunit (Anc.a). Characterization of Anc.a demonstrated that this ancient subunit was able to properly assemble and function within a hybrid V-ATPase complex. While the Vph1p-containing complex localized to the vacuole membrane and the Stv1p-containing complex was present on the Golgi/endosome, incorporation of Anc.a caused the V-ATPase to localize to both types of cellular compartments. Finally, I used ancestral reconstruction to investigate the lineage-specific gene duplication of one of the proteolipid subunits of the V<sub>0</sub> subcomplex that occurred within the fungal clade. I demonstrate that inclusion of a third proteolipid subunit within fungi (as compared to two subunits within metazoans) could have occurred via neutral processes by asymmetric degeneration of subunit-subunit interfaces that "ratcheted" the duplicated subunit with the V<sub>0</sub> ring. These results present a model that describes how macromolecular machines can increase in complexity through evolutionary time. This dissertation includes previously published co-authored material and unpublished co-authored material. / Committee in charge: George Sprague, Chairperson;
Tom H. Stevens, Advisor;
Victoria Herman, Member;
Bruce Bowerman, Member;
Ken Prehoda, Outside Member
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Global identification of human modifier genes of alpha-synuclein toxicityHaider, Ishita 01 September 2020 (has links)
No description available.
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Rie1 and Sgn1 form an RNA-binding complex that enforces the meiotic entry cell fate decisionGaspary, Alec January 2023 (has links)
Budding yeast cells have the capacity to adopt distinct physiological states depending on environmental conditions. Vegetative cells proliferate rapidly by budding while spores can survive prolonged periods of nutrient deprivation and/or desiccation. Whether or not a yeast cell will enter meiosis and sporulate represents a critical decision which could be lethal if made in error. Most cell fate decisions, including those of yeast, are understood as being triggered by the activation of master transcription factors. However, mechanisms that enforce cell fates post-transcriptionally have been more difficult to attain. Here, we perform a forward genetic screen to determine RNA-binding proteins that affect meiotic entry at the post-transcriptional level. Our screen revealed several candidates with meiotic entry phenotypes, the most significant being RIE1 which encodes an RRM-containing protein.
We demonstrate that Rie1 binds RNA, is associated with the translational machinery, and acts post-transcriptionally to enhance protein levels of the master transcription factor Ime1 in sporulation conditions. We also identified a physical binding partner of Rie1, Sgn1, which is another RRM (RNA Recognition Motif)-containing protein that plays a role in timely Ime1 expression. We demonstrate that these proteins act independently of cell size regulation pathways to promote meiotic entry. We propose a model explaining how constitutively expressed RNA-binding proteins, such as Rie1 and Sgn1, can act in cell-fate decisions both as switch-like enforcers and as repressors of spurious cell fate activation.
Chapter 1 serves as a brief overview of the importance cell fate decisions and details how sporulation in the budding yeast Saccharomyces cerevisiae can be used as a model to understand the pathways and mechanisms underlying these decisions. This chapter focuses on the importance of the meiotic master regulator IME1 and the different effectors of regulation that govern its expression at the transcriptional and post-transcriptional levels.
Chapter 2 describes the significance of RNA binding proteins and how they can influence cell fate decisions with a focus on cell cycle modifications that shift mitosis to meiosis. Chapter 3 explains the methodology that I used to discover two RNA-binding proteins that play key roles in meiotic entry: Rie1 and Sgn1. Chapter 4 describes my work to dissect the pathways governed by Rie1 and Sgn1. Chapter 5 discusses the potential mechanisms by which Rie1 and Sgn1 could drive entry into meiosis. Collectively, the studies described in this thesis demonstrate that Rie1 and Sgn1 affect the cell fate decision to enter meiosis in budding yeast by activating as translational activators of IME1.
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Homology-Directed Repair of One- and Two-Ended DNA Double-Strand BreaksKimble, Michael Taylor January 2023 (has links)
DNA double-strand breaks (DSBs) are one of the most dangerous lesions cells encounter, given that DSBs can lead to genomic instability and cell death if not repaired properly. Cells have two primary pathways to repair DSBs: Homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR is the high-fidelity branch of the DSB repair pathway since it employs a process of homology search and synthesis from a homologous template. The homology search is carried out by ssDNA that is generated on either side of the DSB by end resection. End resection occurs via a two-step mechanism involving resection initiation, followed by long-range resection. Previous work has revealed that long-range resection is dispensable for some cases of HR; however, it is currently unclear why the requirement for long-range resection is context- dependent. Furthermore, it is not completely clear how the mechanisms of HR, including requirements for long-range resection, apply to single-ended DSBs (seDSBs) arising during replication. Therefore, we defined the role of long-range resection in two-ended DSB repair in different chromosomal contexts. We also established a Cas9 nickase (Cas9n) system to study seDSB repair and defined genetic requirements for repair.
To study the requirement for long-range resection in HR, we employed inter- and intrachromosomal genetic recombination assays in haploid yeast. We found that long-range resection is required for interchromosomal HR, but not for intrachromosomal HR. This difference is linked to the observation that the DNA damage checkpoint, which is deficient in the absence of long-range resection, is activated in interchromosomal HR, but not intrachromosomal HR. The DNA damage checkpoint has also previously been implicated in promoting chromosome mobility. Therefore, we reason that the requirement for long-range resection in interchromosomal repair is due to a need to activate the DNA damage checkpoint and chromosome mobility, specifically during slower repair events.
To study seDSB repair, we implemented Cas9n, which creates nicks that can cause replication fork collapse. We demonstrated that expression of Cas9n with an efficient gRNA can induce replication fork collapse and that repair of these seDSBs breaks is dependent on the HR machinery. A genome-wide screen using Cas9n revealed a requirement for replication-coupled nucleosome assembly (RCNA) in repair of seDSBs, specifically in replication origin-deplete regions of the genome. Consistent with the model of seDSB repair, we found that Cas9n-induced seDSBs preferentially undergo sister chromatid recombination. This preference was altered in the absence of Mre11, which we hypothesize is due to a role of MRX in sister chromatid tethering. Altogether, the results presented in this thesis offer a different perspective on the role of long-range resection in two-ended DSB repair and establish a Cas9n-based system to better study single-ended DSB repair.
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Analysis of endo-polygalacturonase activity in a recombinant yeast containing a reconstituted PGU1 geneVan Wyk, Herine 03 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / The PGU1 gene encodes an endo-polygalacturonase, an enzyme that degrades pectin. Although the presence and function of this gene is well characterized in Saccharomyces cerevisiae, its regulation is very complex and not yet fully understood. Yeast producing a highly active polygalacturonase (PG) during alcoholic fermentation could potentially improve filtration and turbidity and also enhance extraction of certain aroma compounds. This could replace the addition of expensive commercial enzyme preparations that often contain unwanted enzymes.
The first objective of this study was to evaluate PGU1 expression in recombinant strains of S. cerevisiae that originally lacked the PGU1 gene. A functional PGU1 gene and its promoter were successfully re-introduced into their native position in the genomes of five wine strains. Three of these strains recovered PG activity while two did not transcribe the gene and subsequently lacked activity. The three strains that recovered activity were used in microvinification experiments to determine the effect of PG-producing yeast on the aroma profile of the wine. No significant differences were observed in the volatile compounds production between the recombinants and their respective wild types, but some tendencies arose, especially for the monoterpene geraniol.
The second objective of this study was to analyze the PGU1 gene and promoter from Saccharomyces paradoxus RO88 (a strain that exhibits high PG activity) and to compare it to those of S. cerevisiae S288C in order to identify differences that could potentially be responsible for the difference in their PG activities. Comparison of the gene sequences revealed several amino acid differences, one of which was in the peptide secretion signal. Analyses of the promoters also indicated some potentially important differences. Furthermore, S. cerevisiae strain VIN13, RO88 as well as two interspecies hybrids (all displaying varying PG activities) were compared under winemaking conditions. Clear differences were observed for the production of certain compounds. RO88 and the hybrids produced higher concentrations of certain volatile compounds, although they were not strong fermenters. Two recombinants, each containing a PGU1-overexpressing plasmid (one with the PGU1 gene from S. paradoxus and the other from S. cerevisiae), were also used in vinification to determine the effects of the different PGU1 gene on the aroma profile of the wine. Unfortunately, the plasmids were unstable and lost during the fermentation. Nevertheless, some tendencies were observed that indicated possible higher production of certain compounds by the recombinants compared to their wild types.
This study identified that regulation of the PGU1 gene differs between strains with different genetic backgrounds. Certain differences were observed in the PGU1 gene and promoter
sequences between S. cerevisiae and S. paradoxus that could potentially be the reason for the difference in their PG activities. From an oenological point of view, the presence of PGU1 in the genome of a fermenting strain tends to increase the aromatic potential of wine. These results provide a good platform for further studies on the PGU1 gene.
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Extending chemical complemenation to bacteria and furthering nuclear receptor based protein engineering and drug discoveryJohnson, Kenyetta Alicia 18 May 2009 (has links)
Nuclear receptors (NRs) are modular ligand-activated transcription factors that control a broad range of physiological processes by regulating the expression of essential genes involved in cell physiology, differentiation, and metabolism. These receptors are implicated in a number of diseases and due to their profound role in development and disease progression and their modularity, much emphasis is being put forth into nuclear receptor based drug discovery and engineering these receptors to bind novel small molecules
Chemical Complementation (CC) is a yeast three-hybrid genetic selection system that was developed to aid in the discovery of these engineered receptors by linking the survival of a yeast cell to a small molecules ability to activate the receptor. Due to several advantages, to include faster growth times and higher transformation efficiencies, we have attempted to extend chemical complementation from yeast to E. coli. The bacterial chemical complementation system (BCC) was designed, based on a bacterial two hybrid system, to parallel yeast CC system. However, bacterial chemical complementation did not produce ligand dependent activation due to heterologous protein expression.
In a second project designed to further NR based protein engineering and drug discovery, CC was used to evaluate a library of charge reversal variants rationally designed to gain a better understanding of nuclear receptor function and structure and to produce orthogonal ligand receptor pairs. A library of retinoic acid receptor (RARα) variants were developed based on five residues in the binding pocket known to stabilize the natural negatively charged ligand, all-trans retinoic acid (atRA). We altered the binding selectivity of the receptor to bind positively charged retinoid ligands. We were able to engineer two triple variants capable of activating with the positively charged retinoid but not the natural atRA ligand, however they do not activate as well as RARα wild-type does with atRA.
In a third project we characterized covalently linked tamoxifen and histone deacetylase inhibitor based dual inhibiting compounds as breast cancer therapeutics. Several dual inhibiting compounds were found to decrease the proliferation of ER positive breast cancer cells better than tamoxifen alone, the HDACi alone, or noncovalently linked HDACi and tamoxifen.
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Engineering the pregnane X receptor and estrogen receptor alpha to bind novel small molecules using negative chemical complementationShaffer, Hally A. 05 April 2011 (has links)
Nuclear receptors are ligand-activated transcription factors that play significant roles in various biological processes within the body, such as cell development, hormone metabolism, reproduction, and cardiac function. As transcription factors, nuclear receptors are involved in many diseases, such as diabetes, cancer, and arthritis, resulting in approximately 10-15% of the pharmaceutical drugs presently on the market being targeted toward nuclear receptors. Structurally, nuclear receptors consist of a DNA-binding domain (DBD), responsible for binding specific sequences of DNA called response elements, fused to a ligand-binding domain (LBD) through a hinge region. The LBD binds a small molecule ligand. Upon ligand binding, the LBD changes to an active conformation leading to the recruitment of coactivator (CoAC) proteins and initiation of transcription. As a result of their involvement in disease, there is an emphasis on engineering nuclear receptors for applications in gene therapy, drug discovery and metabolic engineering.
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