Correspondence to Michael Knop: knop{at}embl.de
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Introduction |
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In this study, we provide an in-depth analysis of the molecular mechanism underlying regulated dyad formation. We demonstrate that the formation of any number of spores (14) is subject to regulation in accordance to the nutritional situation of the cell. We describe a new molecular mechanism based on a self-organizing system, which regulates the meiotic SPB function toward spore formation. It provides sporulating cells with a simple way to maximize the number of formed spores. We termed this regulation spore number control (SNC).
Automixis, which is the mating of spores from the same ascus, has been observed occasionally for yeast (Guilliermond, 1905; Winge and Laustsen, 1937). We show that mating of germinating spores occurs with high frequency and mostly involves mating between spores of nonsister origin. We demonstrate that SNC ensures this rate is held constant on the population level over a broad range of sporulation conditions. This ensures the transmission of a constant and high degree of paternal heterozygosity through the meiotic division. We provide indications that this ability is associated with two types of advantages: masking of haploid lethal mutations and enhanced mean fitness of the postmeiotic generation.
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Results |
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The MP resembles a crystal
The rapid relocalization of cytoplasmic Mpc54p-GFP signal to SPBs coincides with the appearance of bright Mpc54p-GFPlabeled MPs inside the cell and the transition of meiosis I to meiosis II. This suggests that the formation of MPs is cell cycle controlled. We used FRAP to investigate the exchange rate of Mpc54p-GFP between the SPBs and the cytoplasm in cells before assembly of MPs (before and during meiosis I) and in cells with assembled MPs (during meiosis II). For cells before and during meiosis I, we found that Mpc54p-GFP exchanged completely within a time span of 30 s (Fig. 4 A). In cells in meiosis II, however, the exchange of Mpc54p-GFP between the SPB and cytoplasm was very low (Fig. 4 B). We made use of
mpc70 cells to directly test the influence of the cell cycle stage on the exchange rate of Mpc54p-GFP. Binding of Mpc54p to the SPB is not impaired in these cells, but MP assembly is blocked as a result of the deletion of one MP component (Knop and Strasser, 2000). FRAP measurements revealed that in
mpc70 cells, Mpc54p-GFP exchanged within
30 s in meiosis II (Fig. 4 C). In addition, the cytoplasmic amounts of Mpc54p-GFP remained high in these cells. This indicates that the cell cycle stage does not, per se, influence the binding of Mpc54p-GFP to the SPB but influences the formation of a fully functional MP.
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SNC is optimized for providing maximal amounts of mating partners within the ascus
In S. cerevisiae, the mating type locus (MAT) is linked to the centromere of chromosome III (CEN3; Hawthorne and Mortimer, 1960). The MAT-CEN linkage causes the spores in nonsister dyads to be of opposite mating types. The frequency of this behavior depends on two factors: the fidelity of nonsister encapsulation in dyads and the genetic MAT-CEN linkage. The fidelity with which dyads contain nonsister spores is 96% irrespective of the acetate concentration used for the experiment and independent of the ady2 mutation (unpublished data). The ability to form dyads that contain spores of opposite mating types suggests that yeast spores are able to undergo direct mating upon germination, which has been reported many years ago (Guilliermond, 1905; Winge and Laustsen, 1937). Dyads as well as triads can theoretically form one diploid cell upon the mating of spores, and tetrads can form two (Fig. 7 A). We used the asci compositions of sporulated wild-type cells as revealed by the sporulation profile (Fig. 6 B) to calculate the amounts of diploids that could be formed via intratetrad sporespore mating. As shown in Fig. 7 B, the SNC mechanism enhances this number especially in poor sporulation conditions. Notably, this requires the MAT-CEN linkage and the spores in dyads to be nonsister (Fig. 7 B).
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Possible influence of intratetrad mating on genome organization and fitness
Our experiments with the heterozygous cdc5 and
pre3 mutants (Fig. 7 C) demonstrate clearly that spores that have haploid lethal mutations in their genomes are still able to mate efficiently. The fidelity of rescuing a lethal mutation depends on its linkage to the MAT locus, as it is the only locus where heterozygosity is reconstituted 100% of the time upon mating of spores from the same ascus. For non-MATlinked loci, the heterozygous state is preserved 66.6% of the time, whereas for MAT-linked loci, this value falls between 66.6% and 100% depending on the strength of the linkage (Zakharov, 1968; Kirby, 1984). In the case in which the MAT locus is linked to a centromere, as in S. cerevisiae, linkage to the MAT locus is expanded to all other centromere-linked sequences in the genome (Fig. 7 A). We calculated the fraction of the yeast genome that exhibits genetic linkage to the mating-type locus based on the genetic map of yeast (www.yeastgenome.org) and found it to be roughly 25% of the yeast genome (
1,440 of the 5,792 genes; see Materials and methods). Moreover, masking of heterozygous deleterious mutations upon mating of spores from the same ascus would be particularly effective if mitotically essential genes exhibited centromere linkage. This idea prompted us to analyze the genome-wide distribution of all essential genes of S. cerevisiae. We found a significant bias for their localization in centromere-adjacent regions (Fig. 8 A).
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Discussion |
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SNC allows the formation of a high number of diploids under different sporulation conditions through the mating of spores from the same ascus (Fig. 7). Our results demonstrate that the majority of S. cerevisiae spores indeed mate directly upon germination and, thus, return to a diploid lifestyle without any haploid mitotic divisions. Being a diploid species offers a number of advantages that are associated with having two copies of each gene present in the genome. Cells are less sensitive to mutagenic conditions (Mable and Otto, 2001), and adaptive evolution may be accelerated by the ability to preserve allelic variations or different genetic traits in the genome. For acquisition of new advantageous mutations, diploidy is a disadvantage, but only in the context of large populations in the absence of meiosis (Zeyl and Bell, 1997; Zeyl et al., 2003; Goddard et al., 2005). Natural S. cerevisiae isolates are always diploid, and they have been reported to often exhibit extremely low spore viability, which indicates the presence of haploid lethal mutations in the genome (Johnston et al., 2000). Such yeast strains would generate very few new diploid cells if they rely on diploidyzation via motherdaughter mating, because this requires at least one haploid mitotic division and leads to homozygous genomes. However, motherdaughter mating may offer an efficient way to occasionally generate diploid strains that have lost all deleterious mutations, which is a process termed renewal of the genome (Mortimer et al., 1994; Mortimer, 2000). In contrast, direct mating of spores from the same ascus after meiosis prevents haploid mitotic divisions and leads to partial reconstitution of the heterozygous stage of the genome as it was before meiosis occurred. This obviously can rescue postmeiotic genomes that are associated with a lethal mutation (Fig. 7 C). In this context, the centromere and, thus, the mating-type linkage of an overrepresented number of essential genes (Fig. 8 A) becomes plausible. It supports the idea that nonsister mating might be associated with a population genetic advantage for natural S. cerevisiae, which is linked to the handling of acquired disadvantageous mutations. This may indicate coevolution of this aspect of global genome organization with increased efficiency of intratetrad nonsister mating. The advantage of intratetrad mating, however, appears to be not only restricted to haploid lethal mutations, as it also provides a fitness advantage to the offspring in situations where genomes, which vary by a high degree of polymorphisms, undergo meiotic mixing (Fig. 8 B). As shown recently, genetic interactions between polymorphisms are widespread and can significantly affect gene expression in yeast (Brem et al., 2005). Thus, it appears that many such interactions are, in fact, not well balanced upon the new recombination of genomes and, therefore, exhibit disadvantageous properties with consequences on the fitness of individuals. Our results also prove that the heterozygous situation can compensate for this. This suggests that many of these polymorphisms are of a recessive nature.
MP assembly: a self-organizing system able to convert a continuous signal into a digital response
We demonstrate that the regulation of spore production depends on the amounts of three proteins: Mpc54p, Mpc70p, and Spo74p. The amounts of these proteins are varied according to externally available amounts of acetate. A transcriptional control of MP component abundance is suggested by the influence of the gene dosage of MP components on the numbers of spores, but we did not rule out experimentally that there are other levels of regulation as well. It might be that protein stability or other posttranslational regulation is involved to adjust the protein concentrations or is used to fine tune the whole system. At first glance, the regulation of spore number formation by protein abundance might appear trivial; the higher the concentration of the proteins, the more spores will be obtained. Complexity is added to the system by the fact that a graded input, namely the increasing amounts of MP components, has to be translated into a digital outcome, which is the assembly of one to four MPs. This is required because exactly four haploid genomes are available for the formation of spores. The SPBs, which correspond in number precisely to the number of genomes, are part of the system. The SPBs provide spatially restricted sites. In this respect, the number of SPBs determines the possible discretization. Moreover, the system requires feedback to generate bistable behavior (Ferrell, 2002) that is necessary to flip an SPB from the off state (not involved in spore formation) to a stable on state (forms a spore). Irreversibility may be another necessary feature of the system because the trigger could be available only for a short period during the cell cycle. Additionally, the system must be able to generate the response locally on the SPBs, and this response must be communicated to other SPBs. The properties of crystal-like assemblies on the SPBs meet all of the requirements of such a system. Thereby, the binding properties of MP components to the SPBs change from exchangeable to nonexchangeable. This rationale suggests a simple, positive feedback provided by the crystal size that leads to a proportional increase in the growth rate.
Currently, we are unsure about the precise mechanism that initiates the formation of MP crystals on selected SPBs. A plausible assumption would be that a regulatory activity is enabling crystal seeding (e.g., via down-regulation of an inhibitory activity or up-regulation of a promoting activity) at a certain time point of meiosis (onset of meiosis II) and that this activity is present at different amounts on the SPBs from different generations. There must be a stochastic process involved because the age bias that directs MP assembly to specific SPBs is not followed strictly. This may relate to very initial events. The observation that the assembly of only one MP is possible with high fidelity, although it occurs on one of two SPBs from the same generation, suggests that digitization does not depend on the age bias of SPBs alone. However, it has been recently shown that not only the age of a particular SPB but also the age of the SPB next to which a new SPB is formed can influence the binding of a protein (Grallert et al., 2004). This would mean that the two new SPBs are also different. We tried to address this question experimentally but could not obtain conclusive results.
What is the role of the age bias of the SPB? Is it needed for faithful digitization of the MP assembly? We think that the age bias generates initial differences between the SPBs that are sufficiently large enough that they can become amplified during MP assembly. This would make the system more robust. Because the molecular basis of the age difference of SPBs or centrosomes is unsolved for all cases in which it was observed to influence the asymmetric localization of proteins (Pereira et al., 2001; Piel et al., 2001; Uetake et al., 2002; Grallert et al., 2004; Maekawa and Schiebel, 2004), the final answer to this question cannot be given.
Positive feedback mechanisms are able to amplify small initial asymmetries. During MP assembly, the initial event might be the formation of a crystal seed. Crystal growth then generates positive feedback because a bigger crystal offers more binding sites for new subunits. Feedback during MP assembly appears to be rather direct and proportional with the size of MP structure. However, it is possible that the MP recruits further enhancing activities in a size-dependent manner to the SPBs. This would lead to stronger feedback provided by the size of the crystal.
The simulation supports the idea that the aforementioned positive feedback circuit in combination with SPBSPB communication via the pool of free subunits is sufficient to digitize MP assembly. The incorporation of population statistics was useful because it enabled fitting of the simulation to real data. It also enabled us to study alterations of SNC upon the introduction of mutations using quantitative predictions and subsequent validation with the sporulation profiles of the corresponding mutations. For the ady2 mutant, the simulation did not require changing the parameters that describe SPBSPB differences; thus, Ady2p is predicted to function only through the regulation of the abundance of available amounts of MP components (e.g., expressional control of the MP genes MPC54, MPC70, and SPO74).
Is acetate-dependent regulation of protein abundance limited to the MP components? We investigated the acetate and ADY2 dependency of protein levels of other proteins, especially components involved in the formation of early intermediates of the spores (Ady3p and Ssp1p; Moreno-Borchart et al., 2001). The levels of these proteins showed a similar acetate-dependent regulation as found for the MP components (unpublished data). These proteins have no contribution during the initiation of spore formation and MP assembly (Moreno-Borchart et al., 2001). Therefore, the acetate-dependent regulation appears to be a more global adaptation, which, in addition to directly controlling spore number formation via protein levels of MP components, also adjusts the amounts of other proteins according to the number of formed spores. Thereby, the cell restricts the production of proteins to the amounts actually needed. Further experiments will be needed to investigate this as well as the underlying signaling processes.
SNC in other yeast species
The MP components, which provide the properties of crystal formation, are conserved within the clade of the hemiascomycete yeasts (www.yeastgenome.org). Many hemiascomycete species have been reported to form asci with variable numbers of spores (Kurtzman and Fell, 1998). In more distant fungi, such as Schizosaccharomyces pombe, no obvious homologues of the MP components can be found. To test whether S. pombe is able to regulate the production of less than four spores per ascus, we tried various starvation conditions for the sporulation of diploid S. pombe cells. We found no indication of SNC in this species (unpublished data). Rather, it appeared that entry into meiosis was subject to regulation, suggesting that this species regulates the spore production on another level. It remains to be answered whether any other fungi evolved a mechanism that regulates spore production in a similar way. If not, one may consider SNC as an evolutionary trait of hemiascomycete yeasts.
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Materials and methods |
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Spore counting was performed using stacks of images acquired from Hoechst 33342stained samples. We used a microscope (IRBE; Leica) equipped with a 63x NA 1.4 oil objective (Leica), a camera (CoolSNAP HQ; Photometrics), and a DAPI filter set (Chroma Technology Corp.). The pictures were recorded using Metamorph software (Molecular Devices). Maximum projections of the Hoechst 33342 images were superimposed with the phase-contrast image using Metamorph software. G0 and nonads (meiosis but no spores) were discriminated based on Hoechst 33342 staining. This spore-counting method was essential to reliably discriminate all of the different species. Counting using only phase-contrast microscopy led to significant systematic errors. Sporulation efficiency was calculated as follows: [(% tetrads x 4) + (% triads x 3) + (% dyads x 2) + % monads]/4.
Microscopic techniques
For live cell imaging, cells were adhered with Concanavalin A on small glass bottom Petri dishes (MaTek). All live cell experiments were performed at RT. Live cell imaging (Fig. 3, A and C) was performed on an imaging system (DeltaVision Spectris; Applied Precision) equipped with GFP and Cy3 filters (Chroma Technology Corp.), a 60x NA 1.4 oil immersion objective (plan Apo, IX70; Olympus), softWoRx software (Applied Precision), and a CoolSNAP HQ camera. For the experiment shown in Fig. 3 F, sporulating cells were inspected on a microscope (IRBE; Leica) equipped with a plan Apo 100x NA 1.4 oil objective (Leica), a CoolSNAP HQ camera, and DAPI, CFP, YFP, and Cy3 filter sets (Chroma Technology Corp.). FRAP was either performed on a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.; Fig. 4, B and C) or on a wide-field epifluorescence microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) equipped with a laser scanner for photobleaching using a high aperture 63x NA 1.2 water immersion objective (C-Apochromat; Carl Zeiss MicroImaging, Inc.; Fig. 4 A). The wide-field microscope was necessary to visualize Mpc54p-GFP at SPBs in cells before meiosis II as a result of the lower amounts of this protein at SPBs. Quantification of videos (Figs. 3, B and D; and 4 A) was performed using MetaMorph software and maximum projections of the videos. Quantification of the experiment in Fig. 4 (B and C) was performed using LSM 510 software (Carl Zeiss MicroImaging, Inc.). Conversion of file formats from 12 to 8 bit was performed using Metamorph software. Photoshop (Adobe) was used to mount the images and to produce merged color images. No image manipulations other than contrast, brightness, and color balance adjustments were used.
For electron microscopy and analysis of the overexpression of MP genes (Fig. 4 D), a diploid strain containing one copy of MPC54, MPC70, and SPO74 under control of the CUP1-1 promoter (strain YCT851) was induced with 10 µm CuSO4 (added 4 h after induction of sporulation on 0.3% acetate). A sample was taken at a time point in which mixed populations of cells in meiosis I and meiosis II were maximally enriched (5.5 h) and were processed for electron microscopy using osmium tetroxide fixation as described previously (Knop and Strasser, 2000). Samples were visualized on an electron microscope (Biotwin CM-120; Philips) using a CCD camera (DualVision; Gatan). Strong overexpression of all three proteins as compared with wild-type cells was validated using Western blotting.
Correlation of MP formation with the age of the SPBs involved
We fused an RFP with a retarded formation of the fluorophore of 26 h (RedStar; Knop et al., 2002) to the integral SPB component Spc42p (Donaldson and Kilmartin, 1996). This allowed the discrimination of SPBs from all three generations (oldest SPB, intermediate SPB [formed before meiosis I], and two new SPBs [formed before meiosis II]; Fig. 3, E and F). (DsRed that was used previously to discriminate SPBs from different generations in yeast [Pereira et al., 2001; Nickas et al., 2004] has a maturation time of the fluorophore of >10 h and, therefore, was not suitable for this application). For live cell imaging of MP assembly, eqFP611 was used as the RFP (Janke et al., 2004). It exhibits properties similar to those of RedStar.
Immunological methods
The antibody specific for Spo74p was produced with bacterially expressed 6HIS-Spo74p and was affinity purified. All of the other antibodies have been described previously (Knop and Strasser, 2000).
Simulation of SNC
Assumptions for the digitization module (assumption A; see supplemental material) are listed as follows: (1) Initially, the free Mpc54p monomers and the Mpc70p/Spo74p heterodimers are homogeneously distributed in the cytoplasm. Diffusion is fast and readjusts a homogeneous distribution in the cytoplasm. Therefore, crystal growth is not diffusion limited. (2) The sizes of the initial crystal seeds at the four different SPBs are different. (3) Mpc54p monomers and Mpc70p/Spo74p heterodimers are incorporated into the crystals at the SPBs if they are both present in some spatial region within some short period of time. (4) A larger crystal provides more binding sites than a smaller one and, therefore, incorporates more protein. Thus, a larger crystal depletes the cytoplasmic pools more rapidly than a smaller one. (5) The crystal size is limited by the size of the SPB. (6) If the crystal size reaches a certain threshold level, the crystal is considered to be a fully functional MP.
Assumptions for the simulation of populations (sporulation profile; assumptions B and C; see supplemental material) are listed as follows: (7) The functional relationship between the number of Mpc54p, Mpc70p, and Spo74p proteins in the cell and acetate concentration (acetate protein function) can be approximated by the experimentally derived functional relationship between sporulation efficiency and acetate concentration. (8) Because of variations within the population, the cellular response of cells in a cell population to a given acetate concentration (i.e., the amount of Mpc54p/Mpc70p/Spo70p) varies according to a second symmetric two-parameter distribution. (9) The acetate concentration available to individual cells in the population is described by a simple symmetric two-parameter distribution (e.g., Gaussian). This accounts for the asynchronicity of the population with regard to progression through meiosis. Cells that perform MP assembly earlier than othersbecause they performed the meiotic divisions fasterhave more acetate available as a result of the simultaneous consumption of acetate by the population.
The simulations were performed using Mathematica software (version 5.0; Wolfram Research Inc.). In the first step, we designed a set of differential equations that models the crystal growth according to assumptions 15 (Fig. 9). To account for assumption 1, we implemented the homogeneously distributed amounts of Mpc54p monomers and Mpc70p/Spo74p heterodimers as time-dependent functions (Mpc54[t] and Mpc70Spo74[t]) that describe the available amounts of protein in the cytoplasm. We defined four time-dependent functions (Crystal1[t]Crystal4[t]) that describe the crystal growth at up to four potential SPBs. To account for assumption 3, the differential equations describing the transition from spore-free to spore-containing cells contain the product of Mpc54[t] and Mpc70Spo74[t]. Assumption 4 is included by a positive feedback in the crystal growths that is proportional to the size of the corresponding crystal. Assumption 5 is implemented by a saturation function that is characterized by the two parameters of slope and maximum crystal size (saturation). The basic differential equations are shown in Fig. 9.
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In the second step, we derived the steady-state crystal sizes from the numerical solution of the differential equations. Corresponding to assumption 6, all crystals with a size above a given threshold (30% of the maximum crystal size, defined by the saturation parameters) were regarded as spores. We defined SporesSum[c] as the function that represents the number of spores resulting from this digitization step depending on the acetate concentration c. Assumption 8 was implemented by convolving this function with the Gaussian distribution that describes the variation of available acetate. As a result, we obtained the statistical appearances of the five possible spore configurations (04 spores) depending on the acetate concentration c.
Finally, we iterated the steps of solving the differential equations and calculating the statistical appearances for a certain interval of maximum amplitudes of Mpc54Metabolism[c] and Mpc70Spo74Metabolism[c]. Considering assumption 9, the statistical appearances were weighted using the Gaussian distribution for the maximum amplitudes that are introduced to model the cellular response. This yielded the theoretical sporulation profile.
Mating assay
Different dominant markers (hphNT1 or kanMX4; Janke et al., 2004) were introduced next to the centromeres of chromosome V between ORFs YER001w and YER002w in a diploid homothallic yeast strain (SK1 background; resulting strains YCT918 and YCT919). The heterozygous CEN5-hph/CEN5-kan strain (YCT930) was selected on Hygromycin B/G418 plates upon the mating of spores of strains YCT918 and YCT919. YCT930 was then used to delete one copy of either CDC5 (ORF YMR001c) or PRE3 (ORF YJL001w) with the natNT2 marker (Janke et al., 2004). After sporulation in liquid medium containing either 0.1 or 0.01% acetate medium, 107 asci were spotted on a YPD plate. Unsporulated cells were killed by ether treatment (Guthrie and Fink, 1991). The ascii were incubated on YPD plates for 18 h. The cells were collected and spread on YPD plates (100150 colonies per plate) and grown for 2 d. The colonies were assayed for the presence of all three markers simultaneously (kan+, hph+, and nat+) as well as for only two or one of the markers by replica plating. 400600 colonies were evaluated for each sample. Colonies, which contained cells with all three markers, were considered to derive from cells (with respect to the deletion of the essential gene) that were formed by mating of nonsister spores. Cells containing the nat+ marker (which marks the deletion of the essential gene) but only the kan+ or hph+ marker in addition were considered to be the result of mating upon germination but not between nonsister spores. Colonies containing the kan+ and/or the hph+ marker but not the nat+ marker were considered to originate from other types of mating.
Fitness experiment
A homothallic YJM145/SK1 (Kane and Roth, 1974; McCusker et al., 1994) hybrid strain was generated by mating spores of strain YCT925 (YJM145 background) that contained one CENV-hphNT1 integration with spores of strain YCT918 that contained one CENV-kanMX integration and selection on Hygromycin B/G418 plates. Upon sporulation of the resulting strain under low acetate concentration (0.01%), a population of heterozygous diploids was generated through the isolation of 80 dyads by micromanipulation (of which 88% did form a colony). For the generation of homozygous diploids, 40 dyads were dissected (spore survival frequency was 68%). Both species were grown on YPD plates for 2 d and independently pooled in water. Equal amounts of the cells (each 5 x 105 cells) were mixed and grown at 30°C in 400 ml YPD for 24 h (1213 generations). For subsequent rounds, 106 cells were transferred to a new flask and grown for another 1213 generations. The composition of the culture was analyzed in the beginning of the experiment and after each round of growth for the content of hph+ or kan+ (homozygous diploids) or hph+ and kan+ cells (heterozygous diploids).
Calculation of the fraction of ORFs in the yeast genome with significant centromere linkage
Significant centromere linkage can be observed up to 35 cM away from the centromere (Sherman and Wakem, 1991), whereas the total yeast genome covers
4,500 cM (Mortimer et al., 1992). With 16 chromosomes present in yeast and a total of 5,792 annotated protein-encoding ORFs (www.yeastgenome.org), an estimated 1,440 ORFs are within the CEN-linked region.
Statistical significance of centromere linkage for groups of genes
Fig. 8 A shows that essential genes are overrepresented close to the centromeres. 70/317 genes found within a 20-kbp distance to a centromere are essential. In comparison, 1,032/5,773 yeast genes are essential. This overrepresentation of essential genes is significant at a P = 0.03 according to a hypergeometric test.
Online supplemental material
Videos 1 and 2 show the transition from anaphase meiosis I to metaphase meiosis II and correspond to Fig. 3 (A and C). Supplemental material provides the Mathematica files for the simulation (Fig. 6) and provides a description of the three parts of the simulation. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200507168/DC1.
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Acknowledgments |
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Submitted: 29 July 2005
Accepted: 19 October 2005
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References |
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