Ethanol improves the transformation efficiency of intact yeast cells

1991 ◽  
Vol 20 (1-2) ◽  
pp. 1-3 ◽  
Author(s):  
Vit Lauermann
Keyword(s):  
Transformation Efficiency ◽  
Yeast Cells

scholarly journals Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

2021 ◽  
Vol 7 (7) ◽  
pp. 520
Author(s):  
Jianmin Fu ◽  
Nohelli E. Brockman ◽  
Brian L. Wickes
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Yeast Cell ◽  
Transformation Efficiency ◽  
Transformation Frequency ◽  
Yeast Cells ◽  
Agar Concentration ◽  
Genetic Tool ◽  
Number Of Factors ◽  
Cryptococcus Spp ◽  
Transformation Systems

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

Cell de-energization prevents plasmid transformation of yeast Saccharomyces cerevisiae: evidence for the requirement of ATP

2010 ◽  
Vol 5 (1) ◽  
pp. 78-82 ◽  
Author(s):  
Larisa Chaustova ◽  
Valė Miliukienė ◽  
Aurelijus Zimkus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transformation Efficiency ◽  
Energy Requirement ◽  
Yeast Cells ◽  
Sodium Arsenate ◽  
Yeast Saccharomyces Cerevisiae ◽  
Plasmid Transformation ◽  
Substrate Level Phosphorylation ◽  
Intracellular Atp ◽  
Substrate Level

AbstractThe dependence of the yeast Saccharomyces cerevisiae transformation on energy requirement was studied. The inhibitory effect of sodium arsenate, used for the depletion of the intracellular ATP pool, was determined. Incubation of the yeast cells in 5 mM sodium arsenate diminished ATP accumulation by 50% and the transformation efficiency decreased by 65%. To discriminate between ATP produced by substrate level phosphorylation and oxidative phosphorylation, the inhibitory analysis of a mutant with defective mitochondria was performed. Sodium fluoride (10–50 mM), as inhibitor of glycolysis, elicited a concentration-dependent decrease in intracellular ATP levels in both parental and mutant cells. The equal transformation efficiency of the mitochondrial mutant and parental strain, in addition to experiments with oligomycin, demonstrated the independence of plasmid transformation on mitochondrial ATP synthesis. This is consistent with our hypothesis that yeast transformation efficiency is associated with ATP produced by substrate level phosphorylation.

scholarly journals Yeast Actin-Related Protein ARP6 Negatively RegulatesAgrobacterium-Mediated Transformation of Yeast Cell

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Yumei Luo ◽  
Zikai Chen ◽  
Detu Zhu ◽  
Haitao Tu ◽  
Shen Quan Pan
Keyword(s):  
Genetic Engineering ◽  
Large Scale ◽  
Transformation Efficiency ◽  
Genetic Diseases ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Scale Production ◽  
Related Protein ◽  
Cellular Mechanisms ◽  
Eukaryotic Organisms

The yeasts, includingSaccharomyces cerevisiaeandPichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values.Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related proteinARP6as a negative regulator of AMT.ARP6is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting thatARP6might regulate AMT via SWR-C. Moreover, knockout ofARP6led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

Metabolic state and cell cycle as determinants of facilitated uptake of genetic information by yeast Saccharomyces cerevisiae

2008 ◽  
Vol 3 (4) ◽  
pp. 417-421 ◽  
Author(s):  
Larisa Chaustova ◽  
Valė Miliukienė ◽  
Aurelijus Zimkus ◽  
Valdemaras Razumas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Exponential Growth ◽  
Transformation Efficiency ◽  
S Phase ◽  
Yeast Cells ◽  
Yeast Culture ◽  
Metabolic State ◽  
Yeast Saccharomyces Cerevisiae ◽  
Exogenous Dna

AbstractThe transformation efficiency of yeast cells during exponential growth might be characterised as undulatory. The aim of the study was to investigate the reason for the fluctuation in transformation efficiency of yeast Saccharomyces cerevisiae p63-DC5 cells during exponential growth. The heightened response to exogenous DNA was observed with the growing yeast culture when budded cells were predominant. To confirm this phenomenon we carried out synchronization of yeast cells with 10 mM hydroxyurea. Results showed that synchronous yeast cells in the S-phase of cell cycle have enhanced transformation efficiency. Furthermore, S. cerevisiae p63-DC5 cells in the S-phase were successfully transformed with plasmid pl13 in the absence of lithium acetate. We indicated that the permeability of yeast cells in the S-phase to tetraphenylphosphonium (TPP) cations was significantly higher than in asynchronous culture. The results of our study showed that the fluctuation in transformation efficiency was strictly dependent on the metabolic state of yeast cells and the capacity of the yeast cells to become competent was related to the S-phase of cell cycle.

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

1977 ◽  
Vol 35 ◽  
pp. 596-597
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Synthetic Medium ◽  
Freeze Fracture ◽  
Translational Diffusion ◽  
Yeast Cells ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
The Matrix ◽  
Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

Ultrathin frozen sections of yeast without aldehyde prefixation

1978 ◽  
Vol 36 (2) ◽  
pp. 58-59
Author(s):  
K. J. Böhm ◽  
a. E. Unger
Keyword(s):  
Aqueous Solutions ◽  
Biological Material ◽  
Yeast Cells ◽  
Frozen Sections ◽  
Good Preservation ◽  
Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

Deformation of Yeast Plasma Membrane by Ethidium Bromide

1988 ◽  
Vol 46 ◽  
pp. 254-255
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Thin Section ◽  
Ethidium Bromide ◽  
Freeze Fracture ◽  
Mutagenic Effect ◽  
Yeast Cells ◽  
Hydrophobic Region ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

Three-Dimensional Immunoelectron Microscopy of Yeast Cells by a New High-Resolution Replica Method

1985 ◽  
Vol 43 ◽  
pp. 562-563
Author(s):  
Hirano T. ◽  
M. Yamaguchi ◽  
M. Hayashi ◽  
Y. Sekiguchi ◽  
A. Tanaka
Keyword(s):  
High Resolution ◽  
Plasma Polymerization ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Replica Method ◽  
Yeast Cells ◽  
Dynamic Aspect ◽  
Replica Technique ◽  
Gaseous Hydrocarbon ◽  
Transmission Electron

A plasma polymerization film replica method is a new high resolution replica technique devised by Tanaka et al. in 1978. It has been developed for investigation of the three dimensional ultrastructure in biological or nonbiological specimens with the transmission electron microscope. This method is based on direct observation of the single-stage replica film, which was obtained by directly coating on the specimen surface. A plasma polymerization film was deposited by gaseous hydrocarbon monomer in a glow discharge.The present study further developed the freeze fracture method by means of a plasma polymerization film produces a three dimensional replica of chemically untreated cells and provides a clear evidence of fine structure of the yeast plasma membrane, especially the dynamic aspect of the structure of invagination (Figure 1).

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