Cell. Vol. 29, 585-594. June 1982, Copyright 0 1982 by MIT

Clonal Lethality Caused by the Yeast Plasmid 2p DNA

Connie Holm * Department of Genetics, SK-50 University of Washington Seattle, Washington 98195

Summary

Strains of Saccharomyces that carry the nib allele of a nuclear gene exhibit a “nibbled” colony mor- phology if they also harbor the plasmid 28 DNA. I have found that the expression of the nibbled phe- notype is correlated with the presence of a subpop- ulation of abnormally large cells that give rise to mortal clones. Large cells apparently become large as a consequence of a defect in DNA replication or nuclear division. Large nib cells contain twice as much 2~ DNA per microgram of total DNA as small nib cells do, and elevated 21.r DNA copy number is the cause, not the effect, of increased cell size. It appears that the NIB allele can prevent an increase in 2~ DNA copy number, but cannot produce a decrease once the copy number has exceeded the normal level. I propose, therefore, that the NIBgene product normally represses the amplification of 2~ DNA copy number, and that the nib allele is partially defective in this function.

Introduction

The yeast plasmid 2~ DNA is potentially useful as a model for chromosomal DNA replication. (For a review of 2~ DNA, see Broach, 1981 .I Like chromosomal DNA, 21.1 DNA is complexed with histones (Livingston et al., 19791, and the plasmid also exhibits a typical nucleosome conformation (Livingston et al., 1979; Nelson et al., 1979). 2~ DNA replication is controlled by the same cell division cycle genes as is chromo- somal DNA replication (Petes et al., 1975; Livingston et al., 19771, and the plasmid is also replicated exclu- sively during the S phase of the cell cycle (Zakian et al., 1979). Each of the approximately 50 molecules in a cell is normally replicated once per cell cycle (Zakian et al., 1979). Thus, the general control of 2~ DNA replication appears to be identical to that of chromo- somal DNA replication.

2~ DNA causes the production of an aberrant “nibbled” colony morphology in nib strains of yeast (C. Helm, submitted); instead of being rounded and homogeneous in size, nibbled colonies are heteroge- neous in size, and they each contain numerous inden- tations. This paper is concerned with the physiological basis of the nibbled colony morphology. Three main conclusions are established. First, the nibbled colony morphology results from the production of sectors of mortal cells, which can be recognized by their abnor- * Present address: Department of Biology 56-743. Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.

mally large size. Second, the large cells are defective at a specific stage of the cell division cycle. Finally, the cause of this defect is an elevated copy number of 2~ DNA. It is proposed that the nuclear gene, nib, plays a role in controlling the replication of 2~ DNA.

Results

nib Strains Are Nibbled at 30°C but Smooth at 38°C Investigation of the nibbled colony morphology was facilitated by the temperature dependence of the nib- bled phenotype. Strains 2129-6-l (NIB), 2129-6-2 (nib), 2129-6-3 (NIB) and 2129-6-4 (nib) were streaked for single colonies on solid medium and allowed to grow for three days at 22’, 30°, 34”, 36’ or 38°C. (All these strains contain 2~ DNA.) As ex- pected, the nib strains grown at 22“, 30” or 34°C exhibited a nibbled morphology (C. Holm, submitted), the colonies were heterogeneous in size and they each contained numerous indentations. When grown at 36’ or 38’C, however, the same strains showed a smooth colony morphology: the colonies were rounded and homogeneous in size. This smooth col- ony morphology was indistinguishable from that ex- hibited by the NIB strains grown at 22”, 30”, 34’, 36” or 38°C. The nibbled phenotype was therefore sup- pressed by growth at high temperature. Suppression was reversible, since when cells grown at 38°C were streaked onto fresh medium at 30°C the resulting colonies were all nibbled; this result was obtained even when the cells were pregrown at 38’C for as many as 50 generations. nib strains grown at 38’C, as well as N/B strains, served as control populations in further experiments.

The Nibbled Phenotype Is Caused by Mortal Clones of Abnormally Large Cells I examined the cells in nibbled and smooth strains in order to investigate the cellular basis of the nibbled colony morphology. When grown at 30°C cells from strain 2129-6-2 (nib) were heterogeneous in size, with 13% of the cells appearing abnormally large. In con- trast, when cells from strain 2129-6-2 (nib) were grown at 38°C or cells from strain 2129-6-3 (NIB) were grown at 30” or 38°C they were homogeneous in size; fewer than 3% of these cells were abnormally large (Table 1). This difference is evident in Figure 1, which shows the size distribution of cells from strains 2129-6-2 (nib) and 2129-6-3 (NIB), grown at 30°C and strain 2129-6-2 (nib), grown at 38°C. These results make it clear that nibbled strains differ from smooth strains in their possession of a subpopulation of abnormally large cells.

The spatial distribution of cells of various sizes in colonies indicated that cell size was clonally inherited. Large cells appeared in clusters on the edges of nibbled colonies (Figure 2). This production of sectors

Cell 586

Table 1. Proportion of Large Cells in NIB and nib Strains

Strain Temperature Large Cells

2129-6-2 (nib) 3ooc 13% n = 500

2129-6-3 (NW 3ooc <1% n = 500

2129-6-2 (nib) 38°C 2% n = 300

2129-6-3 fN/S) 38’C 1% n = 300

a : *‘+-nib- (30’)

CELL SIZE

Figure 1. Distribution of Cell Sizes in Nibbled and Smooth Strains

Strains 2129-6-2 (nib) and 2129-6-3 (NIB) were grown overnight in YM-1 liquid medium. The logarithmically growing cultures were soni- cated briefly and then analyzed for cell size in a Coulter Counter (model Ze) equipped with a Channelyzer. Cell size increases to the right. Solid line: strain 2129-6-3 (NIB) grown at 3O’C. Dashed line: strain 2129-6-2 (nib) grown at 30°C. Dotted line: strain 2129-6-2 (nib) grown at 38°C.

of large cells could be the cause of the nibbled colony morphology; if large cells divided more slowly than small cells, or if clones of large cells stopped dividing after only a few divisions, then the expansion of the colony edge might be blocked by the sectors of large cells.

I investigated the possibility that large cells and small cells differed in division behavior by observing the colonies produced by single cells at 30°C. Strains 2129-6-2 (nib) and 2129-6-4 (nib) were grown at 30°C, and large and small cells were isolated by micromanipulation. Table 2 shows the sizes of the clones produced after 2 days of growth on solid medium. Small cells produced clones that were fairly homogeneous in size; 90% of the cells gave rise to visible colonies (>l O5 cells) in 2 days. In contrast, the large cells produced clones that were heterogeneous in size. In addition, only 5.4% of the large cells gave rise to visible colonies in 2 days. Most large cells, therefore, had a lower division potential than small cells did. This property, together with the clonal pro- duction of large cells, is sufficient to explain why nib colonies are nibbled at 30°C.

The visible colonies produced in the above experi-

Figure 2. Photomicrograph of Cells at the Edge of a Nibbled Colony

A diploid nib/nib strain produced in a complementation test (C. Holm, submitted) was streaked for single colonies on YEPD medium and allowed to grow at 30°C for 2 days. It was photographed by using dark field optics in a Zeiss photomicroscope. with Kodak ASA TriX film. The apposing sectors of large and small cells are typical of those seen in haploid or diploid nibbled colonies.

ment were nibbled regardless of whether they were derived from small cells or large cells. Furthermore, each of the visible colonies contained sectors of both large and small cells (see Figure 2). Thus, although cell size is predominantly clonal, with large cells pro- ducing large cells and small cells producing small cells, small cells can give rise to large cells and large cells can give rise to small cells.

Many Large Cells Can Be Rescued at 38°C Since growth at high temperature was known to yield nib populations containing few large cells, it was in- teresting to examine the effect of temperature on extant large cells. Three possibilities existed. First, elevated temperature might reduce the already low division potential of large cells; in this case, the scar- city of large cells in 38°C populations would be ex- plained by selection. Second high temperature might simply prevent the formation of large cells. In this case, extant large cells would experience their usual reduced division potential, but when they ceased di- viding they would not be replaced. Finally, high tem- perature could actually ameliorate the defect in nib cells. In this case, a shift to high temperature might return the physiology of large cells to normal, and they might subsequently give rise only to cells of normal size.

To distinguish among these hypotheses, strains 2129-6-2 (nib) and 2129-6-4 (nib) were pregrown at 30°C; large and small cells were isolated and then incubated at 38°C. Of the small cells, 87% produced visible colonies after 2 days of growth on solid medium (Table 2). Large cells produced two classes of clones: 56% contained only 1 to 12 cells, and 44% were visible colonies (>l O5 cells). No intermediate size classes were observed. The visible colonies produced at 38’C were smooth whether they were derived from small cells or large cells. Virtually all the cells observed

28 DNA-Induced Lethality 587

Table 2. Colonies Produced by nib Cells after 2 Days of Growth

Colony Size

Cell Size Temperature 1-12 Cells

13-29 Cells

30-60 Cells

70-l 00 Cells

150-l 000 Cells

Visible Colony

Small

Large

Small

Large

30°C 1% 3% 1% 1% 3% 90% n = 87

3ooc 54% 8.3% 19% 6.6% 6.6% 5.4% n = 167

38’C 5% 3% 0% 0% 5% 87% n = 39

38’C 56% 0% 0% 0% 0% 44% n = 85

in these colonies were small. A comparison of the clone sizes produced by large

cells at 38“ and at 30°C indicated that high temper- ature actually rescued many of the large cells. At both temperatures approximately half 64% at 3O’C; 58% at 38’C) of the large cells gave rise to clones of 1 to 12 cells. The balance of the cells produced microcol- onies at 30°C but visible colonies at 38°C. As no such effect was observed in the small cells used as controls, this difference cannot be attributed solely to a generalized increase in growth rate at 38°C. It seems instead that there are two classes of large cells: approximately half of the cells are irreversibly committed to cease dividing within a few generations: the remaining cells have a small division potential at 30°C, but when incubated at 38’C revert to a normal mode of division.

Large Cells Are Defective at a Specific Stage in the Cell Cycle The barrier to normal division in large cells was clari- fied by an examination of cell morphology. Strains 2129-8-2 (nib) and 2129-8-3 (NIB) were grown in liquid medium at 30°C and samples of the exponen- tially growing cultures were compared under the mi- croscope (see Table 3). In the nib strain, 22% of the cells had a bud as large as the mother cell, but in the

N//3 strain less than 1% exhibited this morphology. (Normally, a cell divides before its bud is as large as the mother cell [Hartwell et al., 19771.) A preponder- ance of cells with large buds was not observed when strain 2129-8-2 (nib) was grown at 38’ instead of at 30°C. Thus cells with large buds were prevalent only under conditions in which the nibble phenotype was normally expressed.

When the morphologies of large nib cells and small nib cells were examined separately, it was found that 80% of large cells possessed very large buds, but only 1% of the small cells did so (Table 3). (Cells of intermediate sizes were not examined.) The large buds of large cells were equal in size to the parent cells, and they were inseparable from the parent cells by micromanipulation or sonication; in N/B cells and small nib cells, buds that were two thirds the size of the mother could be detached easily by either method. Thus large cells are abnormal in two respects: first, the cells are abnormally large; second, the bud does not separate from the mother cell until it reaches a proportionally larger than normal size.

Both of these abnormalities could be caused by a defect at a stage-specific event in the cell cycle. Large buds that do not separate from the mother cell are seen in many temperature-sensitive cell division cycle mutants arrested at a variety of specific steps (Hart-

Table 3: Morphology of cells in logarithmically-growing cultures.

Strain Temperature Population Q Qdx2*

2129-6-2 (nib) 3o" all cells 16% 62% 22% n = 600

2129-6-3 (KIB) 3o" all cells 41% 59% <1x n = 700

2129-6-2 (nib) 38' all cells 45% 54% <1x n = 600

2129-6-2 (nib) 3o" small cells 36% 63% 1% n = 200

2129-6-2 (nib) 30° large cells 8% 32% 60% n = 200

* Cells in which the bud was as large as the mother cell.

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well et al., 1973). In cdc mutants, arrested ceils usu- ally display a characteristic nuclear morphology. To determine whether nib strains exhibited this property, I examined cells stained with the fluorescent dye, DAPI.

Strains 2129-6-2 (nib) and 2129-6-3 (NIB) were grown in liquid medium at 30’ and at 38’C. A sample from each exponentially growing culture was stained with DAPI, and the nuclear morphology of the cells was examined by fluorescence microscopy. Table 4 shows the proportion of cells exhibiting various types of nuclear morphology. Of the four cultures, only 2129-6-2 (3O’C) was grown under conditions in which nibbled colonies would be produced. In this latter population, 42% of the cells possessed a single rounded nucleus lodged next to the constriction be- tween the mother cell and its bud. In the other three populations, the proportion of nuclei exhibiting this conformation was only 4-9%, which is typical of a normal population of cells (Hartwell et al., 1970). Thus this unusual morphology was predominant only under conditions in which the nibbled phenotype was ex- pressed.

In the fluorescence microscope it was difficult si- multaneously to distinguish the size of a cell and its nuclear morphology, and so I separated the cells by size before staining them with DAPI in order to deter- mine whether the unusual nuclear morphology was more common among the large nib cells. Strain 2129- 6-2 (nib) was grown in liquid medium at 30°C, and the cells were separated by size on a sorbitol velocity gradient. The gradient was fractionated, and the frac- tions were examined under the microscope; fraction 4 contained large cells, and fraction 6 contained small

cells. (The size distributions of the cells in the two fractions are shown in Figure 3.) A single nucleus, located at the junction between the mother and the bud, was found in 70% of the cells in fraction 4, but in only 22% of the cells in fraction 6 (Table 4). Hartwell et al. (1970) and Culotti and Hartwell (1971) have observed this nuclear morphology in cell cycle mu- tants arrested in DNA replication or mitosis. It seems likely, therefore, that large cells are defective at one of these steps in the cell cycle.

The Defect in Large Cells is Heritable To determine whether the cell cycle defect of large cells was heritable, I attempted to mate them with normal cells. Strains 2129-6-l (a NIB), 2129-6-2 (a nib), 2129-6-3 ((1: NIB) and 2129-6-4 ((Y nib) were grown in liquid medium at 30X Large and small cells from nib strain 2129-6-2 were placed next to cells from NIB strain 2129-6-3, and large and small cells from nib strain 2192-6-4 were placed next to cells from NIB strain 2129-6-l ; the pairs of cells were left undisturbed for 1% to 2 hr. Of the small nib cells (n = 321, 97% mated, and of the large nib cells (n = 73) 38% mated; the low frequency of mating among large cells was probably due to the arrest of many of them at an unreceptive point in the cell cycle (Reid and Hartwell, 1977). All the diploids having a small cell for the nib parent gave rise to a visible colony after 2 days of growth at 3O’C. In contrast, only 64% of the diploids having a large cell as the nib parent did so. (For brevity, I will refer to the diploids as “small-cell diploids” and “large-cell diploids,” respectively.) The large-cell diploids that did not produce visible colonies gave rise to microcolonies containing 9 to 200 large

Table 4: Nuclear morphology of cells in logarithmically-growing liquid

cultures.

0 Strain Temperature Population domeQ@&

2129-6-2 (nib) 30° all cells 32% 42% 8% 17% n=300

2129-6-3 (NIB) 3o" all cells 60% 9% 6% 25% n=300

2129-6-2 (nib) 38' all cells 77% 4% 2% 17% n=300

2129-6-3 (NIB) 38" all cells 78% 5% 3% 14% n=300

2129-6-2 (nib) 3o" fraction 4* 20% 70% 3% 7% n=300

2129-6-2 (nib) 3o" fraction 6* 55% 22% 7% 16% n=300

* Fraction 4 was composed primarily of large cells; fraction 6 was composed

primarily of small cells (cf. Figure 3).

$NA-induced Lethality

CELL SIZE

Figure 3. Distribution of Ceil Sizes in Fractions of a Sorbitoi Velocity Gradient

Strain 2129-6-2 (nib) was grown overnight in liquid medium at 30°C. The ceils were separated by size on a sorbitoi velocity gradient. The gradient was fractionated, and fractions 4 and 6 were analyzed for cell size in a Couiter Counter (model 2s) equipped with a Channeiyzer. Cell size increases to the right. Solid line: unfractionated population. Dashed line: fraction 4. Dotted line: fraction 6.

cells. Thus, in 36% of the nib large cells that mated, the damage to the cell was irreversible; in the other 64%, however, the defect was not.

Nine large-cell diploids and nine small-cell diploids were grown for at least 25 generations and then sporulated in liquid medium. The sporulated cultures were dissected in pairs: tetrads produced by isogenic large- and small-cell diploids were dissected within 2 hr of one another. The dissected tetrads were incu- bated at 30°C on solid medium for 2 days. In eight of the matched dissections, the colonies produced by tetrads derived from the large-cell diploid were distin- guishable from those derived from the small-cell dip- loid. Figure 4 shows a photograph of a pair of dissec- tions in which this distinction was particularly clear; the small-cell diploid produced tetrads in which all four spores gave rise to colonies of equal size, whereas the large-cell diploid produced tetrads in which two of the spores gave rise to colonies of normal size and two of the spores gave rise to very small colonies. Thus, although large- and small-cell diploids did not have detectably different cell or colony mor- phology, they maintained a difference contributed by the original large or small nib parent for over 25 generations.

The cells in the large colonies derived from spores of large-cell diploids were all small in size, whereas the cells in the small colonies were all large in size. The cells in the small colonies were similar in appear- ance to those produced by single isolated nib large cells. In order to determine their nib genotype, the spores from 23 tetrads derived from three independ- ent large-cell diploids were tested by complementa- tion (C. Holm, submitted). In every case, the two

Figure 4. Dissected Tetrads Derived from Dipioids Produced by Ceil-Ceil Matings

Strains 2129-6-1 (NIB) and 2129-6-4 (nib) were grown in liquid medium at 30%. A large nib cell and a small nib cell were each mated individually to a NIB cell. The resulting dipioids were grown for over 25 generations and then sporulated. For each diploid, the four spores in each of 13 tetrads were separated by micromanipulation and placed in rows on slabs of dissection agar. The agar slabs were then placed on YEPD medium, incubated at 3O’C for 2 days and photo- graphed. Left: tetrads derived from the large-ceil dipioid; right: tetrads derived from the small-ceil dipioid. The thick patch of growth on each slab is derived from the tetrads that were not micromanipulated. Colony morphology segregated 2:2 in each of the tetrads shown here. However, as the nibbled phenotype cannot be scored unambig- uously from a single colony without the aid of a microscope (see Figure 2). the 2:2 segregation pattern cannot be observed here.

spores that had given rise to large colonies were N/B, and the two spores that had given rise to small colo- nies were nib. Thus the unexpressed defect carried by large-cell diploids is revealed exclusively in their nib spores.

Either of two hypotheses is sufficient to explain these results. First, the nib large cells could carry a modification on chromosome XVI at or near nib. Be- cause of its close linkage to to nib, this recessive modification would be expressed only in the nib spores derived from large-cell diploids. The insertion of a 2~ DNA molecule at or near nib would be con- sistent with this hypothesis. A second hypothesis is that the large cell parents carry extrachromosomal elements detrimental only to nib cells. These elements would be replicated in the NIB/nib diploids, but their detrimental effect on cell division would be observed only in the nib spores. Changes in the conformation or copy number of 2~ DNA would be congruent with this second hypothesis.

Large Cells Contain an Elevated Copy Number of 2p DNA The two hypotheses discussed above were distin- guished by a direct examination of the DNA from

Cell 590

large-cell diploids and small-cell diploids. As in the genetic experiments, isogenic large- and small-cell diploids were examined in pairs. Equal amounts of DNA from strains 2140 (large-cell diploid) and 2141 (small-cell diploid); 2142 (small-cell diploid) and 2143 (large-cell diploid); and 2144 (small-cell diploid) and 2145 (large-cell diploid) were partially digested with Pst I. The samples were subjected to electrophoresis, transferred to a nitrocellulose filter and hybridized with a 32P-labeled 2~ DNA probe. An autoradiograph of the hybridized filter is shown in Figure 5. In each pair of strains, the large-cell diploid appeared to contain sub- stantially more 2~ DNA than the small-cell diploid did. To quantify this difference, densitometer tracings were made of an autoradiograph for which the expo- sure was in the linear range of the autoradiography film. Although all the diploid cells were the same size (data not shown), it was found that large-cell diploid 2140 had twice as much 2~ DNA relative to total DNA as did small-cell diploid 2141; large-cell diploid 2143 had four times as much 2~ DNA as small-cell diploid 2142; and large-cell diploid 2145 had four times as

Figure 5. Levels of 2~ DNA in Large-Cell Diploids and Small-Cell Diploids

DNA was isolated from large- and small-cell diploids. Of each sample, 0.4 pg was partially digested with restriction endonuclease Pst I, fractionated on a 0.4% agarose gel. transferred to nitrocellulose and hybridized to “P-labeled ~2~141, which contains 2p DNA as its only yeast DNA. DNA from a large-cell diploid and a small-cell diploid of the same genotype was run in adjacent lanes. (Lanes 1 and 2) Large- cell diploid 2140 and small-cell diploid 2141: (lanes 3 and 4) small- cell diploid 2142 and large-cell diploid 2143: (lanes 5 and 6) small- cell diploid 2144 and large-cell diploid 2145. Several bands were obtained in each lane because partially cut multimers of 2p DNA were present and because fully cut multimers produce three discrete bands of 7.0 kb. 6.3 kb and 5.7 kb.

much 2~ DNA as small-cell diploid 2144. Similar re- sults were obtained with uncut DNA isolated by the method of Casse et al. (1979) and with complete Eco RI digests of DNA from strains 2140 and 2141, and 2144 and 2145. Novel bands, which might have re- sulted from integrated copies of 2~ DNA, were not observed in the Eco RI autoradiographs. (The sensi- tivity of the autoradiographs was sufficient to detect the equivalent of a single fragment of 2~ DNA per haploid genome.)

Thus it appears that large-cell diploids contain two to four times more 2~ DNA than small-cell diploids do. Since the only difference between the large-cell dip- loid and the small-cell diploid in each pair of strains was the size of the nib parent cell, it appeared likely that nib large cells differed from nib small cells in containing an elevated number of copies of 2~ DNA. This hypothesis was verified by a direct examination of the DNA from large cells and small cells. Strain 2129-6-2 (nib) was grown at 30°C, and the cells were separated by size on sorbitol gradients (see Figure 3). DNA was isolated from fractions containing large cells and small cells, and equal amounts of DNA were subjected to electrophoresis, transferred to a nitro- cellulose filter and hybridized to a 32P-labeled 2~ DNA probe. Densitometry of the autoradiograph showed that the large cells had twice as much 2~ DNA relative to total DNA as the small cells did (data not shown). Thus it appears that large cells differ from small cells in containing an elevated amount of 2~ DNA.

Discussion

The nibbled phenotype exhibited by nib strains bear- ing 2p DNA results from the production of sectors of cells with a limited division potential at 30%. The mortal cells can be recognized by their abnormally large size and by the fact that a majority exhibit a morphology characteristic of cells arrested at a spe- cific stage in the cell cycle. At 38°C large cells often revert to a normal mode of division, and at the same temperature the morphology of nib colonies becomes normal. Large cells have a higher copy number of 2~ DNA than do small cells in the same population.

An important question that results from these ex- periments is that of causality: does a high copy num- ber of 2p DNA cause nib cells to become large, or do nib cells that are large simply have a high copy number of 2p DNA? The results of the cell-cell mating exper- iments provide a clear answer to this question. When large and small nib cells were mated to N/B cells, the surviving diploids produced colonies that were normal in morphology and cells that were normal in size. In the three pairs of diploids examined, the large-cell diploids had two to four times as much 2~ DNA per microgram of total DNA as the small-cell diploids did. This result showed that 2~ DNA copy number was not determined simply by cell size. Furthermore, in-

2p DNA-Induced Lethality 591

creased 21.1 DNA copy number was shown to have a causative role in nib cell enlargment when the large- cell diploids and small-cell diploids were sporulated. Since approximately 80% of the 2~ DNA in diploid cells is transmitted to their spores (Brewer and Fang- man, 19801, new spores derived from the large-cell diploids had two to four times as much 2~ DNA as those derived from the small-cell diploids. When the spores germinated, the nib spores from the large-cell diploids produced predominantly large cells, and the nib spores from the small-cell diploids produced pre- dominantly small cells. Since the nib spores were derived from diploid cells of identical size, it is clear that elevated 2~ DNA copy number is the cause rather than the effect of nib cell enlargement.

An elevated plasmid copy number causes a similar effect in Escherichia coli. Engberg et al. (1975) have isolated mutants of plasmid Rldrd with two to four times the normal copy number. Strains bearing these plasmids possess a subpopulation of abnormally large cells, which are similar in morphology to cells in which DNA replication has been disrupted. Engberg et al. suggest, therefore, that the excess plasmid DNA may titrate out essential replication products, and that the cells enlarge because DNA replication begins to lag behind cell growth. Uhlin et al. (1978, 1979) have isolated temperature-sensitive mutants that allow copy number to increase even more, and these mutant plasmids actually cause cell death at the restrictive temperature.

In a similar way, intermediate and high levels of 2~ DNA may cause disruption of DNA synthesis and cell death in nib strains of yeast. Many nib cells exhibit a morphology that is characteristic of cells in which DNA replication has been arrested: a single large bud with one nucleus positioned at the junction between the mother cell and the bud (Hartwell, 1971; Hartwell et al., 1973). If we assume for the moment that the copy number of 2~ DNA progressively increases in a subset of nib cells, the types of cells observed in nib strains can be explained as a function of 2~ DNA copy num- ber. First, cells with a low 2~ DNA copy number are relatively normal in morphology and division behavior; these are the small cells that are present in nib strains. Second, cells with a high 21~. DNA copy number are sufficiently disturbed that they are committed to cease dividing; these are the large cells that die even when grown at 38°C or when mated to NIB cells. Finally, cells with an intermediate copy number of 2~ DNA show an intermediate phenotype; they may be delayed in DNA replication, and thus continue to divide slowly. The cells in this group begin small, but disruption of DNA synthesis causes an imbalance between cell growth and cell division, which causes the cells to enlarge (Johnston et al., 1977). Thus this group is composed predominantly of the large cells that are rescued when grown at 38’C or when mated to NIB cells.

How does 2~ DNA copy number progressively in- crease in a subset of nib cells? The copy number of 2~ DNA is amplified in cells into which a single copy is introduced (Sigurdson et al., 1981), and amplifica- tion appears to be mediated by the products of the REP genes, which are carried by 2~ DNA (Broach, 1981). Amplification is clearly repressible, because each molecule of 2~ DNA is replicated only once per cell cycle in an established N/B culture (Zakian et al., 1979). Since 2~ DNA is overproduced in some nib cells, the product of the NIB gene is a good candidate for the repressor of 2~ DNA amplification. The function of the NIB allele may be to limit the copy number of 2~ DNA; for convenience let us assume that the level is held at 50 molecules per cell. If fewer than 50 mole- cules are present, the products of the REP genes amplify plasmid copy number; if more than 50 mole- cules are present, the product of the NIB gene pre- vents REP-mediated amplification. In the nib cell, the defective nib gene product has less than normal effi- ciency, occasionally allowing an upward fluctuation of 2~ DNA copy number. In a subset of nib cells, there- fore, 2~ DNA copy number increases due to a ratchet- like phenomenon. Occasional REP-mediated amplifi- cation nudges 2~ DNA copy number upward, and replication of every molecule in each cell cycle en- sures that the elevated copy number is maintained. Any given increase in 2~ DNA copy number may make further increases more likely, because it is accompa- nied by more REP genes that may escape from repres- sion. At moderate levels, increased 2~ DNA copy number might perturb DNA replication or nuclear di- vision, causing division to lag behind growth, and leading to enlargement of the cells (Johnston et al., 1977). Ultimately, however, 2~ DNA copy number may increase sufficiently to prevent replication or division and to cause cell death.

This model for the interaction between REP and NIB accounts for the tenacity of 2~ DNA in S. cerevisiae. Since 2~ DNA has been maintained for thousands of generations in laboratory strains, it has often been assumed that the plasmid must confer a selective advantage; no such advantage has been conclusively demonstrated, however (Broach, 1981). It is possible, with the model presented here, to adopt an alternative view. Once 2~ DNA is established in a strain, it may be quite difficult for cells to lose the plasmid. Random downward fluctuations in 2~ DNA copy number would be counteracted by the action of the REP gene prod- ucts; a slow upward drift of 2~ DNA copy number would be countered by the NIB gene product. With this view, one would expect an aberrantly high 2~ DNA copy number to be stable for many generations. This is exactly what occurs: nib large cells have an elevated copy number of 2~ DNA, and NIB/nib large- cell diploids maintain this trait for over 25 generations. This observation suggests that the copy number of 2~ DNA is not actively returned to its normal level once

Cell 592

it has increased. Gustafsson and Nordstrom (1980) have observed a similar phenomenon in E. coli cells with an unusually high plasmid copy number.

The effects of a high copy number of 2~ DNA can be ameliorated by growth at 38°C. This rescue of large nib cells at 38% can be explained in one of two ways. First, nib cells may simply be insensitive to 2~ DNA copy number at 38%; in this case, 2~ DNA copy number continues to increase in some cells, but at 38% they have a mechanism for counteracting its detrimental effects. Alternatively, growth at 38% might prevent amplification of 2~ DNA copy number. This could occur by inactivation of the REP gene products or by increased activity of the nib gene product. These two hypotheses are equally consistent with the data presented here. It would be possible to distinguish between them, however, by examining 2~ DNA copy number in nib cells grown at 38%.

Senescence is observed in cultures of Podospora anserina, and it has similarly been associated with the over-replication of small DNA molecules (Cummings et al., 1979; Jamet-Vierny et al., 1980). It is clear, however, that the mechanism of lethality in Podospora is quite different from that in nib strains of yeast. In Podospora it appears that a fragment of mitochondrial DNA is excised and then extensively replicated (Ja- met-Vierny et al., 1980). Lethality is caused by the loss of mitochondrial function, which is essential in this obligate aerobe (Jamet-Vierny et al., 1980).

Clonal senescence is also observed in higher eu- caryotic cells in culture, but the mechanism underlying this phenomenon is unclear (Hayflick and Moorhead, 1961; Cristofalo and Sharf, 1973; Martin et al., 1974; Vincent and Huang, 1976; Holliday et al., 1977). One proposed mechanism bears great resemblance to the model suggested here for 2~ DNA-induced lethality in yeast. Prothero and Gallant (1981) suggest the exist- ence of an autogenously regulated “commitment” molecule. As described, the commitment molecule is a protein, but plasmid DNA with the requisite proper- ties would also fit the model. Prothero and Gallant suggest that the concentration of the commitment molecule is initially low, but that it activates its own synthesis and causes its copy number to increase. The increased concentration leads to a further in- crease in copy number, and the amount spirals up- ward over many generations. Ultimately, the copy number reaches a threshold level, and the cells be- come “committed.” They undergo at most only a few divisions after they reach this point. The similarities between this model and the one I have proposed are obvious.

Experimental Procaduraa

Strains and Cell Growth The construction of the strains used in this study are described elsewhere (C. Helm, submitted); the strains are 2129-6-1 (a his2 met2 cyh2 NIB), 2129-6-2 (a his2 nib), 2129-6-3 (a lysl met2 cyh2

N/S) and 2129-6-4 (a lys 1 nib). The parents of diploid strains 2140 through 2143 are strains 2129-6-l and 2129-6-4; the parents of diploid strains 2144 and 2145 are strains 2129-6-2 and 2129-6-3. All of the strains in this study contain 2p DNA.

Cells were grown in YM-1 liquid medium (Hartwell. 1967) or on YEPD solid medium (Mortimer and Hawthorne, 1969) supplemented with 40 pg/ml adenine and 40 pg/ml uracil. Liquid cultures were growing logarithmically for all experiments. Colony morphology was evaluated after 3 days of growth on YEPD medium.

Examination of Isolated Cells Samples from liquid cultures were placed on dissection agar (1% yeast extract, 2% bactopeptone, 2% glucose, 10 wgg/ml uracil. 10 pg/ml adenine and 2.5% agar). and individual cells were removed to known positions by using a micromanipulator. The agar slabs were then placed on YEPD medium and incubated at 30” or 3S’C for 2 days. In the cell-cell mating experiments, mating was judged to be successful if a dumbbell-shaped zygote was formed within 1 X-2 hr.

Nuclear Staining Logarithmically growing cells were washed in 1% saline and then fixed in 3:l methanol to acetic acid for 30 min or more. They were then centrifuged and washed twice with 1% saline. The cells were stained for 45 min in 1 pg/ml DAPI (4’.6’ diamidino-2-phenylindole. 2HCI). washed in 1% saline and examined with a Leitz Ortholux II fluorescence microscope with front and back filters set at 1.

Sorbitol Gradients Cells were separated by size on linear 20 ml 40% to 60% sorbitol gradients. For each gradient, approximately 10’ to 10”’ cells from a logarithmically growing liquid culture were sonicated briefly, centri- fuged and then resuspended in 0.6 ml of 20% sorbitol. The gradients were centrifuged for 2% min at 3000 rpm in a Sorvall HB4 swinging bucket rotor.

Genetic Techniques Standard genetic techniques for Saccharomyces were used (Morti- mer and Hawthorne, 1969).

DNA Isolation Diploid DNA that was subsequently cut with restriction enzymes was isolated in cesium chloride gradients. Cells were grown at 30°C for seven to nine generations to approximately 5 x 10’ cells/ml in supplemented Y-minimal medium (Zakian et al., 1979) that was 1 @/ml in 5,6-3H-uracil (New England Nuclear). The cells were har- vested by centrifugation and washed twtce with distilled H20. Ap- proximately 5 x 10’ cells were resuspended in 10 ml pretreatment buffer (0.2 M Tris, 0.1 M EDTA. 1.2 M sorbitol [pli 9.11). P-Mercap- toethanol (0.8 ml) was added, and the cells were held at room temperature for 10 min. They were then centrifuged and resuspended in 5 ml SCE (pH 5.8; SCE is 1 M sorbitol, 0.1 M sodium citrate and 0.6 M EDTA). Glusulase (0.1 ml; Endo Laboratories) was added, and the cells were incubated at 37°C until spheroplasting was complete. (Spheroplasting was followed under the microscope by monitoring cell lysis when 10% Sarkosyl was added to the edge of a drop of the glusulase mixture.) The spheroplasts were centrifuged, washed twice with SCE (pH 5.8). lysed in 0.9 ml lysing buffer (9 mM Tris-HCI. 90 mM EDTA and 1% Sarkosyl [pH 81). put on ice for 15 min and then incubated at 65°C for 10 min. Solid cesium chloride (1.21 g) was added to each sample, and the samples were brought to 5.3 ml with a solution of 1.21 g/cc cesium chloride in 10 mM Tris-HCI 100 mM EDTA (pH 6). The lysates were centrifuged in a Beckman VTi65 rotor at 2O’C for 16 hr at 40,000 rpm. The gradients were collected, and the fractions were pooled and ethanol-precipitated as described in Zakian et al. (1979). The DNA was resuspended in 50 ~1 TE (10 mM Tris and 1 mM EDTA [pH 81).

Diploid DNA that was not cut with restriction enzymes was isolated by an adaptation of the method of Casse et al. (1979), which enriches for covalently closed circular DNA molecules, In the modification, cells were first washed and treated with 1% P-mercaptoethanol and

28 DNA-Induced Lethality 593

0.5 mg/ml Zymolyase 60,000 (Kirin Brewery) for 30 min and then they were lysed at pH 12.3.

DNA was prepared from separated large and small cells in micro- fuge tubes. Fractions representing large cells and small cells were pooled, centrifuged and then resuspended in 0.8 ml SCE (pH 7.0) with 0.8% 2-mercaptoethanol and 1 mg/ml Zymolyase 5000 (Kirin Brewery). The cells were incubated at 38°C until spheroplasting was complete. To lyse the cells, 0.4 ml of 0.05 M EDTA. 0.05 M Tris-HCI. 1% Sarkosyl (pH 8) was added. Diethylpyrocarbonate (2 pl) was added, and the mixture was incubated at 65°C for 30 min. Five molar potassium acetate (0.1 ml) was added to the cooled tubes, which were kept on ice for 1 hr. The debris was pelleted in a microfuge. and two 0.5 ml portions of supernatant were taken from each tube. Next, 50 gl of 5 M NaCl were added, and the solutions were phenol- extracted and ethanol-precipitated. The DNA was resuspended in 25 gl of TE.

Electrophoresis and Hybridization Electrophoresis was performed in an 0.4% horizontal agarose gel as described in Zakian et al. (1979), except that the ethidium bromide concentration was reduced to 1.25 X 10-2~g/ml. Phage X DNA cut with endonuclease Eco RI was used as marker DNA. After the gel was photographed, it was prepared for transfer of the DNA to nitro- cellulose filters, as follows. First, the gel was shaken gently in 250 ml of 0.25 M HCI for 10 min; the acid was discarded and this step was repeated. Second, the gel was shaken gently in 250 ml of 0.5 M NaOH and 1 .O M NaCl for 15 min; the base was discarded and this step was repeated. Finally, the gel was shaken in 250 ml of 0.5 M Tris. 3 M NaCl (pH 7.4) for 30 min. The DNA was then transferred to nitrocellulose filters by the method of Southern (1975). Hybridization to the nick-translated 2~ DNA probe was performed as described by Montgomery et al. (1978). A plasmid with l’h copies of 2~ DNA (Nelson and Fangman. 1979) or a plasmid with 2~ DNA inserted into pMB9 (~2~141; a gift from K. Nasmyth) was used as a probe,

Acknowledgments

I thank Leland Hartwell for many timely suggestions and for critically reading the manuscript. I also thank Susan Dutcher for our late-night discussion about the mechanism of 2p DNA-induced lethality and Nancy Gamble for typing the manuscript. I am especially grateful to Lou Holm for expert editorial consultation.

I was supported by a National Science Foundation predoctoral fellowship and by a National Institutes of Health Training Grant. This research was supported by an NIH research grant awarded to Leland Hartwell (GM1 7709).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received November 13, 1981; revised March 9. 1982

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