1. Molecular biology tools for the genetic manipulation of yeast
Plasmids, transformation and working the numbers

3. 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)

4. 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

5. 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; ade2Δ; ade3Δ; leu2-3; his3-11; his3-12; trp1-1; ura3-1 (Rothstein W303 derivative)

6. 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

7. Plasmids/Vectors
What is a Plasmid?
What is a Vector?

8. Genomic DNA Library Genomic 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

9. 3070 fragments/library members required “complexity of the library”
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 F:
Average size of Fragments
Size of genome
Example: yeast about 104 kb; choose P=99%, 15 kb fragment size
ln (1-0.99) ~
3070 fragments/library members required
“complexity of the library” 15
104
Ln ( )

10. Requirements for plasmids and vectors

11. Finding a nutritional marker (e. g
Finding a nutritional marker (e.g. LEU2 involved in leucine biosynthesis)

12. Generation of genetic tools:
How to find the ingredients to make a yeast vector

13. Transform E. coli leuB- mutants with library containing yeast DNA
Selectable marker: Cloning of a yeast gene involved in Leucine biosynthesis
Transform E. coli leuB- mutants with library containing yeast DNA
gene W
ori
bla
LEU2
ori
bla
geneX
ori
bla
Cells are Leu- (cannot grow on media lacking Leucine)
geneY
ori
bla
E. coli cell, with leuB mutation
High frequency transformation!
Select for bacterial transformants on minimal media (lacking Leucine)
Cloning by functional complementation
Isolate plasmid
LEU2
ori
bla

14. Transform Plasmid into Leu- strain
ori
bla
Low frequency transformation!
Recombination event required!
Select for yeast transformants on synthetic media lacking Leucine
ori
SC-Leu
bla
INEFFICIENT!
Integrating Vectors)
LEU2
leu2-
leu2-
LEU2
bla
ori

15. + Double Single Recombination event Reversible! (somewhat unstable)
ori
ori
bla
bla
Double Single
Recombination event
LEU2
LEU2
leu2-
leu2-
ori
bla
LEU2
Leu2- +
bla
leu2-
ori
Reversible! (somewhat unstable)
“Popout” event
Irreversible!
(very stable)
LEU2
leu2-
LEU2
Transformation possible but very low frequency/ inefficient

16. 2. Finding an origin of replication (ARS = automomously replicating sequence)

17. Cloning of ARS fragment
LEU2
ori
bla
Yeast genomic DNA
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

18. Plate cells from non-selective culture on Non-selective plate (e. g
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

19. High frequency of transformation!
Re-transform
LEU2
ori
bla
ARS
leu2-
Transform yeast
SC-Leu
High frequency of transformation!
But: Unstable (Lost at high frequency
 Need centromeric function on plasmid

20. 3. Isolation of a CEN sequence

21. Make new yeast library with marker gene and ARS + genomic DNA insert
LEU2
ori
leu2-
ARS
bla
Transform
Yeast genomic DNA
SC-Leu
High frequency of transformation
(collect large number of colonies)

22.  Isolate plasmids, retransform to confirm stability, sequence…
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 8 2 8 2 8 2 8 8
Transformant with stable plasmid: 50% of cells viable on SC - Leu
Enrichment for cells containing stable plasmid  isolate after large number of generations growing non-selectively! (Plate cells on SC- Leu)
 Isolate plasmids, retransform to confirm stability, sequence…

23. 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

24. Yeast centromeric plasmids, low copy, high stability
EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI, HindIII
Mp13 multiple cloning site
Also: YCp50 (URA3)
Yeast centromeric plasmids, low copy, high stability

25. Yeast episomal (YEp) plasmids (2m origin of replication multicopy intermediate stability)
Yeast integrating (YIp) plasmids

26. 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.

27. - 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

28. Yeast transformation:
Usually done in PEG/DTT/LiAcetate, heat shock (45oC; 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 ( transformants per mg of DNA) (half a day)

29. Generating gene knockouts in yeast

30. 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”

31. + Double Single Recombination event Irreversible Reversible!
ori
bla
Double Single
Recombination event
URA3
Irreversible
Yfg1-1
yfg1-1
YFG1
YFG1
yfg1-1
YFG1
URA3
Yfg1-1 +
Reversible!
ori
bla
URA3
YFG1
YFG1
URA3
yfg1-1
Transformation possible but very low frequency/ inefficient
YFG1

32. One-step gene replacement:
Uses a marker gene with flanking sequences homologous to the gene of interest
Can be generated by PCR:
YFG 5’
YFG 3’
KANMX
YFG 5’
YFG 3’
KANMX
ori
bla
YFG
Integration via homologous recombination
PCR reaction
yfg::KANMX
YFG 5’
YFG 3’
KANMX
Select for geneticin resistance

33. Isolate yeast DNA from transformants - Verify integration by PCR
yfg::KANMX
YFG 1.
1.3 kb
Product 1.7kb 2.
Product (1.2 kb)
No product 3.
Product (0.8 kb)
No product
About 2-3 weeks to create and confirm a yeast knockout! How much in Mouse?

34. Plasmid shuffle: Done when working with essential gene:
Phenotype of mutant versions of the gene can be investigated in a deletion background

35. Introduction into the Diploid and tetrad dissection/selection of the haploid transformant
RFT1
CEN
RFT1
URA3
pGAL1
tCYC1
rft1::KANMX
ura3-
sporulate

36. Dissect! 4 spores of a tetrad rft1::KANMX ura3- rft1::KANMX ura3- RFT1
CEN
RFT1
URA3
pGAL1
tCYC1
ura3-
RFT1
CEN
rft1
URA3
pGAL1
tCYC1
Dissect!

37. Dissect on YPGalactose!!!!
ura3-
rft1::KANMX
ura3-
RFT1
RFT1
ura3-
ura3-
rft1::KANMX
CEN
RFT1
URA3
pGAL1
tCYC1
CEN
RFT1
URA3
pGAL1
tCYC1 or
either
DEAD!
CEN
RFT1
URA3
pGAL1
tCYC1
RFT1
CEN
RFT1
URA3
pGAL1
tCYC1
rft1::KANMX
ura3-
ura3-

38. Select cells that carry the knockout allele plus plasmid
A. Select on SC – ura (Galactose)
CEN
RFT1
URA3
pGAL1
tCYC1
RFT1
CEN
RFT1
URA3
pGAL1
tCYC1
rft1::KANMX
ura3-
ura3-
RFT1
Can grow
Can grow
ura3-
DEAD!

39. B. Select for growth on YPGalactose - geneticin
CEN
RFT1
URA3
pGAL1
tCYC1
RFT1
CEN
RFT1
URA3
pGAL1
tCYC1
rft1::KANMX
ura3-
ura3-
DEAD!
Can grow

40. DEAD! (cannot lose plasmid)
C. Test Growth on SC-Galactose – FOA (5-fluoro-orotic acid)
CEN
RFT1
URA3
pGAL1
tCYC1
RFT1
CEN
RFT1
URA3
pGAL1
tCYC1
rft1::KANMX
ura3-
ura3-
DEAD! (cannot lose plasmid)
Can grow (can lose plasmid)

41. Perform experiments with isolate
CEN
RFT1
URA3
pGAL1
tCYC1
rft1::KANMX
ura3-
Grow on Raffinose – switch to glucose  repression of RFT1  phenotype of Rft1p depletion can be studied
Grow on Raffinose – switch to galactose  activation of RFT1  phenotype of Rft1p overexpression can be studied

42. 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:
b- galactosidase (E. coli lacZ gene):
can be assayed quantitatively (cleavage of ONPG)
cleaves X-gal to produce blue colour (visual assay)
>Transcription
Promoter of YFG
Reporter Gene (e.g lacZ)
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

43. 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