Molecular biology tools for the

genetic manipulation of yeast

Plasmids, transformation and

working the numbers
 Screen vs. Selection

Screen: ALL colonies are looked at, we chose the

ones with the right phenotype (e.g. expressing
galactosidase in a screen designed to identify
mutants in a certain regulatory pathway we pick
the blue colonies)

Selection: Only the colonies we are interested in

will survive (e.g. selecting for transformants)
 Suppression vs. complementation
Definition of complementation: The production
of a wild-type phenotype by re-introduction of
the wild type gene into the mutant (either by
plasmid transformation or mating with a strain
that carries the wild type copy of the gene).
Complementation also by introduction of a functional
homologue
A suppressor is generally defined as a mutation
that completely or partially restores the mutant
phenotype of another mutation
Multicopy suppression: overexpression of one gene
can completely or partially restore the mutant
phenotype of another mutation
 Yeast markers
Marker definition:
A) allele of a gene that allows identification of the
strain itself
e.g. Mutations (marker mutation) in biosynthetic
pathways (in a strain: mutations in ade2,ura3,
leu2, trp1.)
Example:
Yeast strain W1536 5B:
MATa; ade2; ade3; leu2-3; his3-11; his3-12;
trp1-1; ura3-1 (Rothstein W303 derivative)
 Yeast markers
B) Gene on a plasmid/piece of DNA that
allows for the identification of a plasmid in
the cell
a gene that confers a certain ability to the
strain : ADE2, URA3, LEU2, TRP1,
KanMX = geneticin resistance
 Plasmids/Vectors
What is a Plasmid?

What is a Vector?
 Genomic DNA Library
Collection of plasmids/vectors carrying pieces of genomic DNA from
your organism of choice
Genomic library:
Cut into little pieces
with restriction
enzymes
Isolate DNA

Nucleus
with DNA Ligate all different
pieces into vector

Collection of plasmids representing the entire genome (or rather a large fraction
thereof)

cDNA library: containing DNA fragments reverse transcribed from mRNA

Number of fragments of genomic DNA required to
be cloned into vector to cover entire genome
ln (1-P)
N=
ln (1-F)

N: number of library members required

P: Probability that gene is in library

Average size of Fragments

F:
Size of genome

Example: yeast about 104 kb; choose P=99%, 15 kb fragment size

ln (1-0.99)
~ 3070 fragments/library members required
15
Ln (1- )
104 complexity of the library
Requirements for plasmids and
vectors
Plasmid:

Vector:
1. Finding a nutritional marker
(e.g. LEU2 involved in leucine
biosynthesis)
 Generation of genetic tools:
How to find the ingredients to make a
yeast vector
 Selectable marker: Cloning of a yeast gene involved in Leucine
biosynthesis
Transform E. coli leuB- mutants with library containing yeast DNA
gene W
LEU2

geneX
Cells are Leu-
geneY
(cannot grow on
media lacking
E. coli
Leucine)
cell, with
leuB
mutation

High frequency
transformation!
Select for bacterial transformants on
minimal media (lacking Leucine)
Cloning by functional
complementation
Isolate plasmid
LEU2
 Transform Plasmid into Leu- strain

LEU2

leu2-

Low frequency
Select for yeast transformants on
transformation!
synthetic media lacking Leucine
Recombination event
required!

SC-Leu

INEFFICIENT! LEU2

Integrating leu2-
leu2- LEU2
Vectors)
bla
ori
 Double Single

LEU2
Recombination event
LEU2

leu2- leu2-

LEU2 Leu2-

bla
ori

+
leu2-

Reversible!
LEU2
(somewhat
Irreversible! unstable)
(very Popout
stable) event
leu2-

LEU2

Transformation possible but very low frequency/ inefficient

 2. Finding an origin of replication
(ARS = automomously replicating
sequence)
 Cloning of ARS fragment
LEU2

leu2-

(make yeast genomic DNA

library)
Transform yeast
Low efficiency

Pick transformants
(keep a stock)

SC-Leu
Grow non-selectively (e.g. on
YPD) for several generations
 Plate cells from non-selective culture on Non-selective plate (e.g.
YPD)

Replica plate on selective culture (SCD Leu)

SC-Leu SC-Leu SC-Leu SC-Leu

Leu+ - marker lost at high frequency -> plasmid-
borne (not integrated in genome)! Isolate DNA
Re-transform

LEU2

leu2-

Transform yeast

High frequency of transformation!

But: Unstable (Lost at high
SC-Leu frequency
Need centromeric function on
plasmid
3. Isolation of a CEN sequence
 Make new yeast library with marker gene and ARS + genomic DNA
insert
ori LEU2

leu2-

Yeast genomic DNA Transform

High frequency of transformation

(collect large number of colonies)
SC-Leu
 Grow non-selectively (e.g. YPD) will enrich for
stable transformants ( which have CEN fragment)
other cells will lose plasmid if without centromeric
sequence
Transformant
with stable
plasmid: 20%
of cells

After 3 doublings

2 2 2 2 8
8 8 8 8 8
Transformant
with stable
plasmid: 50%
Enrichment for cells containing stable plasmid of cells viable
isolate after large number of generations on SC - Leu
growing non-selectively! (Plate cells on SC- Leu)
Isolate plasmids, retransform to confirm
stability, sequence
 Yeast Plasmids
CEN based vectors: containing yeast centromeric fragment
and yeast ARS, relatively stable (lost~5-10% every
generation)

centromere is important for

Proper segregation
Control of replication (only once during cell cycle)

2 m minicircle based (naturally occuring plasmid in yeast

cir+ cells) multicopy (10-50 copies per cell), relatively
stable (lost at about 5-10% per generation)
Integrating Vectors (low trafo frequency, but very stable
when integrated
 EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI, HindIII

Mp13 multiple
cloning site

Also:
Yeast centromeric
YCp50
plasmids, low
(URA3)
copy, high stability
Yeast episomal (YEp) plasmids (2m origin of replication multicopy intermediate
stability)

Yeast integrating (YIp) plasmids

 YACs (Yeast Artificial Chromosomes)

TEL: The telomere which is located

at each chromosome end, protects
the linear DNA from degradation by
nucleases.
CEN: The centromere which is the
attachment site for mitotic spindle
fibers, "pulls" one copy of each
duplicated chromosome into each
new daughter cell.
ORI: Replication origin sequences
which are specific DNA sequences
that allow the DNA replication
machinery to assemble on the DNA
and move at the replication forks.
It also contains few other specific
sequences like:
A and B: selectable markers that
allow the easy isolation of yeast cells
that have taken up the artificial
chromosome.
Recognition site for the two
restriction enzymes EcoRI and
BamHI.
- While DNA cloning into a plasmid allows the insertion of DNA
fragment of about 10,000 nucleotide base pairs, DNA cloning into a
YAC allows the insertion of DNA fragments up to 1,000,000 nucleotide
base pairs.

- Why is it so important to be able to clone such large sequences?

To map the entire human genome (3x1,000,000,000 nucleotide base
pairs) it would require more than 1,000,000 plasmid clones.
In principle, the human genome could be represented in about 10,000
YAC clones.

- YACs can be isolated in their full size by pulsed field gel

electrophoresis (PFGE)

- Techniques for cloning genomic DNA into yeast artificial

chromosomes (YAC) make it possible to analyze very long DNA
sequences like human genes
 Yeast transformation:
Usually done in PEG/DTT/LiAcetate, heat
shock (45oC; 30-40 minutes)
Simple one-step procedure (102 104
transformants per mg of DNA) (45 minutes)
Or procedure involving growth steps and
transformation at a specific growth stage
(104-106 transformants per mg of DNA)
(half a day)
Generating gene knockouts in
yeast
YIp plasmids were used for gene knockouts/replacements in yeast (2-step
gene replacement)

FOA (5-fluoro-orotic acid) kills cells that carry a functonal URA3 -

gene selection for plasmid popout
 Double Single

Irreversible Yfg1-1
Recombination event

YFG1

YFG1

yfg1-1
YFG1 URA3 Yfg1-1

+
Reversible!

YFG1

YFG1

yfg1-1

Transformation possible but very low frequency/ inefficient

 One-step gene replacement:
-Uses a marker gene with flanking sequences homologous to the gene of interest
-Can be generated by PCR:

YFG 5 KANMX
YFG 3
YFG 5 YFG 3

YFG

Integration via
homologous
recombination
PCR reaction
yfg::KANMX
YFG 5 KANMX
YFG 3

Select for geneticin resistance

 Isolate yeast DNA from transformants - Verify integration by PCR

yfg::KANMX YFG

1.3 kb
1. Product 1.7kb

2.
Product (1.2 kb) No product

3.
No product Product (0.8 kb)

About 2-3 weeks to create and confirm a yeast knockout! How much in Mouse?
 Plasmid shuffle:

Done when
working with
essential
gene:
Phenotype of
mutant
versions of the
gene can be
investigated in
a deletion
background
Introduction into the Diploid and tetrad
dissection/selection of the haploid
transformant

RFT1

RFT1

URA3
rft1::KANMX

rft1::KANMX

ura3-

ura3-

sporulate
 4 spores of a tetrad

ura3-

rft1::KANMX

3
URA

ura3-
ura3-
rft1::KANMX
RFT1

3
URA
Dissect!

ura3-

RFT1
 Dissect on YPGalactose!!!!

3
URA
3
URA
rft1::KANMX

rft1::KANMX

RFT1
ura3-

RFT1
ura3-

ura3-
ura3-

or either
DEAD!

RFT1
URA3

RFT1
rft1::KANMX

URA3
rft1::KANMX ura3-
ura3-
ura3-
ura3-
 Select cells that carry the knockout allele plus plasmid
A. Select on SC ura
(Galactose)

RFT1

RFT1

URA3

URA3
rft1::KANMX

ura3-
rft1::KANMX

ura3-
ura3-

ura3-

RFT1

RFT1

Can grow
Can grow ura3-

ura3-

DEAD!
B. Select for growth on YPGalactose - geneticin

RFT1

URA3
rft1::KANMX RFT1

URA3
rft1::KANMX
ura3-
ura3-

ura3-
ura3-

DEAD!
Can grow
C. Test Growth on SC-Galactose FOA (5-fluoro-orotic acid)

RFT1

URA3
rft1::KANMX RFT1

URA3
rft1::KANMX
ura3-
ura3-

ura3-
ura3-

DEAD!
Can grow
(cannot lose
(can lose
plasmid)
plasmid)
 Perform experiments with isolate

URA3
rft1::KANMX

rft1::KANMX

ura3-

ura3-

1. Grow on Raffinose switch to glucose repression of RFT1

phenotype of Rft1p depletion can be studied
2. Grow on Raffinose switch to galactose activation of RFT1
phenotype of Rft1p overexpression can be studied
 Reporter genes
Reporter genes are heterologous genes that confer and
easily detectable phenotype to the object of study
Reporter genes are often used in the analysis of gene
expression
- examples: >Transcription

Promoter of YFG Reporter Gene (e.g lacZ)

b- galactosidase (E. coli lacZ gene):

can be assayed quantitatively (cleavage of ONPG)
cleaves X-gal to produce blue colour (visual assay)
Use: for example identify genes involved in
repression by glucose; fuse promoter of
glucose-repressed gene to b- galactosidase;
mutagenize screen for blue colonies on
glucose = yeast cells mutant for glucose
repression
 Reporter genes (continued)
examples:
-HIS3 (involved in Histidine biosythesis) can be
competitively inhibited by 3-amino-triazole can be
used in selection for viability on media lacking
histidine; especially used to detect strong activating
activity of transcription factors (e.g. in two-hybrid
screens)

-CAT (chloramphenicol acetyl transferase) Transfers

radioactive acetyl groups to chloramphenicol;
detection by thin layer chromatography and
autoradiography