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A genome – wide screen to identify genes controlling the rate of entry into mitosis in fission yeast

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ABSTRACT

We have carried out a haploinsufficiency (HI) screen in fission yeast using heterozygous deletion diploid mutants of a genome-wide set of cell cycle genes to identify genes encoding products whose level determines the rate of progression through the cell cycle. Cell size at division was used as a measure of advancement or delay of the G2-M transition of rod-shaped fission yeast cells. We found that 13 mutants were significantly longer or shorter (greater than 10%) than control cells at cell division. These included mutants of the cdc2, cdc25, wee1 and pom1 genes, which have previously been shown to play a role in the timing of entry into mitosis, and which validate this approach. Seven of these genes are involved in regulation of the G2-M transition, 5 for nuclear transport and one for nucleotide metabolism. In addition we identified 4 more genes that were 8–10% longer or shorter than the control that also had roles in regulation of the G2-M transition or in nuclear transport. The genes identified here are all conserved in human cells, suggesting that this dataset will be useful as a basis for further studies to identify rate-limiting steps for progression through the cell cycle in other eukaryotes.

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mRNA expression levels for the HI gene set. Graph showing the mRNA expression level for each the HI gene in the heterozygous deletion diploid strain compared to the homozygous control strain (100%) normalized to act1 mRNA. The green line denotes 50% of the control mRNA level.
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f0003: mRNA expression levels for the HI gene set. Graph showing the mRNA expression level for each the HI gene in the heterozygous deletion diploid strain compared to the homozygous control strain (100%) normalized to act1 mRNA. The green line denotes 50% of the control mRNA level.

Mentions: All 17 strains were checked by PCR to confirm they were deleted for the correct gene, and all non-essential gene deletion mutants were checked to confirm that they had not become homozygous at the deletion locus (Table S1C, D). We carried out qPCR to estimate the mRNA levels encoded by a single copy of each HI gene, and found that the transcript level in all cases was reduced by around 50% (Table S1E, Fig. 3). This confirmed that for these genes there is no compensation by up-regulation of transcription from the remaining gene copy. For the 17 HI genes the mean and the median cell lengths were similar (Table S1B, compare columns H and K) and in the following discussion we have used the mean value when referring to cell length. The cell cycle gene set we used for the HI screen consisted of 368 (65.1%) essential genes and 197 (34.8%) non-essential genes (Table S1A). We found that the 17 HI genes had a similar distribution, with 10 (58.8%) essential genes and 7 (41.2%) non-essential genes (Fisher's Exact Test, Odds Ratio = 1.31, p > 0.05), suggesting that haploinsufficient cell cycle genes are no more likely to be essential or non-essential for cell cycle progression than the non-haploinsufficient genes.Figure 3.


A genome – wide screen to identify genes controlling the rate of entry into mitosis in fission yeast
mRNA expression levels for the HI gene set. Graph showing the mRNA expression level for each the HI gene in the heterozygous deletion diploid strain compared to the homozygous control strain (100%) normalized to act1 mRNA. The green line denotes 50% of the control mRNA level.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134717&req=5

f0003: mRNA expression levels for the HI gene set. Graph showing the mRNA expression level for each the HI gene in the heterozygous deletion diploid strain compared to the homozygous control strain (100%) normalized to act1 mRNA. The green line denotes 50% of the control mRNA level.
Mentions: All 17 strains were checked by PCR to confirm they were deleted for the correct gene, and all non-essential gene deletion mutants were checked to confirm that they had not become homozygous at the deletion locus (Table S1C, D). We carried out qPCR to estimate the mRNA levels encoded by a single copy of each HI gene, and found that the transcript level in all cases was reduced by around 50% (Table S1E, Fig. 3). This confirmed that for these genes there is no compensation by up-regulation of transcription from the remaining gene copy. For the 17 HI genes the mean and the median cell lengths were similar (Table S1B, compare columns H and K) and in the following discussion we have used the mean value when referring to cell length. The cell cycle gene set we used for the HI screen consisted of 368 (65.1%) essential genes and 197 (34.8%) non-essential genes (Table S1A). We found that the 17 HI genes had a similar distribution, with 10 (58.8%) essential genes and 7 (41.2%) non-essential genes (Fisher's Exact Test, Odds Ratio = 1.31, p > 0.05), suggesting that haploinsufficient cell cycle genes are no more likely to be essential or non-essential for cell cycle progression than the non-haploinsufficient genes.Figure 3.

View Article: PubMed Central - PubMed

ABSTRACT

We have carried out a haploinsufficiency (HI) screen in fission yeast using heterozygous deletion diploid mutants of a genome-wide set of cell cycle genes to identify genes encoding products whose level determines the rate of progression through the cell cycle. Cell size at division was used as a measure of advancement or delay of the G2-M transition of rod-shaped fission yeast cells. We found that 13 mutants were significantly longer or shorter (greater than 10%) than control cells at cell division. These included mutants of the cdc2, cdc25, wee1 and pom1 genes, which have previously been shown to play a role in the timing of entry into mitosis, and which validate this approach. Seven of these genes are involved in regulation of the G2-M transition, 5 for nuclear transport and one for nucleotide metabolism. In addition we identified 4 more genes that were 8–10% longer or shorter than the control that also had roles in regulation of the G2-M transition or in nuclear transport. The genes identified here are all conserved in human cells, suggesting that this dataset will be useful as a basis for further studies to identify rate-limiting steps for progression through the cell cycle in other eukaryotes.

No MeSH data available.


Related in: MedlinePlus