Biomedical Engineering Reference
In-Depth Information
Schiestl and Petes (1991) developed a method for
forcing illegitimate recombination by transforming
yeast with
Bam
H1-generated fragments in the pres-
ence of the
Bam
H1 enzyme. Not only did this increase
the frequency of transformants but the transformants
which were obtained had the exogenous DNA integ-
rated into genomic
Bam
H1 sites. This technique,
which is sometimes referred to as restriction-enzyme-
mediated integration (REMI), has been extended to
other fungi, such as
Cochliobolus
(Lu
et al
. 1994),
Ustilago
(Bolker
et al
. 1995) and
Aspergillus
(Sanchez
et al
. 1998).
Bcl
I
Aat
II
Mfe
I
Xba
I
Ase
I
Ahd
I
Sna
B
I
Hpa
I
Blp
I
YEp 24
Tth
111
I
Pvu
II
Bsa
B
I
Bsp
E
I
Cla
I
-
Bsp
D
I
Sbf
I
Nco
I
Api
I
/
Bsp
1201
I
Eag
I
Psh
A
I
Sal
I
Eco
N
I
Sph
I
Plasmid vectors for use in fungi
Sma
I
/
Xma
I
Nhe
I
Bam
H
I
If the heterologous DNA introduced into fungi is to
be maintained in an extrachromosomal state then
plasmid vectors are required which are capable of
replicating in the fungal host. Four types of plasmid
vector have been developed: yeast episomal plasmids
(YEps), yeast replicating plasmids (YRps), yeast
centromere plasmids (YCps) and yeast artificial
chromosomes (YACs). All of them have features in
common. First, they all contain unique target sites
for a number of restriction endonucleases. Secondly,
they can all replicate in
E. coli
, often at high copy
number. This is important, because for many exper-
iments it is necessary to amplify the vector DNA in
E. coli
before transformation of the ultimate yeast
recipient. Finally, they all employ markers that
can be selected readily in yeast and which will often
complement the corresponding mutations in
E. coli
as well. The four most widely used markers are His3,
Leu2, Trp1 and Ura3. Mutations in the cognate chro-
mosomal markers are recessive, and non-reverting
mutants are available. Two yeast selectable mark-
ers, Ura3 and Lys2, have the advantage of offering
both positive and negative selection. Positive selec-
tion is for complementation of auxotrophy. Negative
selection is for ability to grow on medium containing
a compound that inhibits the growth of cells ex-
pressing the wild-type function. In the case of Ura3,
it is 5-fluoro-orotic acid (Boeke
et al
. 1984) and
for Lys2 it is
Sgr
A
I
Fig. 9.2
Schematic representation of a typical yeast episomal
plasmid (YEp 24). The plasmid can replicate both in
E. coli
(due to the presence of the pBR322 origin of replication) and
in
S. cerevisiae
(due to the presence of the yeast 2 µm origin of
replication). The ampicillin and tetracycline determinants
are derived from pBR322 and the URA3 gene from yeast.
large and contains sites within the coding sequence
for many of the commonly used restriction sites.
Yeast episomal plasmids
YEps were first constructed by Beggs (1978) by
recombining an
E. coli
cloning vector with the nat-
urally occurring yeast 2
m plasmid. This plasmid is
6.3 kb in size, has a copy number of 50 -100 per
haploid cell and has no known function. A repres-
entative YEp is shown in Fig. 9.2.
µ
Yeast replicating plasmids
YRps were initially constructed by Struhl
et al
. (1979).
They isolated chromosomal fragments of DNA which
carry sequences that enable
E. coli
vectors to replic-
ate in yeast cells. Such sequences are known as
ars
(autonomously replicating sequence). An
ars
is
quite different from a centromere: the former acts
as an origin of replication (Palzkill & Newlon 1988,
Huang & Kowalski 1993), whereas the latter is
involved in chromosome segregation.
-aminoadipate (Chatoo
et al
. 1979).
These inhibitors permit the ready selection of those
rare cells which have undergone a recombination or
loss event to remove the plasmid DNA sequences.
The Lys2 gene is not utilized frequently because it is
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