organization was affected mechanically. Its molecular organization
is controlled by S–S bonds. A pretreatment of the wall by dithiothreitol (DTT) enhanced the cell sensitivity to PEFs. Pretreatment
of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT)
further improved the frequency of transformation by electroporation [11]. Enhanced autolysis was another consequence of PEF on
the yeast wall organization. PEF had a catalytic effect on the events
that occurred during yeast autolysis such as the leakage to the
extracellular buffer of mannoproteins present in the cell wall
[16]. This effect was associated to the electrical damage of the
cytoplasmic membrane when exposed to a strong electric field
pulse lasting several microseconds. Such pulses rendered it permeable to intracellular molecules, such as hydrolytic enzymes, that
would otherwise be unable to cross it. Their action on the cell
wall resulted on the release of wall-associated macromolecules
such as mannoproteins.
The direct consequence of PEF on the yeast envelope was a
transient increase in its permeability [17]. As a consequence it was
shown that small fluorescent dyes could be introduced inside the
cells and that this uptake is very heterogeneous in the pulsed yeast
population and strongly dependent upon the field intensity
[18]. These field-induced conductive wall defects were putative
pathways for the pDNA transfer across the wall. Gene electrotransfer (GET) in intact yeast cells resulted from a two-step process
[19]. Plasmid must be present together with the cells during the
pulse delivery. As the pulsing buffer was not free from divalent
cations, pDNA was bound to the cells by electrostatic forces as
soon as it was added. During the direct field effect (a few milliseconds), DNA was electrophoretically pushed in the electrically
induced wall defects explaining why a long pulse was therefore
more effective. pDNA then crossed the envelope to reach the
cytoplasm in a slow transfer (more than 20 s) where it remained
sensitive to a postpulse addition of DNase. This second step was
neither due to an electrophoretic contribution, nor to free diffusion. It resulted from a still unknown active process due to cell
metabolism [19]. In a systematic investigation of the transfer kinetics when Ca
2+ ions were present, the presence of a DNA molecule
in its whole length in the cell interior appeared clearly as an afterfield event [20]. A cytoplasmic traffic could be responsible for the
final DNA transfer to nucleus.
A step by step protocol for GET on Saccharomyces cerevisiae will
be described. It is applicable to Yarrowia lipolytica, Pichia pastoris,
and Schwanniomyces occidentalis. Transformation frequency is
defined as the ratio of transformed cells (forming CFU) over the
total amount of cells in the sample. Transformation efficiency is the
number of transformants per mg of plasmid DNA. Both parameters
are affected by the cell viability after GET [21].
Saccharomyces
189
is controlled by S–S bonds. A pretreatment of the wall by dithiothreitol (DTT) enhanced the cell sensitivity to PEFs. Pretreatment
of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT)
further improved the frequency of transformation by electroporation [11]. Enhanced autolysis was another consequence of PEF on
the yeast wall organization. PEF had a catalytic effect on the events
that occurred during yeast autolysis such as the leakage to the
extracellular buffer of mannoproteins present in the cell wall
[16]. This effect was associated to the electrical damage of the
cytoplasmic membrane when exposed to a strong electric field
pulse lasting several microseconds. Such pulses rendered it permeable to intracellular molecules, such as hydrolytic enzymes, that
would otherwise be unable to cross it. Their action on the cell
wall resulted on the release of wall-associated macromolecules
such as mannoproteins.
The direct consequence of PEF on the yeast envelope was a
transient increase in its permeability [17]. As a consequence it was
shown that small fluorescent dyes could be introduced inside the
cells and that this uptake is very heterogeneous in the pulsed yeast
population and strongly dependent upon the field intensity
[18]. These field-induced conductive wall defects were putative
pathways for the pDNA transfer across the wall. Gene electrotransfer (GET) in intact yeast cells resulted from a two-step process
[19]. Plasmid must be present together with the cells during the
pulse delivery. As the pulsing buffer was not free from divalent
cations, pDNA was bound to the cells by electrostatic forces as
soon as it was added. During the direct field effect (a few milliseconds), DNA was electrophoretically pushed in the electrically
induced wall defects explaining why a long pulse was therefore
more effective. pDNA then crossed the envelope to reach the
cytoplasm in a slow transfer (more than 20 s) where it remained
sensitive to a postpulse addition of DNase. This second step was
neither due to an electrophoretic contribution, nor to free diffusion. It resulted from a still unknown active process due to cell
metabolism [19]. In a systematic investigation of the transfer kinetics when Ca
2+ ions were present, the presence of a DNA molecule
in its whole length in the cell interior appeared clearly as an afterfield event [20]. A cytoplasmic traffic could be responsible for the
final DNA transfer to nucleus.
A step by step protocol for GET on Saccharomyces cerevisiae will
be described. It is applicable to Yarrowia lipolytica, Pichia pastoris,
and Schwanniomyces occidentalis. Transformation frequency is
defined as the ratio of transformed cells (forming CFU) over the
total amount of cells in the sample. Transformation efficiency is the
number of transformants per mg of plasmid DNA. Both parameters
are affected by the cell viability after GET [21].
Saccharomyces
189