Download et al.

HAPTER 9
Cloning in Saccharomyces
cerevisiae and other fungi
Outline
●Introduction, 156
● Introducing DNA into fungi, 156
● The fate of DNA introduced into fungi, 156
● Plasmid vectors for use in fungi, 158
● Retrovirus-like vectors, 159
● Expression of cloned genes, 163
● Overexpression of proteins in fungi, 165
● Specialist vectors, 166
● Yeast surface display, 168
● Identifying genes encoding particular cellular
activities, 171
● Determining functions associated with particular
genes, 171
1. Introduction

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.

Ideally these eukaryotic genes should be reintroduced into the organism from which they were
obtained. In this chapter we shall discuss the
potential for cloning these genes in Saccharomyces
cerevisiae and other fungi and in later chapters we
shall consider methods for cloning in animal and
plant cells.
2. Introducing DNA into fungi

Like Escherichia coli, fungi are not naturally transformable 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 CaCl2.

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 spheroplasts.
Cells transformed by electroporation can be selected on
the surface of solid media, thus facilitating subsequent
manipulation. Both the spheroplast technique and
electroporation have been applied to a wide range of
yeasts and filamentous fungi.
3. 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
ColEl and a segment of yeast DNA containing the
LEIJ2* gene and is unable to replicate in yeast. Analysis
of the transformants showed that in some of them
there had been reciprocal recombination between the
incoming LEU2+ and the recipient Leu2~ alleles.

The results described above can be confirmed
by restriction-endonuclease analysis, since
pYeLeu 10 contains no cleavage sites for Hindlll.
When DNA from the Leu2~ parent was
digested with endonucle-ase Hindlll and
electrophoresed in agarose, multiple DNA
fragments were observed but only one of these
hybridized with DNA from pYeLeu 10.

During transformation, the integration of
exogenous DNA can occur by recombination
with a homologous or an unrelated sequence. In
most cases, non-homologous integration is more
common than homologous recombination
(Fincham 1989), but this is not so in S. cerevisiae
(Schiestl & Petes 1991).

Schiestl and Petes (1991) developed a method for
forcing illegitimate recombination by transforming
yeast with BamHl -generated fragments in the pre-ence
of the BamHl enzyme. Not only did this increase the
frequency of transformants but the transformants
which were obtained had the exogenous DNA integrated into genomic BamHl sites. This technique, which
is sometimes referred to as restriction-enzymemediated integration (REMI), has been extended to
other fungi, such as Cochliobolus , UstUago and
Aspergillus .
4. Plasmid vectors for use in fungi

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).

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 large and
contains sites within the coding sequence for
many of the commonly used restriction sites.
5. Yeast episomal plasmids

YEps were first constructed by Beggs (1978) by
recombining an E. coli cloning vector with the
naturally occurring yeast 2 |xm 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 representative YEp is shown in Fig.
9.2.
6. Yeast replicating plasmids

YRps were initially constructed by Struhl et al.
(19 79). They isolated chromosomal fragments
of DNA which carry sequences that enable B.
coli vectors to replicate 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.
7. Yeast centromere plasmids

Using a YRp vector, Clarke and Carbon (1980) isolated
a number of hybrid plasmids containing DNA
segments from around the centromere-linked leu2, cdclO
and pgk loci on chromosome III of yeast. As expected
for plasmids carrying an ars, most of the recombinants
were unstable in yeast. However, one of them was
maintained stably through mitosis and meiosis. The
stability segment was confined to a 1.6 kb region lying
between the leu2 and cdc10 loci and its presence on
plasmids carrying either of two ars tested resulted in
those plasmids behaving like minichromosomes .
8. Yeast artificial chromosomes

All three autonomous plasmid vectors described above
are maintained in yeast as circular DNA molecules even the YCp vectors, which possess yeast centromeres.
Thus, none of these vectors resembles the normal yeast
chromosomes which have a linear structure. The ends
of all yeast chromosomes, like those of all other linear
eukaryotic chromosomes, have unique structures that
are called telomeres. Telomere structure has evolved as a
device to preserve the integrity of the ends of DNA
molecules, which often cannot be finished by the
conventional mechanisms of DNA replication .

Thus, there is no practical limitation to the size
of a YAC and they are essential tools in any
genome-sequencing project. The method for
cloning large DNA sequences in YACs
developed by Burke et al. (1987) is shown in Fig.
9.3.
9. Retrovirus-like vectors

The genome of S. cerevisiae contains 30-40
copies of a 5.9 kb mobile genetic element called
Ty (for review see Fulton et al. 1987). This
transposable element shares many structural and
functional features with retroviruses (see p. 193)
and the copia element of Drosophila. Ty consists
of a central region containing two long open
reading frames (ORFs) flanked by two identical
terminal 334 bp repeats called delta (Fig. 9.4).

The Ty element has been modified in vitro by
replacing its delta promoter sequence with promoters derived from the phosphoglycerate
kinase or galactose-utilization genes (Garfinkel et
al. 1985, Mellor et al. 1985). When such
constraints are introduced into yeast on highcopy-number vectors, the Ty element is
overexpressed. This results in the formation of
large numbers of virus-like particles (VLPs),
which accumulate in the cytoplasm (Fig. 9.5).

The Ty element can also be subjugated as a
vector for transposing genes to new sites in the
genome. The gene to be transposed is placed
between the 3' end of ORF 2 and the 3' delta
sequence (Fig. 9.6).
10. Choice of vector for cloning

There are three reasons for cloning genes in yeast. The
first of these relates to the potential use of yeast as a
cloning host for the overproduction of proteins of
commercial value. Yeast offers a number of advantages,
such as the ability to glycosylate proteins during
secretion and the absence of pyrogenic toxins.
Commercial production demands overproduction and
the lactors aliecting expression ol genes in yeast are
discussed in a later section (see p. 165). Yeast is also
used in the production of food and beverages.

For many biologists the primary purpose of
cloning is to understand what particular genes
do in vivo. Thus most of the applications of
yeast vectors have been in the surrogate genetics
of yeast. One advantage of cloned genes is that
they can be analysed easily, particularly with the
advent of DNA-sequencing methods.

The availability of different kinds of vectors
with different properties (see Table 9.1) enables
yeast geneticists to perform manipulations in
yeast like those long available to E. coli
geneticists with their sex factors and transducing
phages. Thus cloned genes can be used in
conventional genetic analysis by means of
recombination using YIp vectors or linearized
YRp vectors
11. Plasmid construction by
homologous recombination in yeast

During the process of analysing a particular
cloned gene it is often necessary to change the
plasmid's selective marker. Alternatively, it may
be desired to move the cloned gene to a
different plasmid, e.g. from a YCp to a YEp.
Again, genetic analysis may require many
different alleles of a cloned gene to be
introduced to a particular plasmid for
subsequent functional studies.

Suppose we wish to move the HIS 3 gene from
pBR328, which cannot replicate in yeast, to
YEp420 (see Fig. 9.7). Plasmid pRB328 is cut
with PvuI and PvuII and the HIS3 fragment
selected. The HIS3 fragment is mixed with
YEp420 which has been linearized with EcoRI
and the mixture transformed into yeast.
12. Expression of cloned genes

When gene manipulation in fungi first became
possible, there were many unsuccessful attempts
to express heterologous genes from bacteria or
higher eukaryotes. This suggested that fungal
promoters have a unique structure, a feature first
shown for S. cerevisiae (Guarente 1987). Four
structural elements can be recognized in the
average yeast promote (Fig. 9.8).


First, several consensus sequences are found at the
transcription-initiation site. Two of these
sequences, TC(G/A)A and PuPuPyPuPu, account
for more than half of the known yeast initiation
sites . These sequences are not found at
transcription-initiation sites in higher eukaryotes,
which implies a mechanistic difference in their
transcription machinery compared with yeast.
The second motif in the yeast promoter is the
TATA box (Dobson et al. 1982). This is an AT-rich
region with the canonical sequence
TATAT/AAT/A, located 60-120 nucleotides before
the initiation site. Functionally, it can be
considered equivalent to the Pribnow box of E.
coli promoters .

The third and fourth structural elements
are upstream activating sequences (UASs)
and upstream repressing sequences (URSs).
These are found in genes whose
transcription is regulated.
13. Qverexpressiort of proteins in
fungi

The first overexpression systems developed were
for S. cerevisiae and used promoters from genes
encoding abundant glycolytic enzymes, e.g.
alcohol dehydrogenase (ADH1), PGK or
glyceraldehyde-3-phosphate dehydrogenase
(GAP).

These are strong promoters and mRNA
transcribed from them can accumulate up to 5%
of total. They were at first thought to be
constitutive but later were shown to be induced
by glucose (Tuite et al. 1982). Now there is a
large variety of native and engineered promoters
available (Table 9.2), differing in strength,
regulation and induction ratio. These have been
reviewed in detail by Romanos et al. (1992).

The ideal promoter is one that is tightly regulated so
that the growth phase can be separated from the
induction phase. This minimizes the selection of nonexpressing cells and can permit the expression of
proteins normally toxic to the cell. The ideal promoter
will also have a high induction ratio. One promoter
which has these characteristics and which is now the
most widely used is that from the GAL1 gene.
Galactose regulation in yeast is now extremely well
studied and has become a model system for eukaryotic
transcriptional regulation (see Box 9.1).

Following addition of galactose GAL1 mRNA is
rapidly induced over 1000-fold and can reach
1% of total mRNA. However, the promoter is
strongly repressed by glucose and so in glucosegrown cul-tures this induction only occurs
following depletion of glucose. To facilitate
galactose induction in the presence of glucose,
mutants have been isolated which are insensitive
to glucose repression (Matsumoto et al. 1983,
Horland et al. 1989).

In recent years, methylotrophic yeasts, such as
Pichia pastoris, have proved extremely popular as
hosts for the overexpression of heterologous
proteins. There are a number of reasons for this.
First, the alcohol oxidase (AOX1) promoter is
one of the strongest and most regulatable
promoters known.

Second, it is possible to stably integrate
expression plasmids at specific sites in the
genome in either single or multiple copies. Third,
the strains can be cultivated to very high density.
To date, over 300 foreign proteins have been
produced in P. pastoris (Cereghino & Cregg
1999,2000). Promoters for use in other yeasts
are shown in Table 9.2.
14. Specialist vectors

Many different specialist yeast vectors have been
developed which incorporate the useful features
found in the corresponding E. coli vectors (see p.
70), e.g. an fl origin to permit sequencing of
inserts production of the cloned gene product
as a purification fusion, etc. Some representative
examples are shown in Fig. 9.9. Two aspects of
these vectors warrant further discussion:
secretion and surface display.

Fig. 9.9 Two specialized vectors for use in Saccharomyces
(YES vectors) and Pichia (pPICZ). V5. Express, 6XHis and
c-myc encode epitopes which can be readily detected and
purified by affinity chromatography. The YES vectors offer
a choice of 2 ųm origin for high copy or CEN6/ARSH4
origin for low copy in yeast, a choice of UR A 3 or TRP1
genes for auxotrophic selection on minimal medium,
blasticidin resistance for dominant selection in any strain
and an f 1 origin to facilitate DNA sequencing. In the
pPICZ vectors, the zeocin-resistance gene is driven by the
EM-7 promoter for selection in E. coli and the TEF1
promoter for selection in Pichia. More details of these
features can be found in Chapter 5.

In yeast, proteins destined for the cell surface or
for export from the cell are synthesized on and
translocated into the endoplasmic reticulum.
From there they are transported to the Golgi
body for processing and packaging into
secretory vesicles.

Fusion of the secretory vesicles with the plasma
membrane then occurs constitutively or in response to
an external signal (reviewed by Rothman & Orci 1992).
Of the proteins naturally synthesized and secreted by
yeast, only a few end up in the growth medium, e.g. the
mating pheromone a factor and the killer toxin. The
remainder, such as invertase and acid phosphatase,
cross the plasma membrane but remain within the
periplasmic space or become associated with the cell
wall.

Polypeptides destined for secretion have a
hydrophobic amino-terminal extension, which is
responsible for translocation to the endoplasmic
reticulum (Blobel & Dobberstein 1975). The
extension is usually composed of about 20
amino acids and is cleaved from the mature
protein within the endoplasmic reticulum.
15. Yeast surface display

S. cerevisiae can be used to elucidate and dissect
the function of a protein in a manner similar to
phage-display systems. Either can be used to
detect protein ligand interactions and to select
mutant proteins with altered binding capacity
(Shusta et al. 1999). However, phage-display
systems often cannot display secreted eukaryotic
proteins in their native functional conformation,
whereas yeast surface display can.

In practice, the gene fusion is somewhat more
complicated. Usually the cloned gene product is
sandwiched between two simple epitopes to
permit quantitation of the number of fusion
proteins per cell and to determine the
accessibility of different domains of the fusion
protein (Fig. 9.10).
16. Detecting protein-protein
interactions

Chien et al. (1991) have made use of the
properties of the GAL4 protein to develop a
method for detecting protein-protein
interactions. The GAL4 protein has separate
domains for the binding to UAS DNA and for
transcriptional activation. Plasmids were constructed which encode two hybrid proteins.

The second consisted of residues 768-881 of
the GAM protein, representing the activation
domain, fused to protein sequences encoded by
a library of yeast genomic DNA fragments.
Interaction between the test protein (bait) and a
protein encoded by one of the library plasmids
(prey) led to transcriptional activation of a
reporter gene (Fig. 9.11).

A major operational problem with the twohybrid system has been the high frequency of
false positives, i.e. cells in which the reporter
gene is active even though the bait and prey do
not interact in nature and/or in yeast. There are
two general solutions to this problem. In the
first of these (James elal. 1996), a host strain is
used that contains three reporter genes under
the control of different GAL4-inducible
promoters.
17. Identifying genes encoding
particular cellular activities

As a result of a major genome-sequencing
project, the sequences of genes encoding every
biochemical activity of S. cerevisiae are known.
The next task is to connect specific biochemical
activities with particular genes. Martzen et al.
(1999) have developed a rapid and sensitive
method for doing this which is applicable to
almost any activity.

To correlate genes with activity, the different
trans-formants were dispensed into 64 96-well
microtitre plates (64 x 96 = 6144). Initially, all
the clones from each plate were pooled and the
64 pools analysed for a specific biochemical
activity. Analysis is facilitated since the GSTORF fusions are readily purified from other
cellular activities by affinity chromatography
using immobilized glutathione.

Once a plate was identified as having the desired
activity, pools of clones were made from each
of the eight rows and 12 columns of wells and
these 20 pools reassayed. If only one clone
expresses the desired activity, then one row and
one column should be identified (Fig. 9.13) and
the point of intersection identifies the wanted
clone. Sequencing of the ORF then allows
identification of the gene corresponding to the
activity.
18. Determining functions associated
with particular genes

Determining the function of a particular gene
product usually involves determining the
expression profile of the gene, the subcellular
location of the protein, and the phenotype of a
null strain lacking the protein. Conditional alleles
of the gene are often created as an additional
tool. These procedures usually require multiple
independent manipulations.

To use this transposon system, yeast genes are
cloned in an E. coli strain that overexpresses the
Tn3 transposase. The transposon, carried on a
derivative of the sex factor F, is introduced by
mating and transposition ensues. Yeast DNA
containing the transposon is excised from the E.
coll vector and transformed back into yeast, with
selection made for URA3 activity.

An alternative approach to the large-scale mutational analysis of the yeast genome is to
individually delete each gene using a polymerase
chain reaction (PCR)-based methodology
(Winzeler et al. 1999). For each gene, a deletion
cassette is constructed (Fig. 9.15) that contains a
kanamycin resistance gene, two 'molecular barcodes' and yeast sequences homologous to the
upstream and downstream flanking sequences.

Fig. 9.15 'Knockout' strain collection, (a) The bar-coded
deletion cassette is generated in two rounds of PCR. It
contains a kanamycin-resistance gene for selection in yeast
and two molecular bar-codes 20 bp long that are unique to
each deletion. The molecular bar-codes are flanked by
common sequence elements that allow for the PCR
amplification of all the bar-codes in a population of yeast
strains. Atthe end of the cassette are45 bp sequences that are
homologous to sequences flanking the gene of interest, (b)
The deletion cassette is transformed into diploid yeast strains.
The 45 bp of yeast sequences allows one copy of the gene of
interest to be replaced with the deletion cassette via
homologous recombination. The resulting heterozygous
strains (c) can then be sporulated and dissected to generate a
collection of haploid mutant strains, if viable (d).
Homozygous mutant strains (e) are made by the selective
matings of haploid segregants.

The molecular bar-codes are particularly important
since they are unique to each deletion strain within a
pool of many different strains (Shoemaker et al. 1996).
The bar-code can be amplified by PCR, using common
primer sequences that flank the bar-code. A pool of
yeast mutant strains can thus be analysed in a single
experiment by amplifying the bar-codes and probing a
DNA microarray (p. 116) containing sequences
complementary to the bar-codes. Variations on this
technique have been used in other organisms to identify
key functions associated with particular activities.
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