Biomedical Engineering Reference
In-Depth Information
CHAPTER 9
Cloning in
Saccharomyces
cerevisiae
and other fungi
Introduction
Introducing DNA into fungi
The analysis of eukaryotic DNA sequences has been
facilitated by the ease with which DNA from eukary-
otes can be cloned in prokaryotes, using the vectors
described in previous chapters. Such cloned sequences
can be obtained easily in large amounts and can be
altered
in vivo
by bacterial genetic techniques and
in
vitro
by specific enzyme modifications. To determine
the effects of these experimentally induced changes
on the function and expression of eukaryotic genes,
the rearranged sequences must be taken out of the
bacteria in which they were cloned and reintroduced
into a eukaryotic organism. Despite the overall unity
of biochemistry, there are many functions common
to eukaryotic cells which are absent from prokary-
otes, e.g. localization of ATP-generating systems to
mitochondria, association of DNA with histones,
mitosis and meiosis, and obligate differentiation of
cells. The genetic control of such functions must be
assessed in a eukaryotic environment.
Ideally these eukaryotic genes should be reintro-
duced into the organism from which they were
obtained. In this chapter we shall discuss the poten-
tial for cloning these genes in
Saccharomyces cere-
visiae
and other fungi and in later chapters we shall
consider methods for cloning in animal and plant
cells. It should be borne in mind that yeast cells are
much easier to grow and manipulate than plant and
animal cells. Fortunately, the cellular biochemistry
and regulation of yeast are very like those of higher
eukaryotes. For example, signal transduction and
transcription regulation by mammalian steroid
receptors can be mimicked in strains of
S. cerevisiae
expressing receptor sequences (Metzger
et al
. 1988,
Schena & Yamamoto 1988). There are many yeast
homologues of human genes, e.g. those involved in
cell division. Thus yeast can be a very good surrog-
ate host for studying the structure and function of
eukaryotic gene products.
Like
Escherichia coli
, fungi are not naturally trans-
formable and artificial means have to be used for
introducing foreign DNA. One method involves the
use of spheroplasts (i.e. wall-less cells) and was first
developed for
S. cerevisiae
(Hinnen
et al
. 1978). In
this method, the cell wall is removed enzymically and
the resulting spheroplasts are fused with ethylene
glycol in the presence of DNA and CaCl
2
. The
spheroplasts are then allowed to generate new cell
walls in a stabilizing medium containing 3% agar.
This latter step makes subsequent retrieval of cells
inconvenient. Electroporation provides a simpler and
more convenient alternative to the use of sphero-
plasts. Cells transformed by electroporation can be
selected on the surface of solid media, thus facilitat-
ing subsequent manipulation. Both the spheroplast
technique and electroporation have been applied to
a wide range of yeasts and filamentous fungi.
DNA can also be introduced into yeasts and
filamentous fungi by conjugation. Heinemann and
Sprague (1989) and Sikorski
et al
. (1990) found that
enterobacterial plasmids, such as R751 (IncP
) and
F (IncF), could facilitate plasmid transfer from
E. coli
to
S. cerevisiae
and
Schizosaccharomyces pombe.
The
bacterial plant pathogen
Agrobacterium tumefaciens
contains a large plasmid, the Ti plasmid, and part
of this plasmid (the transfered DNA (T-DNA)) can be
conjugally transferred to protoplasts of
S. cerevisiae
(Bundock
et al
. 1995) and a range of filamentous
fungi (De Groot
et al
. 1998). T-DNA can also be
transferred to hyphae and conidia.
β
The fate of DNA introduced into fungi
In the original experiments on transformation of
S.
cerevisiae,
Hinnen
et al
. (1978) transformed a leucine
auxotroph with the plasmid pYeLeu 10. This plasmid
is a hybrid composed of the enterobacterial plasmid
