scholarly journals Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae

2006 ◽  
Vol 72 (8) ◽  
pp. 5266-5273 ◽  
Author(s):  
Elke Nevoigt ◽  
Jessica Kohnke ◽  
Curt R. Fischer ◽  
Hal Alper ◽  
Ulf Stahl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Fine Tuning ◽  
Limited Information ◽  
Promoter Replacement ◽  
Pathway Gene ◽  
Glycerol Yield ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
The Impact ◽  
Mutant Promoter

ABSTRACT The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.

scholarly journals Effects of Low-Shear Modeled Microgravity on Cell Function, Gene Expression, and Phenotype in Saccharomyces cerevisiae

2006 ◽  
Vol 72 (7) ◽  
pp. 4569-4575 ◽  
Author(s):  
B. Purevdorj-Gage ◽  
K. B. Sheehan ◽  
L. E. Hyman
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Cell Function ◽  
Yeast Cells ◽  
Lag Phase ◽  
Limited Information ◽  
Cellular Functions ◽  
Modeled Microgravity ◽  
Expression Of Genes ◽  
Low Shear

ABSTRACT Only limited information is available concerning the effects of low-shear modeled microgravity (LSMMG) on cell function and morphology. We examined the behavior of Saccharomyces cerevisiae grown in a high-aspect-ratio vessel, which simulates the low-shear and microgravity conditions encountered in spaceflight. With the exception of a shortened lag phase (90 min less than controls; P < 0.05), yeast cells grown under LSMMG conditions did not differ in growth rate, size, shape, or viability from the controls but did differ in the establishment of polarity as exhibited by aberrant (random) budding compared to the usual bipolar pattern of controls. The aberrant budding was accompanied by an increased tendency of cells to clump, as indicated by aggregates containing five or more cells. We also found significant changes (greater than or equal to twofold) in the expression of genes associated with the establishment of polarity (BUD5), bipolar budding (RAX1, RAX2, and BUD25), and cell separation (DSE1, DSE2, and EGT2). Thus, low-shear environments may significantly alter yeast gene expression and phenotype as well as evolutionary conserved cellular functions such as polarization. The results provide a paradigm for understanding polarity-dependent cell responses to microgravity ranging from pathogenesis in fungi to the immune response in mammals.

The impact of exogenous abscisic acid on carotenoid composition and carotenoid pathway gene expression in carrot callus in vitro

2016 ◽  
Vol 33 ◽  
pp. S158
Author(s):  
Tomasz Oleszkiewicz ◽  
Magdalena Klimek-Chodacka ◽  
Anna Kostyn ◽  
Aleksandra Boba ◽  
Jan Szopa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abscisic Acid ◽  
Pathway Gene ◽  
Carotenoid Composition ◽  
The Impact ◽  
Carotenoid Pathway

scholarly journals Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Liping Xu ◽  
Pingping Liu ◽  
Zhubo Dai ◽  
Feiyu Fan ◽  
Xueli Zhang
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Fine Tuning ◽  
Minimal Promoter ◽  
Pathway Engineering ◽  
Gfp Expression ◽  
Chimeric Promoter ◽  
Promoter Library ◽  
Pathway Gene ◽  
Chimeric Promoters

Abstract Background Tailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering. Results In this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L). Conclusions All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

scholarly journals Histone H3Q5 serotonylation stabilizes H3K4 methylation and potentiates its readout

2021 ◽  
Vol 118 (6) ◽  
pp. e2016742118
Author(s):  
Shuai Zhao ◽  
Kelly N. Chuh ◽  
Baichao Zhang ◽  
Barbara E. Dul ◽  
Robert E. Thompson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Posttranslational Modification ◽  
Histone H3 ◽  
Fine Tuning ◽  
H3k4 Methylation ◽  
Serotonergic Neurons ◽  
Phd Finger ◽  
Downstream Effectors ◽  
Binding Preference ◽  
The Impact

Serotonylation of glutamine 5 on histone H3 (H3Q5ser) was recently identified as a permissive posttranslational modification that coexists with adjacent lysine 4 trimethylation (H3K4me3). While the resulting dual modification, H3K4me3Q5ser, is enriched at regions of active gene expression in serotonergic neurons, the molecular outcome underlying H3K4me3–H3Q5ser crosstalk remains largely unexplored. Herein, we examine the impact of H3Q5ser on the readers, writers, and erasers of H3K4me3. All tested H3K4me3 readers retain binding to the H3K4me3Q5ser dual modification. Of note, the PHD finger of TAF3 favors H3K4me3Q5ser, and this binding preference is dependent on the Q5ser modification regardless of H3K4 methylation states. While the activity of the H3K4 methyltransferase, MLL1, is unaffected by H3Q5ser, the corresponding H3K4me3/2 erasers, KDM5B/C and LSD1, are profoundly inhibited by the presence of the mark. Collectively, this work suggests that adjacent H3Q5ser potentiates H3K4me3 function by either stabilizing H3K4me3 from dynamic turnover or enhancing its physical readout by downstream effectors, thereby potentially providing a mechanism for fine-tuning critical gene expression programs.

scholarly journals Fitness effects of altering gene expression noise in Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Fabien Duveau ◽  
Andrea Hodgins-Davis ◽  
Brian P.H. Metzger ◽  
Bing Yang ◽  
Stephen Tryban ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Expression Level ◽  
Gene Expression Noise ◽  
Expression Levels ◽  
Fitness Effects ◽  
Expression Noise ◽  
Average Expression Level ◽  
The Difference ◽  
The Impact

AbstractGene expression noise is an evolvable property of biological systems that describes differences in gene expression among genetically identical cells in the same environment. Prior work has shown that expression noise is heritable and can be shaped by natural selection, but the impact of variation in expression noise on organismal fitness has proven difficult to measure. Here, we quantify the fitness effects of altering expression noise for the TDH3 gene in Saccharomyces cerevisiae. We show that increases in expression noise can be deleterious or beneficial depending on the difference between the average expression level of a genotype and the expression level maximizing fitness. We also show that a simple model relating single-cell expression levels to population growth produces patterns that are consistent with our empirical data. We use this model to explore a broad range of average expression levels and expression noise, providing additional insight into the fitness effects of variation in expression noise.

scholarly journals Investigation of the transcriptional profile of human kidneys during machine perfusion reveals potential benefits of haemoadsorption

2019 ◽  
Author(s):  
John R. Ferdinand ◽  
Sarah A. Hosgood ◽  
Tom Moore ◽  
Christopher J. Ward ◽  
Tomas Castro-Dopico ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cold Storage ◽  
Inflammatory Mediators ◽  
Organ Shortage ◽  
Machine Perfusion ◽  
Inflammatory Genes ◽  
Pathway Gene ◽  
Potential Benefits ◽  
End Stage ◽  
The Impact

AbstractTransplantation is the optimal treatment for most patients with end stage kidney disease but organ shortage is a major challenge. Normothermic machine perfusion (NMP) has been used to re-condition marginal organs but the mechanisms by which NMP might benefit transplant kidneys are not fully understood. Furthermore, the question of whether removal of pro-inflammatory mediators from the perfusate might offer additional benefits in optimising kidneys prior to transplantation has not been addressed. Using pairs of human kidneys obtained from the same donor, we compared the effect of NMP with that of cold storage on the global transcriptome of kidneys, and then went on to investigate the impact of adding a haemoadsorption device to the NMP circuit. We found that cold storage significantly reduced the expression of inflammatory genes, but also of genes required for energy generation such as those encoding oxidative phosphorylation (OXPHOS) enzymes. In contrast, during NMP, there was marked upregulation OXPHOS genes, as well as a number of immune and inflammatory pathway genes. The induction of inflammatory genes during NMP was substantially attenuated by the addition of a haemoadsorber to the perfusion circuit, which also further increased OXPHOS pathway gene expression. Together, our data suggest that absorption of pro-inflammatory mediators from the perfusate represents a useful intervention that may further improve organ viability and should be tested in clinical practice.Single sentence summary: The use of a haemoadsorber during machine perfusion reduces inflammatory gene expression, with potential benefits for kidney transplantation.

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