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A New Genetic Method for Isolating Functionally Interacting Genes: High plo1+-Dependent Mutants and Their Suppressors Define Genes in Mitotic and Septation Pathways in Fission Yeast
C. Fiona Cullena,b, Karen M. Maya, Iain M. Haganc, David M. Gloverb,d, and Hiroyuki Ohkuraa,ba Institute of Cell and Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom,
b Department of Anatomy and Physiology, Medical Sciences Institute, The University of Dundee, Dundee DD1 4HN, United Kingdom,
c School of Biological Sciences, The University of Manchester, Manchester M13 9PT, United Kingdom
d Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom
Corresponding author: Hiroyuki Ohkura, Institute of Cell and Molecular Biology, The University of Edinburgh, Mayfield Rd., Edinburgh EH9 3JR, United Kingdom., h.ohkura{at}ed.ac.uk (E-mail)
Communicating editor: P. RUSSELL
| ABSTRACT |
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We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1+ gene that encodes a fission yeast homologue of the polo-like kinases. plo1+ regulates both spindle formation and septation. We have isolated 17 high plo1+-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1+, sce3+, cdc8+, and rho1+, and two previously uncharacterized genes, mpd1+ and mpd2+. mpd1+ exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2+ resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16+, dma1+, byr3+, byr4+ and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.
THE family of polo-like kinases (plks) is conserved from yeast to humans. With the exception of a small subfamily of mammalian homologues that may function early in the cell cycle, polo kinase function is required at various steps in mitosis such as G2/M transition, bipolar spindle formation, APC (anaphase promoting complex)-mediated proteolysis, and cytokinesis in various eukaryotic systems (![]()
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In contrast, the existing mutants of the Saccharomyces cerevisiae plk gene, cdc5, arrest at a late stage of mitosis with separated chromosomes and an elongated spindle (![]()
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There is also evidence that human plk1 and Xenopus plx function even earlier at the onset of mitosis/meiosis (![]()
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The activity and localization of plks undergo major changes during the cell cycle. The abundance of mammalian and S. cerevisiae polo-like kinases peaks during mitosis (![]()
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The multifunctional nature of an individual polo kinase was first demonstrated by work on the fission yeast polo kinase encoded by plo1+ (![]()
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Although the abundance of plo1 kinase appears constant through the fission yeast cell cycle, plo1 kinase localizes to SPBs in a cell cycle-dependent manner (![]()
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To identify potential regulators or downstream targets of plo1 kinase we have devised a genetic screen to identify functionally interacting genes. Because plo1 kinase has more than one essential function, a suppressor screen that relies on restoring the viability of a mutant may not be ideal for this purpose unless alleles affecting one specific function are used. In our screen we isolate mutants that are dependent upon high levels of Plo1 protein for viability. The rationale behind this approach is that the lethality caused by the partial loss of function of regulators or effectors may be rescued by elevated plo1 kinase activity. We describe the results of such a screen that has identified mutations in genes required for both mitosis and septation.
| MATERIALS AND METHODS |
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Handling of fission yeast and cytological methods:
Basic handling of fission yeast was carried out as described in ![]()
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DNA manipulation:
General DNA manipulations were carried out according to ![]()
Isolation and genetic characterization of pld mutants:
h- leu1 int[nmt1-plo1+, LEU2] was selected as a stable transformant of h- leu1 with pHN204 (![]()
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Isolation and characterization of the genomic fragments that complement cdc7-PD1:
Temperature-sensitive (ts-) h- leu1-32 ura4-D18 cdc7-PD1 was transformed with a genomic library in the ura4+ marked multicopy vector pUR19 (![]()
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Isolation and characterization of chromosomal suppressors of cdc7-PD1:
To isolate chromosomal suppressors, single colonies of temperature-sensitive cdc7-PD1 grown at 25° were plated out on YE and incubated at 35°. Ts+ revertants from each plate were tested for cold sensitivity (Cs-) at 22°. Three independent Cs- revertants were studied further. Temperature-sensitive progeny were recovered from all three revertants after crossing with wild type, indicating that extragenic mutations caused the suppression. To determine whether these suppressor mutations were allelic, the three revertants were crossed with each other. Recombination between suppressor mutations would give progeny with the cdc7-PD1 single mutation. None of the crosses produced temperature-sensitive progeny, indicating that the three suppressor mutations were allelic to each other. Given the similarity of the phenotype to byr4 and cdc16, we tested allelism to these loci. A strain carrying the suppressor mutation and cdc7-PD1 were crossed with a ura4+ integrant at the byr4 locus or cdc16-116. Recombinants (Ura- Ts+ or Ura+ Cs- progenies) between byr4 and the suppressor mutation were isolated. No recombinants (wild-type or cdc7-PD1 progeny) between the suppressor mutation and cdc16 were isolated, indicating the suppressor mutation is allelic to cdc16.
We also found that in the absence of the cdc7-PD1 mutation all of the suppressor mutations were lethal, even at 35°. To confirm this, the ura4+ gene was integrated at the cdc7 locus, and the integrant was crossed with h- leu1-32 ura4-D18 cdc7-PD1 cdc16-sp1. All Cs- progenies were Ura- (and therefore have cdc7-PD1), indicating that the cdc7-PD1 mutation is required for survival of the suppressor mutation. h- leu1-32 ura4-d18 cdc7-PD1 cdc16-sp1 was transformed with a S. pombe genomic library (![]()
| RESULTS |
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Isolation of high plo1+-dependent mutants:
The wild-type plo1 gene was placed under the control of the conditional nmt1+ promotor (![]()
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This strain was mutagenized with EMS or NTG. Mutagenized cells were then plated onto selective media without thiamine to induce high levels of plo1+ expression and incubated at 30° for 2 or 3 days. The resulting colonies were replicated twice onto media containing thiamine to switch off gene expression from the nmt1+ promotor and onto media without thiamine as a control. To facilitate the identification of mutants dependent upon high-level expression of plo1+ (high plo1+-dependent mutants), these plates also contained Phloxine B, which stains dead cells dark red. Colonies that grew well in the absence of thiamine but grew poorly or not at all or stained red in the presence of thiamine were selected. These mutants were tested further by streaking to give single colonies in the presence or absence of thiamine, and the size of colonies was compared. Strains showing better growth in the absence of thiamine at 30° were selected as high plo1+-dependent (pld) mutants for further analysis. Under these conditions the original strain without mutagenesis shows slightly slower growth on EMM without thiamine.
Basic characterization of pld mutations:
From a total of 600,000 colonies screened, 19 high plo1+-dependent mutants that showed better growth on plates containing thiamine (the condition where cells produced high levels of Plo1) than on plates with thiamine (the condition where cells produced normal levels of Plo1) at 30° were identified. All 19 strains were tested for growth at various temperatures (22°35°) both in the presence and absence of thiamine (Table 2). The degree of dependency on elevated levels of Plo1 varied in different mutants and at different temperatures. At normal levels of plo1+ expression most of the mutants showed temperature-sensitive growth that could be completely or partially rescued by high levels of expression of plo1+. One example is shown in Fig 2A. Two mutants, PD2 and PD26, were not temperature sensitive but were dependent on high plo1+ expression at all temperatures tested.
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To eliminate the possibility that the nmt1-plo1+ construct was essential for the apparent high plo1+-dependent phenotype, we crossed out nmt1-plo1+ from these strains to obtain temperature-sensitive mutants whenever possible. The wild-type plo1+ gene, under the control of its own promoter on a multicopy vector, was then introduced into the resulting temperature-sensitive mutants. In all cases examined, the temperature-sensitive lethality of these mutants could be fully or partially rescued by the introduction of the wild-type plo1+ gene on a multicopy vector, but not by the introduction of an empty vector (Fig 2B). In other words plo1+ acted as a multicopy suppressor of pld mutations. This confirmed that the differential growth in the presence or absence of thiamine observed in the original strains was due to suppression of the pld mutations by a high level of plo1+ expression.
The cytological phenotype of each of the pld mutants was determined to identify the particular pathways on which each of the pld genes functioned. The mutants that showed temperature sensitivity without high plo1+ expression were grown in liquid culture at the permissive temperature and then shifted to the restrictive temperature. Cells were fixed at different time points following the temperature shift and stained with the DNA dye, DAPI. To examine the defective phenotype of those mutants that did not show temperature sensitivity, cells were first cultured in media lacking thiamine and the high level expression of plo1+ was switched off by addition of thiamine to the culture. This approach was straightforward, but as plo1 kinase is not degraded in a cell cycle-specific fashion (![]()
This cytological analysis identified three distinct phenotypic classes. The first class consisting of 14 mutants showed only septation defects. The second class (3 mutants) had mitotic defects. The final class consisted of 2 mutants that showed neither septation nor mitotic or other notable defects. The mutations were not linked to each other. In summary, 17 out of 19 pld mutants had defects in the pathways in which plo1 kinase has been shown to be involved, thus suggesting that this screen was effective in identifying interacting genes.
pld mutants defective in septation:
All of the 14 pld mutants that had a septation defect produced elongated multinucleate cells that failed to form septa under the conditions inhibiting their growth. Mitosis was not affected in any of these pld mutants. ![]()
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Linkage analysis revealed that these early septation pld mutants fell into six complementation groups, pld1pld6 (Table 2). pld1 (six alleles: PD1, PD3, PD9, PD17, PD23, and PD32) was allelic to cdc7, pld2 (two alleles: PD2 and PD12) was allelic to cdc15, and pld3 (1 allele: PD21) was allelic to cdc11. None were linked to cdc14. PD37 was found to contain more than one mutation; therefore allelism could not be determined. In addition to the original mutants identified by ![]()
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To determine whether any of the remaining strainspld4-PD19, -PD20, pld5-PD10, pld6-PD11, and PD37contain mutations in spg1, PCR was used to amplify a genomic fragment containing the spg1 gene from all five strains. The nucleotide sequences of the amplified spg1 genes were determined. The two allelic mutants of pld4 (-PD19 and -PD20) contained different single-point mutations in the coding sequence of the spg1 gene (Fig 4), while the other three mutants had a sequence that was identical to the reported wild-type spg1. The mutations in both pld4-PD19 and pld4-PD20 caused a conversion from G to A (or C to T), which is expected from alkylating mutagens, such as the EMS or NTG we used in this screen. The mutation in PD19 changed the 65th amino acid residue from D (Asp) to N (Asn), and the PD20 mutation changed the 97th amino acid residue from T (Thr) to I (Ile). The aspartate residue is conserved through all known G-proteins and is implicated in GTP binding. The threonine is also conserved among all G-proteins, with some exceptions that have a serine residue at the equivalent position. These results indicated that the two mutations pld4-PD19 and -PD20, which confer high plo1+ dependency, were allelic to spg1 and are likely to affect general properties shared by all G-proteins rather than Spg1-specific function.
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pld5-PD10 and pld6-PD11 were crossed with sid1, sid2, and sid4. A cross between pld5 and sid2 did not produce recombinant progeny, indicating that pld5 is alleleic to sid2. pld6 is not allelic to any of the tested early septation mutants.
pld mutants defective in mitosis:
Three pld mutants, PD24, PD26, and PD36, showed defects in chromosome segregation. Genetic analysis indicated that they were not allelic to each other and therefore defined three independent loci, pld8, pld9, and pld10. pld8-PD24 was temperature sensitive in the presence of thiamine, but removal of thiamine to allow high expression of the plo1+ gene improved growth at the semirestrictive temperature (Table 2). The nmt1-plo1+ construct was therefore crossed out to obtain a temperature-sensitive mutant. To examine the cytological defects, pld8-PD24 cells were cultured at the permissive temperature (25°), then shifted to the restrictive temperature (35°), and then processed for DAPI staining (Fig 5A). Although the culture was asynchronous in terms of cell cycle progression prior to the temperature shift, cells with condensed chromosomes temporarily accumulated after the shift (Fig 5B). In such cells chromosomes were condensed so that three individual chromosomes were sometimes clearly visible, indicating that chromosome separation was prevented in this mutant. After the frequency of the cells with condensed chromosomes had peaked, the frequency of "cut" cells increased, and cell separation occurred in the absence of nuclear division. The cut phenotype occurs when the septum bisects the nucleus and cells complete separation (![]()
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pld9-PD26 and pld10-PD36 were inviable at all temperatures tested unless plo1+ was expressed at an elevated level. Therefore, we examined the defective phenotype by switching off plo1+ gene expression from the nmt1 promoter by the addition of thiamine to the culture. Microscopic analysis of cells fixed and stained with DAPI 18 to 26 hr after the addition of thiamine revealed that both mutants become considerably elongated and accumulated overcondensed chromosomes; however, they rarely showed a cut phenotype (Fig 5C and Fig D). In summary the three mitotic pld mutants we isolated are defective in chromosome separation.
A pld mutant cdc7(pld1)-PD1 is a useful tool to isolate genes that positively regulate septation:
The septation class of pld mutants contained six new alleles of cdc7, two new alleles of cdc15, and one new cdc11 allele. While each of these new mutations conferred dependency upon elevated Plo1 levels, we found that the original temperature-sensitive alleles (cdc7-24, cdc11-119, and cdc15-136; ![]()
Two different genetic approaches were taken to use the cdc7(pld1)-PD1ts mutant to screen for suppressors. In the first case multicopy suppressors were isolated from an S. pombe genomic library based on a multicopy vector (pUR19; ![]()
The cdc7+ and plo1+ genes could rescue the temperature sensitivity of cdc7-PD1 (data not shown). The cdc7+ gene was isolated twice in this screen, but the plo1+ gene was not isolated, indicating that the screen was not saturated. Among the remaining six multicopy suppressors four were known genes that have been implicated in septation in fission yeastspg1+, sce3+, cdc8+, and rho1+ (Table 3). spg1+ (septation promoting G-protein) encodes a small G-protein that associates with Cdc7 (![]()
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Two genes, which we have called mpd1+ and mpd2+ (multicopy suppressor of pld1), were previously uncharacterized in fission yeast; however, the amino acid sequences of their predicted gene products showed that similar proteins have been identified in other organisms. mpd1+ encodes a protein that shares significant similarity (31 and 53% identity in 426 residues) with mammalian PIP kinase and S. cerevisiae MSS4p (![]()
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Plasmids carrying suppressor genes were introduced into the other temperature-sensitive pld mutants to test whether the multicopy suppressors of cdc7-PD1 could suppress other pld mutants (Table 4). Some of the multicopy suppressors could rescue more than one mutant, suggesting that the suppression of cdc7-PD1 represented a functional interaction between the genes rather than an indirect consequence of changes in general cell physiology, such as nonspecific stabilization of protein structure. In contrast, the original cdc7-24 allele that does not show high plo1+ dependency was not rescued by any of these multicopy suppressors.
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A chromosomal suppressor of cdc7-PD1 induces hyperactivation of septum formation:
The multicopy suppressor screen successfully identified genes positively involved in septation. As a complementary approach, chromosomal revertant mutants of cdc7-PD1 were isolated in the hope that mutations that promote septation and counteract the septation defect of cdc7-PD1 would be identified. We isolated a number of Ts+ revertants, which grew well at 35°, from the temperature-sensitive cdc7-PD1 mutant. For ease of subsequent analysis we limited our analysis to the studies of three independent cold-sensitive revertants that do not grow at 22°. Genetic analysis indicated that all three suppressor mutations were extragenic and mapped to the cdc16 locus (we call them cdc16-sp1, sp2, and sp3). In the absence of the cdc7-PD1 mutation these individual cdc16 mutations were lethal at all temperatures tested. In other words, cdc7-PD1ts can suppress the lethality of these cdc16 mutations (and vice versa) at 35° but fails to do so at 22°. For this reason we examined the phenotype of the cdc16 mutations in the presence of cdc7-PD1ts.
At 35°, cells containing both cdc7-PD1 and the cdc16 mutations grew at nearly the same rate as wild type and looked relatively normal (Fig 7A). After shifting down to the restrictive temperature (22°), the frequency of septated cells gradually increased, reaching 70% 4 hr after the shift to 22° (Fig 7B and Fig C). (The generation time of wild-type cells was about 4 hr under these conditions.) The majority of septated cells had two nuclei separated by a septum, but a significant number of septated cells had only one interphase nucleus that was bisected by the septum or located to one side of the septum. Such cells did not complete cell separation, but rather accumulated more than one septum after longer incubation (Fig 7B and Fig C). As this phenotype is unlike that of the cdc7-PD1 single mutant, this septation phenotype is most probably due to the presence of the cdc16 mutation. Similar phenotype has been observed in the original cdc16-116 allele and the gene deletion (![]()
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From an S. pombe genomic library (![]()
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cdc16+ is required to limit septation to once per cell cycle (![]()
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Unexpectedly, the cdc7 gene, a septation inducer, was also isolated in the screen. However, close examination revealed that the genomic fragment responsible for cdc16-sp1 suppression lacked the first exon of the cdc7+ gene. This would produce an amino-terminal truncated protein, lacking an essential subdomain required for protein kinase activity. It is likely that this truncated Cdc7 protein acts as a dominant-negative protein inhibiting septum formation, as a kinase-null cdc7 mutant has been shown to act in this way (![]()
| DISCUSSION |
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Here we have described the use of a novel genetic approach to identify functionally interacting genes and have demonstrated the effectiveness of the method using the plo1+ gene in fission yeast. A screen was carried out for mutants dependent upon a high level of expression of plo1+ for growth. Nineteen high plo1+-dependent mutants were isolated. Seventeen of these had defects in either mitotic progression or septation, both pathways in which plo1+ gene function is implicated. We have also shown that pld mutants have potential as tools for identifying other interacting genes in these pathways. We identified a number of genes involved in septation, starting with one of the pld mutants, cdc7-PD1, using screens for both chromosomal and multicopy supressors. By combining these screens, we have isolated most of the known regulators of septum formation, demonstrating that this is a powerful means to identify a group of genes functioning in a related process.
plo1 and septation:
The isolation and characterization of high plo1+-dependent mutants revealed a close genetic interaction between plo1+ and the early septation genes. Gene disruption indicated that plo1+ is required for both medial actin ring formation and septation, as well as formation of the bipolar spindle (![]()
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Many of the genes identified in this screen, cdc7, cdc11, cdc15, spg1, and sid2, are known to be involved in the regulation of septation. The cdc7+ gene encodes a protein kinase that is required to initiate septum formation (![]()
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As a reflection of the close functional interactions in vivo, it has been reported that changes in dosage or activity of some of these genes can suppress mutations in others (![]()
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In S. cerevisiae a parallel genetic interaction is reported among the late mitotic genes including CDC5, CDC15, and TEM1 (![]()
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Suppressors of cdc7-PD1 mutant and regulation of actin dynamics:
A screen for suppressors of one pld mutant, cdc7-PD1, was highly fruitful. Four known genes, spg1+, sce3+, cdc8+, and rho1+, were isolated as multicopy suppressors, all of which have been shown to be involved in septation in S. pombe (![]()
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Cdc7 kinase is not required for medial actin ring formation but is essential for subsequent septum formation (![]()
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The chromosomal suppressors of cdc7-PD1 identified one locus, cdc16. cdc16+ is required for limiting septum formation to once per cycle by inhibiting the Spg1 cascade. Multicopy suppressors of the mutation include another known gene involved in the inhibition of septum formation, byr4+, together with a dominant-negative cdc7 gene encoding a truncated protein lacking part of the kinase domain. In addition two known genes, dma1+ and byr3+, have been isolated, neither of which is essential for the regulation of septation. However, there are some observations that link these genes with those that inhibit septum formation. High-level expression of dma1+ can complement the multiple septation phenotype of cdc16-116 (![]()
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Advantages of high dose-dependent mutant screening:
The methodology used for the high plo1+-dependent mutant screen can be applied to any genes. It is of interest to compare this method to which we assign the generic term "high dose-dependent mutant screen" to multicopy suppressor screens and other conventional methods, such as a chromosome suppressor screen or synthetic lethal screen. We see that it offers three main advantages, each derived from the fact that a cloned gene is used as the starting point for the screen. First, because the complete genomes of many organisms have been or are in the process of being sequenced, it is increasingly common to initiate a study from cloned genes. Second, it has been estimated that two-thirds of genes are dispensable for growth in yeast, and a significant proportion of those are probably due to functional redundancy (![]()
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The end product of this approach is a bank of mutants. This provides two advantages. The mutant phenotype can be used in deciding which of those should be studied further. In addition, high dose-dependent mutants should be ideal for suppressor screening, because they are, by definition, suppressed by at least one other gene. Our results suggest that the pld allele of cdc7 is superior for suppressor screening.
Finally, multifunctional genes can be studied. If genes are required for two or more essential pathways, suppression of one pathway cannot suppress the lethality of the mutation. Therefore suppressor screening would be powerless for these genes, unless mutant alleles that affect only one essential pathway are used. In contrast, high dose-dependent mutant screens provide a powerful alternative method for investigating multifunctional genes. This was illustrated recently when an attempt was made to isolate genomic DNAs that suppress temperature-sensitive alleles of plo1 mutants (![]()
Application to other systems:
We believe high dose-dependent mutant screening can be adapted to any genetic system, with minor modification, because the only requirements are a conditional high-expression system and effective mutant isolation methods. In the budding yeast S. cerevisiae, there are well-developed conditional expression systems (![]()
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We hope that this report will trigger the application of this new method in a wide variety of genes and organisms and that this method will be proven to be an effective genetic method in various systems.
| ACKNOWLEDGMENTS |
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We thank Drs. A. Carr, K. Maundrell, P. Nurse, and K. Gould for a genomic library, expression vectors, cdc mutants, and sid mutants. This work is supported by the Wellcome Trust and the Cancer Research Campaign.
Manuscript received February 2, 2000; Accepted for publication April 10, 2000.
| LITERATURE CITED |
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BALASUBRAMANIAN, M. K., D. M. HELFMAN, and S. M. HEMMINGSEN, 1992 A new tropomyosin essential for cytokinesis in the fission yeast S. pombe. Nature 360:84-87[Medline].
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) Interphase and mitotic cells with normal appearance, (
) cells with condensed nuclei, (
) cells showing a cut phenotype. pld9-PD26 (C) and pld10-PD36 (D) grown in the presence (+T) and absence (-T) of thiamine and stained with DAPI, which visualizes cell outlines and septa as well as DNA.

) Cells with multiple septa.