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O’Keefe and Beggs, Page 1
Summary: Genetic analysis in yeast by plasmid shuffle
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O’Keefe and Beggs, Page 2
Title: Yeast genetics to investigate the function of core pre-mRNA splicing factors
Raymond T. O’Keefe1 and Jean D. Beggs2
1Faculty
2Wellcome
of Life Sciences, The University of Manchester, Manchester, M13 9PT, United Kingdom
Trust Centre for Cell Biology, University of Edinburgh, King’s Buildings, Edinburgh, EH9 3JR, United
Kingdom.
*Address correspondence to: Raymond T. O’Keefe, Faculty of Life Sciences, The University of Manchester,
Michael Smith Building, Oxford Road, Manchester, M13 9PT, Fax 44 (0)161 275 5082, E-mail:
[email protected]
1. Abstract
The yeast Saccharomyces cerevisiae has been used extensively to investigate functions of
RNA and protein components of the spliceosome. As many of the yeast core splicing factors
are evolutionarily conserved with human counterparts, information gained in yeast can be
directly applied to humans. The ease with which budding yeast can be manipulated
genetically has resulted in numerous resources that make genetic analysis in this organism
routine. The function of any gene that codes for a spliceosomal component can be analyzed
in yeast and its genetic interactions investigated using genetic screens. Synthetic
enhancement or suppression occur when a mutation in one gene respectively exacerbates or
alleviates the severity of a mutation in another gene, indicating a functional interaction. In the
case of splicing factors this can lead to new information on the mechanisms of splicing. This
chapter describes methods for functional analysis and synthetic enhancement/lethality
screens in yeast.
Keywords: Saccharomyces cerevisiae, synthetic enhancement, synthetic lethality
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2. Theoretical background
2.1 The use of yeast Saccharomyces cerevisiae as a model system for studying
spliceosomal factors
The spliceosome is composed of over a hundred protein factors and five small nuclear RNAs
(snRNAs) (see chapters 5 and 6 by Lührmann and Rymond). Compositions of both human
and yeast core spliceosomes are now known, and many protein factors and all the snRNAs
are evolutionarily conserved. Yeast have has been used to investigate mutations in core
spliceosome factors known to cause disease in humans [1], and are being used to dissect the
network of genetic interactions between different core spliceosomal components [2]. Overall,
using yeast has complemented work in humans and has provided key information on
spliceosome function.
2.2 Yeast genetics
S. cerevisiae can be maintained in diploid and haploid states can you please enter this in the
glossary and induced from one to the other easily. Yeast/bacterial shuttle plasmids are
available with marker genes such as URA3, TRP1, HIS3 and LEU2, suitable for selection in
auxotrophic yeast strains. The pRS series of plasmids is one of the most versatile [3] Ray, can
you please give the constructs a name and add them to the web companinon
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. Yeast strains are available with these marker genes deleted, including the BY strain used in
the large scale yeast deletion project [4]. Transformation of yeast and homologous
recombination between added DNA and endogenous chromosomes are both efficient
processes making genetic manipulation straight forward. Since sequencing of the yeast
genome many resources have been assembled to investigate yeast gene function including
deletion of most of the known and predicted protein coding genes [5]. These “knockout”
strains are useful for functional and genetic analysis of genes. Deletions in essential genes
are maintained in diploid strains that can be complemented with a plasmid-encoded copy then
O’Keefe and Beggs, Page 4
sporulated to produce a haploid strain. A URA3-containing plasmid is usually the
complementing plasmid as it allows positive and negative selection with plates lacking uracil
or containing 5-fluoro orotic acid (5-FOA) respectively [6]. By using negative selection of a
URA3 plasmid functional analysis of essential genes can be investigated by plasmid shuffle
where a haploid knockout strain complemented with a URA3-containing wild-type plasmid is
transformed with a second plasmid with a different marker and carrying a mutant allele.
Growth on 5-FOA selects for cells that lose the URA3 plasmid and mutant phenotype can be
tested. LYS2 is another counter selectable marker and ADE2/ADE3 act as visual markers
through red/white colony colour. See [7] for more background on yeast genetics, recipes for
yeast medium/plates and other yeast methods.
2.3 Synthetic genetic interactions
Availability of deletions in non-essential genes and conditional mutants in essential genes has
allowed investigation of genetic interactions between the core splicing machinery. Synthetic
enhancement/lethality occurs when one gene mutation exacerbates the severity of another
gene mutation. Alternatively, a second mutation may suppress the phenotype of the first
mutation. Traditionally this type of analysis was used to uncover genetic interactions between
different genes where the pathway or complex was uncharacterized. More recently, with
molecular complexes like the spliceosome where the purified complex has been identified by
mass spectrometry, more directed analyses can be employed to determine genetic interaction
networks between core spliceosomal factors. This analysis can provide important insights into
mechanistic aspects of core factors. There are many examples of synthetic lethality between
genes encoding splicing factors, e.g. this approach identified an extensive genetic interaction
network between SNU114 and spliceosomal snRNAs that suggests how snRNA interactions
may be sensed by Snu114p [2]. In a highly informative example of suppression, requirement
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for Prp28p, an essential DExH/D-box splicing factor, was suppressed by mutations in U1
snRNP protein Yhc1p, providing support for Prp28p in destabilising U1 snRNP at the 5’splice
site [8]. Furthermore, effects of various prp8 mutant alleles in synthetic enhancement or
suppression of mutations in other splicing factors that affect the first or second catalytic step
of splicing led to the proposal that first and second step spliceosome conformations are in
equilibrium [9,10].(see chapter 5, Luhrmann)
High through-put genome-wide synthetic genetic screens can be performed, in which a
mutation of interest is combined with the >4,000 deletions of non-essential yeast genes or
with conditional alleles of essential genes [11]. Such approaches have aided in
characterisation of core spliceosomal proteins of unknown function [12,13], however, these
large scale screens require access to high through-put robotic equipment. We describe here
methods to perform genetic screens between pairs of genes in any laboratory.
3. Protocol
3.1 Plasmid shuffle for functional analysis and production of conditional mutations in
essential yeast genes
The basis of plasmid shuffle is construction of a haploid yeast strain where a gene of interest
is deleted and complemented by a wild-type gene copy on URA3 plasmid pRS416 (Figure 1).
This strain is then transformed with another pRS plasmid (usually with HIS3 or LEU2 marker)
containing a mutant allele of the gene to test functionally (Figure 1). This strain is grown on 5FOA to counter select the URA3 plasmid and growth phenotype of the mutation is assessed
(Figure 1).
3.1.1 Construction of complementing URA3 plasmid and a plasmid for mutagenesis
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Design PCR primers to amplify 500 nucleotides upstream and downstream of open reading
frame of gene. This is usually sufficient to complement a gene knockout. PCR primers should
also contain at their 5’ ends recognition sequences for restriction enzymes compatible with
PCR product cloning into multi-cloning site (MCS) of pRS416 plasmid. PCR amplify yeast
genomic DNA using a high fidelity polymerase following manufacturers instructions. An
aliquot of PCR product is checked by agarose gel electrophoresis and, if of predicted size,
purify with commercially available PCR cleanup kit. Digest PCR product with appropriate
restriction enzymes, purify and ligate into a similarly cut pRS416 plasmid. Candidate colonies
are cultured and plasmid isolated by miniprep then sequenced to identify correct plasmid. The
gene is then transferred to a HIS3 or LEU2 plasmid for mutagenesis.
3.1.2 Transformation of diploid knockout strain
Diploid knockout strains are available from Open Biosystems or EUROSCARF with KanMX4
cassette gene replacement which is selected for by growth on Geneticin (G418). These
strains are transformed with a complementing pRS416 plasmid using an established method
[14].
Protocol 1: Transformation of yeast cells
Use diploid knockout strain freshly streaked on YPD-G418 for single colonies.
1. Inoculate single colony into 5ml YPD broth in 15 ml sterile tube and incubate 16-20hrs
with shaking at 300C.
2. Dilute cells in 25ml YPD broth in 50 ml sterile tube to A600 of 0.5units
3. Incubate with shaking at 300C until A600 = 2 (approximately 4 hrs.)
4. Pellet cells at 2000 g for 5 minutes then decant supernate.
5. Resuspend pelleted cells in 25ml sterile water then pellet cells at 2000 g for 5 minutes
and decant supernate.
6. Resuspend cells in 700µl sterile 0.1M lithium acetate and transfer to a sterile 1.5ml
microcentrifuge tube.
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7. Pellet cells 10,000 g for 10 seconds then aspirate supernate.
8. Resuspend cells in 200µl 0.1M lithium acetate.
9. For each transformation aliquot 50µl of cells to a new sterile 1.5ml microcentrifuge
tube.
10. Pellet cells 10,000 g 15 seconds then aspirate supernate.
11. Add to pelleted cells the following:
240µl 50% polyethylene glycol 3500
36µl 1M lithium acetate
50µl 2mg/ml salmon sperm DNA (boiled for 5 mins, then on ice)
0.2-1.0µg DNA in a volume of 34µl
12. Vortex to resuspend cells.
13. Incubate at 300C for 30 minutes.
14. Incubate at 420C for 30 minutes.
15. Pellet cells 2000 g for 15 seconds.
16. Gently resuspend cells in 1ml sterile water and gently spread 50-200µl aliquots on the
appropriate selective plate (SD-Ura).
17. Grow cells at 300C for 3 days
3.1.4 Yeast sporulation and tetrad dissection
Transform diploid knockout strain with complementing pRS416 URA3 plasmid, sporulate then
identify haploid knocked out for the gene of interest and containing complementing plasmid.
Sporulation
Single colonies from transformation are resuspended in 20µl of sterile water and dribbled
down separately onto a sporulation plate. Plates are left at room temperature for 5 – 10 days
to allow sporulation to occur.
Tetrad dissection
Resuspend sporulated cells in 10µl sterile water. Mix with equal volume of lyticase (2.5mg/ml)
and incubate at room temperature for 5-15 minutes to digest cell wall. Dribble mixture down
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side of YPD plate and identify spore tetrads using a light microscope. Separate four spores of
several tetrads on YPD plate. Incubate plate 30ºC for 3 days. If URA3 plasmid with wild-type
gene complements the gene knockout then three or four viable spores should be recovered.
Transfer spores to different plates to test for markers. Haploid knockout strains with
complementing URA3 plasmid should grow on G418 YPD and SD–Ura. If the original diploid
strain was knocked out for HIS3 and LEU2 then you would expect no growth on SD–His
and/or SD–Leu. The haploid strains identified should be tested for growth on 5-FOA plates. A
knockout strain should not grow on 5-FOA whereas a non-knockout will. The identified
knockout strain with complementing URA3 plasmid can be used for functional analysis of the
essential
gene
or
for
construction
of
a
double
knockout
strain
for
synthetic
enhancement/lethality analysis.
3.1.5 Functional analysis of essential yeast genes by plasmid shuffle
Haploid knockout strain with complementing URA3 plasmid identified above is transformed
(Protocol 1) with a HIS3 pRS plasmid with wild-type gene as positive control, HIS3 pRS
plasmid without wild-type gene as negative control or HIS3 pRS plasmid with mutation in
gene of interest. Single colonies obtained from transformations are streaked on 5-FOA plates
in triplicate and incubated at 16ºC, 30ºC and 37ºC with a positive and negative control on the
same plate. This method can provide viable mutants for the synthetic enhancement/lethal
analysis below.
3.2 Genetic interaction analysis
By combining non-lethal mutations in two essential genes one can determine whether they
interact genetically. This analysis can be carried out by plasmid shuffle with a haploid double
knockout strain where two knockouts are complemented by a single URA3 plasmid containing
both wild-type genes. Transformation of this strain with two non-lethal mutants on separate
plasmids with different markers (HIS3 and LEU2), then growth on 5-FOA, will determine
O’Keefe and Beggs, Page 9
whether there is synthetic enhancement/lethality between the two genes (Figure 2). If one of
the mutations causes a growth phenotype by itself, such as cold- or heat-sensitivity,
suppression of the defect by the second mutation is also possible.
3.2.1 Construction of double gene URA3 plasmid
A URA3 marked plasmid containing two wild-type genes for complementation of the two gene
deletions can be constructed as described above for a URA3 plasmid containing a single
gene (3.1.1) by inserting two genes into the MCS instead of one.
3.2.2 Construction of double knockout strains
The construction of a double knockout strain with a complementing URA3 plasmid with the
two wild-type genes can be carried out by two different methods. The first method involves
mating two haploid knockout strains of opposite mating types where each knockout is
complemented by a single URA3 plasmid. Mating can be simply induced by mixing the two
opposite mating types on a YPD plate. Following mating the resulting diploids are grown on 5FOA to cure this term is not clear to me, to remove the URA3? them of the single URA3
plasmids. The heterozygous double knockout diploid (containing one wild-type and one
deleted allele of each gene) is then transformed (Protocol 1) with another URA3 plasmid with
the two wild-type genes for each knockout. The resulting strain is then sporulated and tetrads
dissected (3.1.4). Haploid spores are then identified that contain the double knockout with a
complementing URA3 plasmid with both wild-type genes. This strain will be the basis for the
synthetic enhancement/lethality experiments. The second method for the construction of a
double knockout strain with a complementing URA3 plasmid would be to start with a
heterozygous or homozygous single knockout diploid strain and delete the second gene by
homologous recombination with a knockout cassette that has a different selectable marker
such as hphNT1 which provides resistance to hygromycin [15]. The double knockout diploid is
then transformed (Protocol 1) with another URA3 plasmid with the two wild-type genes for
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each knockout. The resulting strain is then sporulated and tetrads dissected (3.1.4). Haploid
spores are then identified that contain the double knockout with a complementing URA3
plasmid with both wild-type genes. This strain will be the basis for synthetic
enhancement/lethality experiments.
3.2.3 Genetic screens
Synthetic enhancement/lethal screens use a haploid double knockout strain with
complementing URA3 plasmid encoding both wild-type genes, transformed (Protocol 1) with
plasmids with markers like HIS3 and LEU2 and containing mutations in each gene. One
control transformation should be a negative control of a HIS3 and LEU2 plasmid without
either test gene. Another control transformation should be a positive control of a HIS3 and
LEU2 plasmid with wild-type copies of both genes. Single colonies obtained from these
transformations are streaked out on 5-FOA plates in triplicate and incubated at 16ºC, 30ºC
and 37ºC.
4. Example of an experiment
Figure 3 is a typical plate from a synthetic enhancement/lethality experiment. The plate
includes positive and negative controls as well as an example of a synthetic lethal interaction.
5. Troubleshooting
Problem
No colonies obtained from transformation
No cell growth on 5-FOA plates
Reason + Solution
 Not enough DNA used in
transformation, increase DNA
concentration especially if transforming
two plasmids
 Medium used to plate cells incorrect,
check genotype of cells and be sure to
add all appropriate supplements
 Plating technique too vigorous, gently
spread cells onto plates following
transformation
 Medium used to plate cells incorrect,
O’Keefe and Beggs, Page 11

check genotype of cells and be sure to
add all appropriate supplements
Too few cells streaked onto 5-FOA
Figure Legends
Figure 1 – Plasmid shuffle for functional analysis of essential yeast genes
Haploid knockout strain with complementing wild-type gene on URA3 plasmid is transformed
with a HIS3 plasmid with mutation in gene of interest. Growth on 5-FOA then selects for cells
that have lost the URA3 plasmid and the growth phenotype of the mutation is assessed.
Figure 2 – Genetic interaction analysis by plasmid shuffle
Haploid double knockout strain with complementing double wild-type gene URA3 plasmid is
transformed with HIS3 and LEU2 plasmids with mutations in the genes of interest. Growth on
5-FOA then selects for cells that have lost the URA3 plasmid and the genetic interaction
phenotype of the two mutations combined is assessed.
Figure 3 – Example of the analysis
5-FOA plate showing a typical synthetic genetic analysis of mutations in two essential yeast
genes. The plate displays all the controls that should be included and provides an example of
a synthetic lethal interaction. Plate supplied by Rebecca Hogg.
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Abbreviations
5-FOA (5-fluoro orotic acid)
multi-cloning site (MCS)
small nuclear RNAs (snRNAs)
O’Keefe and Beggs, Page 14
Open Biosystems
O keefe
EUROSCARF
O keefe
Synthetic
one gene mutation exacerbates the
enhancement/lethality severity of another gene mutation.
O”keefe
Synthetic
one gene mutation exacerbates the
enhancement/lethality severity of another gene mutation.
Diploid yeast
Haploid yeast
O”keefe
O”keefe
O”keefe