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.

Visual Assay For YAC Loss

Analysis Of Candidate Genes That Suppress Chromosome Loss In Analysis Of Candidate Genes That Suppress Chromosome Loss In SaccharomycesSaccharomyces cerevisiaecerevisiae Mutants With Defects In Chromosome Transmission Mutants With Defects In Chromosome Transmission

Naomi Adjei, Alyssa Ellis, Lanie Feigenbutz, Traci Gwinn, James Kolnik, Lindsey Miller, Neel Patel, Kevin Peterson, Alexandra Richardson, 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 UniversityChristine Setsodi, Whitney Michaels, and Heidi Sleister. Biology Department, Drake University

ResultsResults

Experimental Question & MethodsExperimental Question & Methods

Blast search to determine which candidate yeast genes are present in each suppressor plasmid.

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

BackgroundBackground

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 BamHI-PstI, Sau3A, and EcoRI, respectively. Lanes 1 and 8 contain a 100bp plus ladder.

1 2 3 4 5 6 7 8

• Many of the candidate suppressor genes have known roles related to chromosome transmission. For example, CAK1 and OCA1 are involved in cell cycle regulation.

• 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.

• Suppressor plasmid ysm83-p41 contains full-length genes OCA1, RAS2, and PHO23. All three genes were isolated by PCR, ligated to BamHI-linearized 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 BamHI-linearized 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.

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 BamHI-linearized vector YEplac181 (lane 1). Lanes 4 and 5 contain markers: 100bp plus ladder and Lambda-HindIII.

Wildtype (YSM+) yeast cells have low rates of YAC loss, resulting in white colonies with little

to no red sectoring.

ResultsResults

3.0 kb

2.0 kb

1.5 kb

1.0 kb

PCR (primers- add Bam HI

sites to ends of product)

Restriction digest (enzyme)

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

ysm84 – p57 SMF1 HS202 & HS203 --- 2647 bp p366 Bam HI

ysm83 – p71

ysm84 – p11

Candidate suppressor

plasmid

Yeast genes present in

suppressor plasmid

Molecular CloningMethod for isolating individual genes from

candidate suppressor plasmids

ysm83 – p26

ysm83 – p41

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.

* * * * * *

1 2 3 4 5

ysm83-p194967 bp

YDL218W

DTD1

TIM22

partial CDC13 (~5' half)

Bam HI (4963)

Pst I (3626)

Eco RI (473)

Eco RI (1850)

Eco RI (1904)

Eco RI (2154)

Eco RI (3948)

Sau 3A (1)

Sau 3A (387)

Sau 3A (970)

Sau 3A (1026)

Sau 3A (1041)

Sau 3A (2911)

Sau 3A (2944)

Sau 3A (3142)

Sau 3A (3485)

Sau 3A (3560)

Sau 3A (4016)

Sau 3A (4714)

Sau 3A (4852)

Sau 3A (4963)

YEplac1815741 bp

LEU2

Ampicillin resistance gene

2 micron origin of replication

pBR322 origin of replication

Bam HI (264)

Eco RI (285)

Pst I (250)

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.

Figure 2. Yeast DNA insert from suppressor plasmid ysm83-p26.The ysm83-p26 insert contains three full-length yeast genes: DTD1, YDL218W, and TIM22.

Table 1. Summary of molecular cloning.

Which candidate yeast genes are responsible for suppressing YAC loss in mutants with increased YAC loss?

Conclusions & Current WorkConclusions & Current Work

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.

ysm83 ysm84ysm76 ysm83 [p26]

YSM+

Mutant strains ysm76, ysm83, and ysm84 have increased rates of YAC loss, resulting in white

colonies with red sectors.

Mutant ysm strains containing plasmid

suppressors of YAC loss (e.g.,

ysm83 [p26]) have few red sectors.