A Nuclear Gene of Saccharomyces cerevisiae Needed for Stable Maintenance of Plasmids
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MOLECULAR AND CELLULAR BIOLOGY, Nov. 1986, p. 4053-4059 Vol. 6, No. 11
0270-7306/86/114053-07$02.00/0
Copyright © 1986, American Society for Microbiology
A Nuclear Gene of Saccharomyces cerevisiae Needed for Stable
Maintenance of Plasmids
YOSHIKO KIKUCHI1t* AND AKIO TOH-E2
Laboratory of Molecular Genetics, Keio University, School of Medicine, Shinjuku-ku, Tokyo,l and Department of
Fermentation Technology, Hiroshima University, Saijo, Higashi-Hiroshima, Hiroshima,2 Japan
Received 18 April 1986/Accepted 8 August 1986
We have isolated host mutants of Saccharomyces cerevisiae in which the 2,um plasmid is poorly maintained.
AU the mutants tested constituted one complementation group, which was designated map] (maintenance of
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plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were also affected in
the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the
mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2,um
inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of
replication, probably at the initiation of replication.
The nuclear genome of Saccharomyces cerevisiae is orga- Our primary interest was to elucidate the replication
nized into 17 chromosomes, each of which replicates once at control in eucaryotic cells, and we chose the 2,um plasmid as
the S phase during each cell cycle. Replication control and a a model system to analyze genetically this complex process.
partitioning mechanism ensure the stability of the genomic Our first step was to isolate mutants defective in some stage
constitution. Many autonomously replicating sequences of chromosome duplication. In a previous study (39), we
(ARS) have been cloned (9, 21, 36, 37), and most of them are constructed pSLel, a derivative of the 2,um plasmid carrying
probably replication origins in each contiguous replicon. the LEU2 gene and ORI, which is maintained stably in the
Centromeric DNAs which confer stability on chromosomes wild-type host. In this report, we describe the isolation and
by donating the partition mechanism to them have also been characterization of chromosomal mutations (mapi) which
cloned (10, 17, 35). However, the mechanisms of DNA cannot maintain pSLel stably. We also identified several
replication and partition are largely unknown. In spite of the plasmids generated both in vivo and in vitro which overcame
genetic and biochemical utility of S. cerevisiae, dealing with or compensated for the loss of the MAP product. Analysis of
chromosomal DNA is still difficult. the structure of such plasmids suggested the function of the
The 2I,m plasmid of S. cerevisiae is a circular double- MAP gene in chromosomal replication. Similar mutants
stranded DNA containing 6,318 base pairs (bp) (15). It were isolated by Maine et al. (26) by using YCp plasmids as
carries a pair of inverted repeats (IR1 and IR2) consisting of selective markers.
599 bp which separate two unique sequences. Site-specific
recombination between the two IRs results in isomeric and MATERIALS AND METHODS
multimeric forms. Nucleotide sequence analysis reveals that Strains and plasmids. The S. cerevisiae and Escherichia
there are at least three open reading frames, Able (A), Baker coli strains used in this study are listed in Table 1. YAT274
(B), and Charlie (C) (15). The A protein is required for (leu2 karl MAP') was constructed by cytoducing pSLel
site-specific recombination and is called the FLP protein (1, from YAT234 to YAT226. pSLel is a self-annealed circular
6). The plasmid is maintained at a copy number of 50 to 100 DNA of the 3.2-kilobase (kb) HindIIl fragment from
as an extrachromosomal element in the nucleus (23, 33), and pJDB219 (2) which carries IR1, the origin, and the STB locus
plasmid DNA is organized into a nucleosomal structure (24, of the 2,um plasmid and LEU2. The Leu+ phenotype of
29, 41). Furthermore, replication control of this plasmid is YAT274 was quite stable: less than 1% of cells were Leu-
similar to that of chromosomal DNA (25): replication de- after seven generations under nonselective conditions. Con-
pends on some CDC genes required for chromosomal repli- struction of hybrid plasmids has been described previously
cation, and each plasmid duplicates itself once per cell cycle (20).
(44). Replication of the plasmid starts at the ORI site located Media. SD, YPD, and sporulation media were prepared
within 75 bp spanning a part of IR1 and the large unique and used for cultivation of S. cerevisiae (32). L-broth and M9
sequence contiguous to it (5, 6) and proceeds bidirectionally media for E. coli were prepared by the method of Miller (28).
(22). For stable maintenance of the plasmid, both B and C Appropriate amounts of amino acids, nucleic acid bases, or
proteins, in conjunction with the STB locus on the plasmid, antibiotics were added as necessary. Agar (2%) was added to
are required (19, 20). These factors are probably involved in prepare solid media. Yeast cells were grown at 30°C and
partitioning of plasmid molecules (20). When the plasmid bacteria at 37°C.
reduces its copy number, there seems to exist some mech- Enzymes. Restriction endonucleases, T4 DNA ligase,
anism for recovering the original copy number (33). DNA polymerase I, and DNase I were purchased from
Takara Shuzo Co. (Kyoto, Japan), Toyobo Biochem.
(Kyoto, Japan), Bethesda Research Laboratories (Bethesda,
* Corresponding author. Md.), or Boehringer Mannheim (Mannheim, West Ger-
t Present address: Laboratory of Molecular Biology, Toho Uni- many). Reaction conditions were those recommended by the
versity, School of Medicine, Omori-Nishi 5-21-16, Ohta-ku, Tokyo manufacturers. Zymolyase was from Kirin Brewery Co.
143, Japan. (Takasaki, Japan).
40534054 KIKUCHI AND TOH-E MOL. CELL. BIOL.
TABLE 1. List of strains electrophoresis in 40 mM Tris hydrochloride (pH 8)-5 mM
Strain Relevant genotype Reference or source sodium acetate-1 mM trisodium EDTA. The DNA frag-
ments were transferred to nitrocellulose membrane filters
S. cerevisiae (Schleicher and Schuell) by the procedure of Southern (34).
YAT274 a leu2 karl [cir+, pSLel] This study Plasmid X was nick-translated with [32P]dATP (Amersham;
TM68 YAT274 mapl-TM68 This study 3,000 Ci/mmol) by the method of Rigby et al. (30). Immobi-
TM83 YAT274 mapJ-TM83 This study lized DNA was prehybridized for 1 h at 42°C with 400 ,ug of
TM87 YAT274 mapl-TM87 This study
TM92 YAT274 mapl-TM92 This study denatured fragmented salmon testis DNA per ml in 50%
TM102 YAT274 mapl-TMI02 This study (vol/vol) deionized formamide-0.9 M NaCl-50 mM sodium
TM114 YAT274 mapl-TMIJ4 This study phosphate (pH 7)-5 mM trisodium EDTA-1% glycine-0.1%
YAT234 a leu2 his4 [cir+, pSLel] This study polyvinylpyrrolidone. It was then hybridized with [32P]DNA
YAT226 a leu2 cyh2 karl [cir+] This study probe at 42°C overnight in 50% formamide-0.9 M NaCl-50
YAT381 a leu2 his4 adel cyh2 This study mM sodium phosphate (pH 7)-5 mM trisodium EDTA-
[cir', pSLel] 0.02% bovine serum albumin-0.02% Ficoll-0.02% polyvinyl-
YAT374 a leu2 his4 karl [cir+, This study pyrrolidone-0.3% sodium dodecyl sulfate (SDS)-100 ,ug of
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pSLel] denatured fragmented salmon testis DNA per ml. Hybrid-
YAT243 a leu2 cyh2 pho3 [cir+] Lab stock
SA-9 a leu3 Lab stock ized bands were located by autoradiography.
917 a leul cdc5 ROCI his2 R. Wickner
trpi met4 rna3 gall RESULTS
YAT399 a leu2 [cir+, pJDB219] This study
YAT519 a leu2 his4 mapl-TM83 TM83 x YAT381 Isolation of map mutants. The scheme for the isolation of
YAT525 ot leu2 adel mapl-TM92 TM92 x YAT381 mutants defective in maintenance of pSLel is described in
YK9-2 a ura3 leu2 trpl his mapl- YAT519 x YH4-1A Materials and Methods. Ninety-seven leucine-requiring mu-
TM83 tants were isolated and then classified by complementation
YK9-4 ura3 leu2 YAT519 x YH4-1A tests with the standard leul, leu2, and leu3 tester strains.
YH4-1A a ura3 leu2 trpl his Lab stock Among those that were Leu-, 68 were leul mutants, 3 were
D13-IA a LEU+ trpl his3 [cir'] 36 leu3 mutants, and 21 did not complement the standard leu2
E. coli JA221 recA leuB trpE hsdR 2 strain. Four isolates did not complement more than one of
hsdM+ lacY the tester strains. The 21 mutants which did not complement
the leu2 strain could be explained in one of two ways: (i)
pSLel was cured from YAT274 during mutagenesis, or (ii) a
gene was mutated which was required for maintenance of
Mutagenesis with EMS. Conditions for ethyl methane- pSLel. In the second case, we predicted that the endoge-
sulfonate (EMS) and nystatin treatment were as described nous 2p,m plasmid of the mutants would be lost or the copy
(14). Briefly, YAT274 cells grown in 20 ml of SD medium number of it would be reduced, because pSLel replicates by
supplemented with histidine were washed with water and the same replication machinery as that of the 2,um plasmid.
suspended in 10 ml of 0.2 M potassium phosphate buffer, pH DNA was prepared from each Leu- strain which failed to
8.0. Then, 0.3 ml of EMS (Kodak) was added, and the complement the leu2 testers and analyzed by agarose gel
suspension was incubated at 25°C for 60 min without shak- electrophoresis. All the strains had lost pSLel, and 10
ing. After the cells were washed and suspended in 5 ml of strains gave very faint bands of 2,um on the gel, whereas 11
water, one drop of the suspension was distributed to small strains retained 2,um at a copy number comparable to that of
test tubes containing 1 ml of YPD. The tubes were kept at the wild type (data not shown). To examine whether the
25°C overnight without shaking. Each culture was subjected phenotype of these 10 mutants was due to mutations occur-
to the concentration procedure for leucine-requiring mutants ring in chromosomal genes, each of 6 mutants was crossed
with nystatin. Genetic analysis, dissection of asci, and
scoring genetic markers were carried out as described pre- TABLE 2. Segregation of Map phenotype
viously (32).
Strain Crossa Segregation
Transformation. Preparation of competent cells of E. coli phenotype (no.ofofLeu
asci)
and the procedure for E. coli transformation were described
(27). Yeast transformation was carried out with either W667 TM68 x YAT381 2+:2- (11)
W675 TM68 x YAT374 2+:2- (9)
protoplasts (16) or competent cells (18). W668 TM83 x YAT381 2+:2- (5)
Plasmid stability. Plasmid stability was checked qualita- W673 TM83 x YAT374 2+:2- (7)
tively and quantitatively as described previously (20). W669 TM87 x YAT381 2+:2- (6), 3+:1- (1),
Isolation and construction of plasmids. For detection of the 1+:3- (1)
2,um plasmid and its derivatives, a plasmid-enriched DNA W674 TM87 x YAT374 2+:2- (12)
fraction was prepared (8) from cells grown in 5 ml of YPD W670 TM92 x YAT381 2+:2- (4)
overnight and analyzed by 1% agarose gel electrophoresis. W671 TM102 x YAT381 2+:2- (2), 3+:1- (1)
Large-scale preparation of yeast plasmids was done as W672 TM114 x YAT381 2+:2- (2)
described previously (40). Plasmid DNA was extracted from W704 W667-3C x YAT399 2+:2- (6)
E. coli by the alkali lysis method (3) and used for construc- (pJDB219)
W678b TM83 x D13-1A 2+ :2-c (8)
tion and characterization of hybrid plasmids as described W681 W678-2B x YAT374 2+:2- (6), 3+:1- (1)
previously (20). Plasmids were purified by CsCl-ethidium W682 W678-3D x YAT374 2+:2- (7)
bromide centrifugation when necessary.
Southern hybridization. Total DNA was prepared by the
a
Ieu2
map mutants were crossed with leu2 MAP+ [LEU] strains, except as
noted. [LEU] denotes [cir', pSLell.
method of Cryer et al. (11), cleaved with appropriate restric- b Cross was
leu2 map x LEU+ MAP+ [cir+].
tion enzymes, and fractionated by 0.8 or 1% agarose gel c Map phenotype identified by amount of plasmid on agarose gels.VOL. 6, 1986 YEAST NUCLEAR GENE NEEDED FOR PLASMID MAINTENANCE 4055
with YAT374 or YAT381 (leu2 his4 adel)(pSLel). These TABLE 3. Complementation tests between map mutants:
mutants were derived from different subcultures in the EMS interpretation
mutagenesis, so that the mutants should be independent of Relevant genotypes of crossed strains Leu phenotype
each other. All the diploids were Leu+, indicating that the
mutations were recessive. The 2+:2- segregation of the Leu Sporesa Haploids X ¢ Y X = Y
phenotype in every ascus demonstrated that a single muta- a leu2 his4 adel mapX (pSLel) a leu2 mapY +
tion in a chromosomal gene conferred the mutant phenotype a leu2 HIS4+ adel mapX ot Ieu2 map Y +
on the cell (Table 2). The Leu- phenotype no longer showed (pSLel)
linkage with the his4 locus. When DNA was prepared from a Ieu2 his4 adel MAP' (pSLel) a leu2 mapY + +
each spore clone of three tetrads in the cross of W674 and a leu2 HIS4+ adel MAP+ a Ieu2 mapY + +
analyzed by agarose gel electrophoresis, two spore clones of (pSLel)
each tetrad had clear bands of pSLel as well as 2,um and its a Spores were derived from the heterozygous diploid strain: a/a leu2/leu2
derivatives, while the other two had lost pSLel and gave his4/+ adelladel mapXl+ pSLel.
very faint bands at the position of 2,um (Fig. 1). The 2+:2-
segregation of the plasmid pattern exactly corresponded to
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the segregation of the Leu phenotype: plasmid-positive grown on YPD plates with grids. Then the Leu phenotype
clones were always Leu+ and plasmid-negative clones were was tested for each isolate by replica plating onto SD. When
Leu-. Therefore, we concluded that the mutations occurred the spores were crossed with Map+ cells, essentially all the
in the chromosome. diploids showed the Leu+ phenotype: Map+/Map-
Complementation tests between mutants. Since mutant heterozygous diploids could stably maintain pSLel. This
strains lost pSLel and transformation of yeast cells with result also confirmed that every spore received pSLel during
pSLel DNA was not practical, we adopted the method meiosis. If a map mutation in a spore and a map mutation in
developed by Wickner (43) which had been used for the a haploid cell can complement each other, none of the
classification of the mak genes. Diploid strain TM68/ diploids should be Leu-. In contrast, half of diploid clones
wt(pSLel) or TM83/wt(pSLel) was placed on sporulation would be Leu- if these two map mutations are located in the
medium for 2 days. The sporulated culture was scraped off same gene. As shown in Tables 3 and 4, the appearance of
the plate and digested for 30 min at 30°C with zymolyase to Leu- diploids was consistently higher than expected for
remove the ascus wall. A portion of this digested culture was diploids of complemented pairs. The reason we found less
mixed for mating with the following haploid leu2 strains with than 50o Leu- in noncomplementing pairs is not clear, but
an appropriate marker for diploid selection: (i) a Map+ this was frequently observed in the complementation test of
strain, to check whether all the spores received pSLel; (ii) a the mak genes (43). Thus, we concluded that all the map
Map- strain carrying the same mutant map allele used for mutations tested so far occurred in the same gene, which
the construction of the map-lmap- diploid, to check was designated mapi (maintenance of plasmid).
whether diploids required the MAP gene for maintenance of Unstable maintenance of other minichromosomes. As de-
pSLel; and (iii) a Map- strain, to test whether these two scribed above, the phenotype of the mutants appeared to be
map mutations occurred in the same gene. Mating mixtures a reduction of copy number of the 2,um plasmid. Apparent
containing spores and haploid cells were spread on SD plates defects in host cells could not be detected, although the
containing leucine, and 96 or fewer colonies were picked and growth rate of some of the mutants, for example, TM83, was
slower than the parent. When DNA was prepared from
several clones after single-colony isolation and the 2,um
plasmid was detected by Southern analysis, the cultures of
W674- the mutants were found to be a mixture of cells containing
2,um and those without it (data not shown). Thus, the
>- lAlBIClD2A2B22D3A3B3C3D mutations may cause the plasmid to be unstable because of
3'5; w
ta7IT
:W
either low levels of replication of uneven partitioning of the
wpM IFW W Imn plasmid. To distinguish these cases, various hybrid plasmids
were introduced into the mutants and the stability of those
plasmids was tested. YEp13, a YEp vector containing the
origin of the 2p.m plasmid and the STB locus (7) was less
stable (13%) in TM83 [cir+] than in the wild type (wt) (57%)
pSLe4. (Table 5). The STB locus is a cis-acting site on 2,um which is
2A1 - required for its stable maintenance and is probably respon-
pSLel - TABLE 4. Complementation tests between map mutants: data
Leu-/total diploids
Haploid Spores from Spores from
TM68/wt TM83/wt
TM68 14/46, 11/26 12/%, 42/%
TM83 31/%
FIG. 1. 2+:2- segregation of the Map phenotype. DNA was TM87 25/96 9/16
prepared from each spore clone of three tetrads in the cross of W674 TM92 10/% 30/96
and electrophoresed on a 1% agarose gel. YAT374 served as a TM102 12/96
positive control. pSLe4 is a recombinant product between pSLel TM114 22/96 31/%
and the 2,um plasmid. wt 0/%, 2/% 0/%, 2/%4056 KIKUCHI AND TOH-E MOL. CELL. BIOL.
TABLE 5. Stability of minichromosomes in Map- and tion origin to compensate for the mutation. It should be
Map' hostsa noted that one IR sequence resided in an opposite orienta-
Stability (%) tion to the other IR in pMAI, like the original 2,um.
Plasmid TM83 [cir+J, YK9-4 [cir+J, Isolation of stable plasmnids in the map] mutant. From a
Map- Map' gene library, we searched for plasmids which could over-
come the mutation. Total S. cerevisiae DNA was partially
YEp13 (ORI, STB) 13 57 digested with the restriction enzyme Sau3A and cloned into
pYK2107 (ORI, CEN4) 10 72 the BamHI site of YEp13, which contained LEU2 as a
pYK2100 (ARSI, CEN4) 16 87 selective marker. About 2,000 Leu+ transformants of TM83
a
Plasmid stability was checked as described in Materials and Methods. The [cir+] were pooled and grown in nonselective medium for
structure of the plasmids pYK2107 and pYK2100 is shown in Fig. 2a. about 40 generations. Individual colonies of Leu+ survivors
were checked for plasmid stability, and from two transform-
ants which gave high stability, plasmids were recovered and
sible for the partitioning of the plasmid (20). Even though the characterized further.
plasmid carried a functional centromere sequence, another Plasmid harboring additional ARSs. The stability of
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cis-acting component for stability, the plasmid, pYK2107, pYK2162, carrying a 3.7-kb insert, was 63%, compared with
was unstable (Fig. 2a, Table 5); a circular minichromosome 13% for YEp13 in map- strain YK9-2 (Fig. 3a). When this
as well as 2,um was also affected in the mutant. Thus, inserted fragment was connected with the YCp vector, the
replication of extrachromosomal elements may be defective resulting plasmid pYK2171 was stabler (90%) than the par-
in the mutant, because their stability was independent of ent, YCp19 (31%). Subcloning experiments demonstrated
partitioning systems. Furthermore, ARSl, a chromosomal that a 1.4-kb HindIII-EcoRI fragment from pYK2162 was
replication origin, was also sensitive to the mutation, since responsible for the plasmid stability (pYK2173). When the
the plasmid pYK2100 carrying ARSI and CEN4 was unsta- replication origin of the vector was removed by deleting the
ble. KpnI fragment from pYK2162 to make YIp-type plasmid
Plasmids produced in vivo bypassing mqpl mutation. Dur- pYK2167 and the linear plasmid was integrated into the
ing tetrad analysis of Map+/Map- diploids, Leu+ papillae chromosome, it did not suppress the mutation: YEp24(4)
arose in the lawn of Map- clones. Tests were performed to was unstable in YK9-2(pYK2167). Thus, it acted only in cis.
determine whether the Leu+ papillae resulted from reversion The transformation efficiency of pYK2167 itself was high,
of the map) mutation or from a compensating alteration of suggesting that it carried ARS activity. And ARS was located
the plasmid DNA. W667-1A (1eu2 his4 mapJ-TM68) clones within the 1.4-kb EcoRI-HindIII fragment (pYK2176; see
containing Leu+ papillae were crossed with W667-1D (leu2 Fig. 4b), which was tentatively called ARSX.
adel mapJ-TM68) cells, and Leu+ diploid W683 was iso- Replication at ARSX was not insensitive to the mutation,
lated. When the sporulated diploid was dissected, four spore because pYK2176 carrying ARSX instead of ARS) of YCp19
clones were Leu+ among four asci tested. Ade and His
markers segregated 2+:2- in the same cross. Two spore
clones from W683, W683-1A and 1D, were characterized a
R STB R
further. When these two strains were grown overnight in
YPD, Leu- cells arose in the cultures: 13 Leu- per 96 clones ORI
of W683-IA and 2 Leu- out of 96 clones of W683-1D. This pMB9 H
mitotic instability of the Leu+ phenotype suggested that the 2 107 (B/Bg)
Leu+ phenotype was inherited by a Leu+ plasmid. How-
ever, no pSLel band could be detected by electrophoresis in CEN4
agarose gels in either strain. Instead, a plasmid DNA band S (B/Bg)
appeared at the position of 2,im (data not shown). To prove LEU 2
that W683-lA retained the map) mutation, Leu- clones from LEU 2
W683-1A obtained by curing the Leu+ plasmid were crossed b
with YAT374. The Leu phenotype segregated 2+ :2- in 11 x HR x
complete asci dissected, indicating that W683-1A retained ORI LEU2 H
the map) mutation. Therefore, a new plasmid carrying the B
LEU2 gene must arise in vivo, whose maintenance was no p pMAI 21 5 5
longer dependent on the MAP) gene. R R I
\ / ~LEU2
The plasmid remaining in W683-1A was named pMAI H H
(map independent) and its sequence was deduced by South- H
ern blotting experiments. For the blotting experiments, FIG. 2. Structure of various plasmids. (a) pYK2107 and
pMAI was purified from one clone which appeared to pYK2100. Plasmid pYK2100 was constructed by joining the follow-
contain a low amount of endogenous 2,um DNA. This clone ing DNA fragments: EcoRI-BamHI fragment of pYK2060 (20)
was chosen to reduce the signal of 2,um DNA in the blotting carrying ARSI; BgIII-BglII fragment of YCpl9 (35) containing
experiments, in which both 32P-labeled 2,um DNA and CEN4; BamHI-SalI fragment of YIp32 (4) harboring LEU2; and
pSLel DNA were used as probes. As shown in Fig. 2b, the SaII-EcoRI fragment of pMB9. Plasmid pYK2107 was constructed
size of pMAI was similar to that of 2,um, and the plasmid by replacing the EcoRI-HindIII fragment of pYK2100 (ARSI) with
must be derived from a recombination event between 2pRm the EcoRI-HindIII fragment of pYK2035 carrying the 2,um ORI (20).
and pSLel, followed by extensive structural rearrange- (b) Compensating plasmid and its derivative. Structure of pMAI (6.3
ments. When we cloned the PstI-HindIII fragment contain- kb) was deduced from restriction enzyme mapping and Southern
analysis as described in Materials and Methods. pYK2155: the
ing the replication origin of pMAI, the plasmid pYK2155 HindIII-PstI fragment containing ORI from pMAI was inserted
(Fig. 2b) was found to be unstable, like YEp13, in YAT525 between the HindIlI and Pstl sites of YIp32 (4). R, EcoRI; H,
[cir+]. Apparently, no change was observed in the replica- HindlIl; B, BamHI; X, XbaI, S, Sall; P, PstI; and Bg, BgIII.VOL. 6, 1986 YEAST NUCLEAR GENE NEEDED FOR PLASMID MAINTENANCE 4057
(Fig. 4b). was unstable in the mutant. These facts may
suggest that plasmid stability is affected by the number of
a
ARSs on its contiguous DNA chain. To check this possibil- TRPARS1
ity, we constructed the following plasmids: pYK2180, har-
boring three copies of ARSI; pYK2179, bearing two copies
of ARS1; and YCp19, carrying one copy of ARSI (Fig. 4a).
These had plasmid stabilities of 98, 91, and 31%, respec- Cp19 179
tively.
Plasmids containing a set of 2,m IR sequences. The other
type of plasmid which was stably maintained in the mutants
had a set of 2,um IR sequences. One pair of plasmids,
pYK2181 and pYK2161, were isolated from a transformant
(Fig. 3b). Southern analysis showed that the 2.6-kb insert of
b
pYK2181 was derived from 2p.m (data not shown). Struc-
tural analysis by restriction enzyme showed that it contained CEN4
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IR2 of the A type from 2p.m, and pYK2161 must be a
recombination product between IR2 in the insert and IR1 on Yp {2176 A
the vector YEp13, mediated by FLP protein from endoge- /URA3
nous 2p.m. When a DNA portion carrying IR2 was removed
by deleting the HindIII fragment of pYK2161, which gave FIG. 4. Plasmids carrying various numbers of copies of ARSI.
52% stability in TM83, the plasmid pYK2165 (9%o) was now (a) One or two copies of the 1.4-kb EcoRI fragment of YRp7 (36)
as unstable as YEp13. were inserted into the EcoRI site of YCp19 to make pYK2179 and
pYK2180, respectively. Arrows indicate one unit of the EcoRI
fragment and its direction. (b) The HindIII-EcoRI fragment contain-
DISCUSSION ing ARSI of YCp19 was replaced with the 1.4-kb EcoRI-HindIII
fragment of pYK2173 carrying ARSX to make pYK2176. H,
We have isolated mutants of S. cerevisiae in which pSLel, HindIII; R, EcoRI.
a derivative of the 2,um plasmid, was poorly maintained.
Other minichromosomes as well as 2t.m were mitotically
unstable in those strains, although the extent of stability for diploids, all the spores received pSLel, but mutant clones
various plasmids differed. For example, YCp5O (61%) was lost pSLel after the germination step so that the segregation
stabler than YCp19 (31%) in YK9-2: these two plasmids have pattern was 2+:2-. In contrast, the segregation pattern of
the same structural elements (ARSJ, CEN4, URA3, and YEp13 was 4+ :0- (unpublished result). Subsequently,
pBR322), but the arrangement on the plasmids is different. YEp13 was mitotically unstable in the mutant clones as
Nonetheless, plasmids carrying ARSJ or the 2,um origin and above. One copy of YEp13 should be enough for Leu+,
CEN4 or the STB system were always mitotically less stable while many copies of pSLel are required, because the LEU2
in the mutant than the wt. At meiosis of heterozygous gene on pSLel is defective and the expression of the gene is
about 5% of the wt (12).
All the mutants isolated by the present selection were in
a the same complementation group, mapi. Tye and her col-
laborators had isolated similar mutants in which YCp plas-
K ORI ARSI H N mids containing centromeres were poorly maintained and
classified them into 16 complementation groups (26). Among
LE jH211B those, YEp13 was rather unstable in one of the groups. That
mutation could be the same as map]. It is not clear why we
R isolated only one type of mutation. We used pSLel as a
selective marker plasmid instead of YCp plasmids. The copy
S number of pSLel should be much higher than YCp plasmids.
The mutant would be deficient in replication, because
b IR1 4 plasmids containing a functional centromere sequence as a
LEU partitioning apparatus were also unstable, although we can-
not rule out the possibility that the MAP] gene regulated
R both partitioning systems, CEN and STB of 2,um. The
(2181J 211)-H----- structural analysis of compensating plasmids supports the
notion, however, that the MAP] gene modulated replication,
R2 w. A\ 2165 probably at the level of the initiation of replication. As
described above, one of the compensating plasmids carried
FIG. 3. Stable plasmids in map mutants. (a) Plasmids carrying the replication origin, ARSX, in addition to ORI of 2pum.
ARSX. Plasmid pYK2162 has a DNA insert at the BamHI site of Moreover, plasmids containing more than one copy of ARSI
YEp13 (7). The BamHI-SalI fragment of pYK2162 was inserted appeared to be stabler than the plasmid harboring only one
between the BamHI and SalI sites of YCp19 (35) to produce copy. ARSI on the plasmid is known to be responsible for
pYK2171. Plasmids pYK2172, pYK2174, and pYK2173 carry initiating duplication once every cell cycle (13). In the
subfragments of the insert. (b) Plasmids containing a set of IR mutant every ARSI would not function well, so that the copy
sequences. Plasmid pYK2181 has a 2.6-kb insert at the BamHI site number of the plasmid containing only one ARSI would
of YEp13. Plasmid pYK2161 is an inverted type mediated by the
FLP system. Plasmid pYK2165 was constructed by deleting the gradually decrease and it would finally be lost. On the other
HindIII fragment containing IR2 in pYK2161. H, HindIII; B, hand, those having more replication origins should have
BamHI; S, Sall; R, EcoRI; X, XbaI; K, KpnI. more chances to initiate replication under this circumstance.4058 KIKUCHI AND TOH-E MOL. CELL. BIOL.
Thus chromosomal DNA replication would not be sensitive 11. Cryer, D. R., R. Eccleshall, and J. Marmur. 1975. Isolation of
to this mutation, because multiple replication origins reside the yeast DNA. Methods Cell Biol. 12:39-44.
in one polynucleotide chain. Probably the replication does 12. Erhart, E., and C. P. Hollenberg. 1983. The presence of a
not have to start at every origin in every cell cycle. For
defective LEU2 gene on 2pL DNA recombinant plasmids of
Saccharomyces cerevisiae is responsible for curing and high
example, there must be a lot of potential origins in rDNA copy number. J. Bacteriol. 156:625-635.
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ment (38). One out of several units actually starts replication 14. Fink, G. R. 1970. The biochemical genetics of yeast. Methods
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are introduced into the cells, they occasionally form the yeast plasmid. Nature (London) 286:860-864.
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vivo and isolated from a gene library, carried two IR 18. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transfor-
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one of the duplicated IRls is recombined with unreplicated Saccharomyces cerevisiae plasmid 2,um circle. Mol. Cell. Biol.
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ACKNOWLEDGMENTS plasmid DNA replication in vitro: origin and direction. Proc.
Natl. Acad. Sci. USA 78:7261-7265.
Y.K. would like to thank T. Fukasawa of Keio University, in 23. Livingston, D. M. 1977. Inheritance of the 2,um DNA plasmid
whose laboratory a part of this work was done, for his encourage- from Saccharomyces. Genetics 86:73-84.
ment and generosity, H. Shimatake of Toho University for use of 24. Livingston, ID. M., and S. Hahne. 1979. Isolation of a condensed,
the facility to prepare the manuscript, and Akihiko Kikuchi for intracellular form of the 2-p.m DNA plasmid of Saccharomyces
critical reading of the manuscript. cerevisiae. Proc. Natl. Acad. Sci. USA 76:3727-3731.
25. Livingston, D. M., and D. M. Kupfer. 1977. Control of Saccha-
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