Transcript Slide 1

Analysis Of Candidate Genes That Suppress Chromosome Loss In
Saccharomyces cerevisiae Mutants With Defects In Chromosome Transmission
Naomi Adjei, Alyssa Ellis, Lanie Feigenbutz, Traci Gwinn, James Kolnik, Lindsey Miller, Neel Patel, Kevin Peterson, Alexandra Richardson,
Christine Setsodi, Whitney Michaels, and Heidi Sleister. Biology Department, Drake University
Background
Results
Results
 Goal:
To identify yeast gene products important for accurate
chromosome transmission in mitosis.
 Importance: Errors during chromosome transmission in humans can
lead to cell death, genetic disorders (e.g., Down Syndrome), and cancer.
 Experimental Strategy: Plasmids containing yeast genes that suppress
YAC loss in ysm83 and ysm84 mutant strains were identified in previous
studies. The multiple genes present in each suppressor plasmid are
being subcloned and introduced into yeast cells to determine their
abilities to suppress YAC loss.
Yeast genes
present in
suppressor
plasmid
Candidate
suppressor
plasmid
ysm83 – p26
Visual Assay For YAC Loss
ysm83 – p41
ysm83 – p71
Method for isolating individual genes from
candidate suppressor plasmids
PCR (primersRestriction
add Bam HI
sites to ends of digest (enzyme)
product)
Molecular Cloning
Size of
isolated
gene
Vector
used for
cloning
Restriction
enzymes used
to linearize
vector
DTD1
---
Sau 3A
1903 bp
YEplac181
Bam HI
TIM22
---
Bam H1 + Pst 1
1337 bp
YEplac181
Bam H1 + Pst 1
YDL218W
---
Eco R1
1794 bp
YEplac181
Eco R1
OCA1
HS206 & HS207
---
1127 bp
YEp13
Bam HI
RAS2
HS208 & HS209
---
1980 bp
YEp13
Bam HI
PHO23
HS76 & HS77
---
1703 bp
YEp13
Bam HI
SSF1
HS70 & HS71
---
1942 bp
YEp13
Bam HI
HTD2
HS200 & HS201
---
1635 bp
YEp13
Bam HI
RRP3
---
---
---
---
---
AGX1
HS204 & HS205
---
1967 bp
YEp13
Bam HI
CAK1
HMS31 & HMS32
---
1374 bp
YEp13
Bam HI
SMF1
HS202 & HS203
---
2647 bp
p366
Bam HI
** * *
Conclusions & Current Work
ysm84 – p11
ysm84 – p57
Wildtype (YSM+)
yeast cells have low
rates of YAC loss,
resulting in white
colonies with little
to no red sectoring.
YSM+
ysm76
ysm83
ysm84
ysm83
[p26]
Mutant ysm strains
containing plasmid
suppressors of
YAC loss (e.g.,
ysm83 [p26]) have
few red sectors.
• Many
of the candidate suppressor genes have known roles related to
chromosome transmission. For example, CAK1 and OCA1 are involved in
cell cycle regulation.
Table 1. Summary of molecular cloning.
• Suppressor
plasmid ysm83-p26 contains full-length genes DTD1,
YDL218W, and TIM22 (Figure 2). These genes were isolated from ysm83-p26
using restriction digests (Figure 3) and ligated to vector YEplac181 (Figure
1). Correct vector + insert constructs for TIM22 and YDL218W were created
(Figure 5), transformed into yeast ysm84 cells (similar to ysm83), and
assayed for suppression of chromosome loss. Preliminary results suggest
YDL218W at least partially suppresses YAC loss in ysm84 cells.
Sau 3A ( 291 1 )
P stI (2 5 0 )
Sau 3A ( 2944)
Ba mHI (2 6 4 )
Sau 3A ( 31 42)
EcoRI (2 8 5 )
pBR3 2 2 origin of re plic a tio n
YDL218W
Sau 3A ( 3485)
Mutant strains ysm76, ysm83, and ysm84 have
increased rates of YAC loss, resulting in white
colonies with red sectors.
Sau 3A ( 3560)
Ps tI ( 3626)
Sau 3A ( 1 041 )
2 m ic ron origin of re plic a ti on
YE p lac1 8 1
Am pic illin re s is ta nc e ge ne
E coR I ( 3948)
Sau 3A ( 1 026)
57 41 bp
Sau 3A ( 401 6)
Sau 3A ( 970)
partial CDC13 (~5' half)
E coR I ( 473)
Sau 3A ( 387)
Sau 3A ( 1 )
TIM22
E coR I ( 21 54)
Sau 3A ( 471 4)
E coR I ( 1 904)
Sau 3A ( 4852)
E coR I ( 1 850)
Sau 3A ( 4963)
DTD1
B amHI ( 4963)
LEU2
Experimental Question & Methods
Which candidate yeast genes are responsible for
suppressing YAC loss in mutants with increased YAC loss?
ysm83-p19
Figure 1. Vector YEplac181.
YEplac181 is a high copy yeast shuttle vector
with selectable markers LEU2 and ampR.
Cloning sites are BamHI, PstI, and EcoRI.
1
Blast search to determine which candidate yeast genes are present in
each suppressor plasmid.
2
3
4
5
6
7
8
3.0 kb
2.0 kb
1.5 kb
Clone each candidate suppressor gene into vector (YEplac181, YEp13
or p366).
• Isolate vector and suppressor plasmid DNAs
• Linearize vector (restriction enzyme) and dephosphorylate
• Isolate yeast genes by PCR or restriction digest
• Ligate vector + insert (candidate suppressor gene)
• Transform ligation mixture into E. coli
• Screen transformants for correct constructs
Assay subcloned yeast genes for suppression of YAC loss in ysm
mutant strains.
• Transform correct constructs into ysm mutant strains
• Screen transformed cells for suppression of YAC loss
1.0 kb
Figure 3. Restriction digests and gel
electrophoresis to isolate candidate
suppressor genes.
Digestion of suppressor plasmid ysm83-p26
with BamHI-PstI (lane 2), Sau3A (lane 4),
and EcoRI (lane6) resulted in DNA bands
containing TIM22 (1337bp), DTD1
(1903bp), and YDL218W (1794bp),
respectively (marked with yellow arrows).
DNA extracted from these bands was
ligated to vector YEplac181 cut with BamHIPstI, Sau3A, and EcoRI, respectively. Lanes
1 and 8 contain a 100bp plus ladder.
**
Figure 5. Screening E. coli cells
transformed with ligation of vector
YEplac181 + insert candidate
suppressor gene (TIM22 or
YDL218W).
Transformants were “cracked,” and
resulting plasmid DNA was run on a
0.6% agarose gel. Plasmids containing
an insert are larger than the vector
alone (YEplac181, lane 1). Lanes 2-8:
YEplac181 + TIM22 transformants;
Lanes 9-16: YEplac181 + YDL218W
transformants. Plasmids larger than
the vector (*) were screened further.
Figure 2. Yeast DNA insert from suppressor
plasmid ysm83-p26.
The ysm83-p26 insert contains three full-length
yeast genes: DTD1, YDL218W, and TIM22.
4967 bp
Figure 4. PCR
amplification to
isolate candidate
genes.
Candidate
suppressor plasmid
ysm83-p71 was used
as a template for
amplifying candidate
suppressor genes
HTD2 (1635bp, lane
2) and SSFI (1942bp,
lane 3). PCR
products were
digested with BamHI
and ligated to BamHIlinearized vector
YEplac181 (lane 1).
Lanes 4 and 5
contain markers:
100bp plus ladder
and Lambda-HindIII.
1
2
3
4
5
• Suppressor
plasmid ysm83-p41 contains full-length genes OCA1, RAS2,
and PHO23. All three genes were isolated by PCR, ligated to BamHIlinearized YEp13, and transformed into E. coli. Transformants are now being
screened for presence of the insert.
• Suppressor
plasmid ysm83-p71 contains full-length genes SSF1, RRP3
and HTD2. Two of these genes were isolated by PCR (Figure 4) and ligated
to BamHI-linearized YEp13. Transformants are now being screened for
presence of the insert.
• Suppressor
plasmid ysm84-p11 contains full-length genes AGX1 and
CAK1. These genes were isolated by PCR, separately ligated to BamHIlinearized vector YEp13, and transformed into E. coli. Transformants are
now being screened for presence of the insert.
• Suppressor
plasmid ysm84-p57 contains a single gene, SMF1. To
determine if SMF1 can suppress YAC loss in a single copy plasmid, the gene
was isolated by PCR, ligated to BamHI-linearized single copy vector p366,
and transformed into E. coli. Transformants are currently being screened.
• The
BLAST analysis and restriction digests revealed that suppressor
plasmid ysm76-p152 is rearranged. A primer walking approach is underway
to determine the genes present in this plasmid.
We thank previous Drake BIO106L and BIO124L (Genetics Research)
students for isolation of the candidate suppressor plasmids described
here. This work was supported, in part, by a Drake research grant.