JOURNAL OF BACTRIOLOGY, Jan. 1981, p. 382-3900021-9193/81/010382-09$02.00/0 Vol. 145, No. 1

Isolation and Characterization of Linear DeoxyribonucleicAcid Plasmids from Kluyveromyces lactis and the Plasmid-

Associated Killer CharacterNORIO GUNGE,l* ATSUKO TAMARU,' FUMIKO OZAWA,' AND KENJI SAKAGUCHI

Central Research Laboratories, Mitsubishi Chemical Industries, Midori-ku, Yokohama 227,' andMitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya Machida-shi, Tokyo 194,2 Japan

Two linear deoxyribonucleic acid plasmids, designated pGKll and pGK12, wereisolated from the yeast Kluyveromyces lactis IFO 1267. pGKll and pGK12 hadmolecular weights of 5.4 x 106 and 8.4 x 106, respectively. Both plasmids possessedthe same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map ofpGKll was constructed from digestions by EcoRI, HindIII, PstI, and BamHI.pGK12 was cleaved by EcoRI into seven fragments and by BamHI into twofragments. K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killerstrains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromycesthermotolerans, and K. vanudenii. All K. lactis strains lacking the pGKl plasmidswere nonkillers. A hybrid was constructed between K. lactis IFO 1267 and anonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysisafter sporulation. The killer character was extrachromosomally transmitted in alltetrads in association with the pGKI plasmids. The double-stranded ribonucleicacid killer plasmid could not be detected in any K. lactis killer strains. It is thushighly probable that the killer character is mediated by the linear deoxyribonu-cleic acid plasmids. A single chromosomal gene was found which was responsiblefor the resistance to the K. lactis killer.

The presence of a 2-,um circular DNA plasmidin the yeast Saccharomyces cerevisiae has beenreported previously (4, 11, 15, 27). Attempts toisolate 2-,um DNA from nuclei or mitochondriawere unsuccessful (2, 5). Recently, however, theassociation of 2-rum DNA with folded chromo-somes has been reported (32). The replication ormaintenance is under the control of nucleargenes, whereas the mode of inheritance is non-Mendelian (18, 19). The 2-,um DNA was joinedwith bacterial plasmids, and the resulting hybridDNA molecules were used as shuttle vectors forgenetic manipulation in yeast and Escherichiacoli systems (1, 6, 9, 30). The existence of 2-tumor 2-Am-like DNA in Saccharomyces italicus(16) and Schizosaccharomyces pombe (6) hasalso been demonstrated; however, no otherDNAplasmids have been detected in yeasts. We havesearched for plasmids among 70 strains of yeastsbelonging to 17 different genera. As a result, astrain of the petite negative yeast Kluyvero-myces lactis was found to harbor two novellinear DNA plasmids, designated pGKll andpGK12. The K. lactis strain with the pGKl plas-mids expressed a killer phenotype toward all ofthe Saccharomyces cerevisiae strains tested andcertain strains of S. italicus and Kluyveromycesspp. The present paper describes the isolation

and characterization of the linear DNA plasmidsfrom yeasts and the possible control of the killercharacter by the plasmids.

MATERIALS AND METHODSStrains used for the plasmid detection. The

following species (and strains) were used: Saccharo-myces cerevisiae (G706, 1035-186, 0708-11-16A, F38-4A, B060 AF-1, 3026-1, 3027-1, KL-88, 55R5-3C/1, andH-1), Saccharomyces italicus (IFO 0253, IFO 0725,and IFO 1049), Saccharomyces rouxii (NISL-A31),Schizosaccharomycespombe (IFO 0346, M210, M216,and SG55), Saccharomycodes ludwigii (M10, M15,M18, M35, and M38), Candida krusei (IFO 0839 andIFO 1395) Candida lipolytica (NRRL-Y-6795), Can-dida solani (MCI 0632), Candida tropicalis (IFO1400), Torulopsis famata (RIFY-7455), Torulopsiscandida (IFO 0664), Citeromyces matritensis (IFO0954), Pachysolen tannophilus (IFO 1007), Debary-omyces hansenii (IFO 0855), Metschunikowia pul-cherima (IFO 1407 and IFO 1405), Metschunikowiabicuspidata (MCI 1027), Pichia membranefaciens(IFO 0864), Rhodosporidium toruloides (IFO 8766),Leucosporidium scotti (IFO 1212 and MCI 1026),Kluyveromyces lactis (IFO 0433, IFO 0648, IFO 1090,IFO 1267, IFO 1903, WM37, W600B, L3a, K43, L4, K5,and K51), Kluyveromyces fragilis (ATCC 10022),Kluyveromyces thermotolerans (IFO 0662, IFO 1050,IFO 1674, IFO 1778, IFO 1779, and IFO 1780), Kluy-veromyces drosophilarum (IFO 1012), Kluyveromyces

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LINEAR DNA PLASMIDS IN KILLER YEAST 383

marxianus (IFO 0219), Kluyveromyces africanus (IFO1671), Kluyveromyces phaffi (IFO 1672), Kluyvero-myces vanudenii (IFO 1673), Kluyveromyces wicker-hamii (IFO 1675), Botryoaswcu synaedendrus (MCI1025), Sporidiobolusjohnsonii (MCI 1030), Endomy-copsis fibuligera (MCI 2200), Hansenula anomala(MCI 2321), and Hansenula capsulkta (MCI 2325).

Detection and preparation of plasmid DNA.Plasmid DNA was detected by a modification of theprocedure of Hirt (14). Cells of yeast grown in 40 ml ofYEPD medium (2% peptone, 1% yeast extract, 2%glucose) were converted to protoplasts with Zymolyase60,000 as described previously (12). The protoplastswere washed and suspended in 4 ml of 0.1 M citrate-phosphate buffer (pH 6) containing 0.8M sorbitol and0.01 M EDTA and then lysed by adding 0.5 ml of 10%sodium dodecyl sulfate solution. After the addition of5 M NaCl at a final concentration of 1 M, the mixturewas kept on ice ovemight and subjected to centrifu-gation at 17,000 rpm for 30 min. To the supernatantwere added two volumes of cold (-20°C) effianol andone-tenth volume of 3 M sodium acetate. The mixturewas incubated at -20°C for 2 h. The precipitate wascollected by centrifugation at 17,000 rpm for 20 minand dissolved in 100 pl of TES buffer (30 mM Tris-hydrochloride [pH 8.0], 50 mM NaCl, 5 mM EDTA)containing 0.4% Sarkosyl. RNase A (Sigma ChemicalCo.; preheated for 5 min at 100°C) was added at a finalconcentration of 50 pg/ml, and the mixture was incu-bated for 30 min at 37°C. A portion of the DNAsample thus obtained was subjected to agarose gelelectrophoresis. Plasmid DNA bands were visualizedunder UV (365-nm) light.

Plasmid DNA was separated from gels by the reezeand squeeze method described by Tanaka and Weis-blum (33), and the recovered DNA was purified byremoving ethidium bromide with butanol treatmentand dialyzing against 0.1x SSC (0.15 M NaCl, 0.015 Msodium citrate) containing 1 mM EDTA.

Detection of double-stranded RNA (daRNA)plasmids. Nucleic acid fraction was collected fromthe sodium dodecyl sulfate lysate of yeast protoplastsby adding two volumes of cold ethanol as described indetection of plasmid DNA. A portion of the samplewas digested with DNase (20,ug/ml) and subjected to0.7% agarose. gel electrophoresis for the detection ofRNA plasmids. Cytoplasmic polyhedrosis virus RNA,which consists of 10 dsRNA segments of known mo-lecular weights (8), was used for molecular weightmarkers.

Digestion with restriction endonucleases.EcoRI, HindJII, BamHI, and PstI were purchasedfrom Boehringer Co., and HpaI was purchased fromBethesda Research Laboratories, Inc. Digestions werecarried out for 1 h at 37°C in the following buffers:EcoRI buffer (10 mM MgC12, 50 mM NaCl, 100 mMTris-hydrochloride at pH 7.5), HindrII buffer (7 mMMgCl2, 60 mM NaCl, 7 mM Tris-hydrochloride at pH7.4), BamHI buffer (10 mM MgC12, 100 mM T-is-hydrochloride at pH 7.5), PstI buffer (10 mM MgSO4,90 mM Tria-hydrochloride at pH 7.5), and HpaI buffer(20 mM KCI, 10 mM MgC12, 1 mM dithiothreitol, 100pg of bovine serum albumin per ml, 10 mM Tris-hydrochloride at pH 7.4). Double digestions were donewith EcoRI plus HindIII in HindIII buffer, with

EcoRI plus PstI in EcoRI buffer, with HindIII plusPstI in Hindli buffer, and with PstI plus BamHI inBamHI buffer. Double digestions with EcoRI plusBamHI and with BamHI plus HindIII were first donewith BamHI in BamHI buffer and then redigestedafter adjusting the NaCl concentration to 50mM withEcoRI and HindIII, respectively. The reactions wereterminated by the addition of BJ solution (60% su-crose, 0.005% bromophenol blue, 0.08 M Tris, 0.04 Msodium acetate, 0.002 M sodium EDTA, and 0.5 pug ofethidium bromide per ml at pH 7.9) and heated for 5min at 650C before agarose gel electrophoreois. Fordetermination of molecular weights of digested DNA,HindlI fragments of X DNA and Hindc fragments of*X174 DNA were used as molecular weight m4rkers.

Test of killer phenotypes. The killer phenotypewas asayed by the methylene blue agar techniquedescribed by Somers and Bevan (29). Cells of testerstrains grown in YEPD medium were suspended insterile water, 0.2 ml of the suspension (107 cells perml) was spread on an assay medium (1% yeast extract,2% peptone, 2% glucose, 3 mg of methylene blue per100 ml, and 2.5 to 3.5% agar) adjusted to pH 4.5, 5.0,6.0, and 7.0 with 0.05 M citrate-phosphate buffer. Forexamination of the Illing activity, cells of variousyeast strains grown on YEPD agar were streaked ontothe above assay medium on which cells of a testerstrain were spread. After 2 to 3 days of incubation at250C, a clear killing zone was observed around thestreaks of killer yeasts. The following S. cerevisiaestrains were used as tester strains: killer strains B060(a haploid), F38-4A (a haploid), and B511-4C (a/adiploid) and sensitive strains G102D (a haploid) andM1-7C (a/a diploid).

Genetic analysis. Tetrad analysis was conductedwith the aid of a de Fonbrune micromanipulator bythe procedure of Johnston and Mortimer (17).

RESULTS

Detection ofplasmid DNA. Sodium dodecylsulfate lysates of protoplasts from various yeastswere examined for the existence of plasmids byagarose gel electrophoresis. In investigating 70strains ofyeasts belonging to 33 different speciesof 17 genera listed above, 2-tum DNA was foundin all of 10 S. cerevisiae strains, except for straKL-88, and in two S. italicus strains (IFO 0253and IFO 1049); two novel-type plasmids werefound in K. lactis IFO 1267. No DNA plasmidswere detected among the remaining yeasts.

Figure 1 shows the result of electrophoreticanalysis of plasmid samples from K. lactis IFO1267 (lane A), S. cerevisiae 0708-11-16A (lane B)and S. italicus IFO 0253 (lane C). In lane A, twodiscrete plasmid bands (pGKl1 and pGK12) withdifferent mobilities are revealed, in addition tothe top band DNAs corresponding to nuclearand mitochondrial DNAs. All of these bandsdisappeared when treated with DNase. Theplasmid and top band fractions from K. lactisIFO 1267 were separately isolated from a num-

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384 GUNGE ET AL.

A B C

pGKI-2-b-

pGKI-1 -

-2o-2pDNA(OC)

-2 pm DNA(CC)

FIG. 1. Agarose gel electrophoresis of DNA ex-tracts. Ekectrophoresis is from top to bottom. Photo-graph shows DNA extracts from K lactis IFO 1267(lane A), S. cerevisiae 0708-11-16A (lane B) and S.italicus IFO 0253 (lane C). The top band ofeach lanecorresponds to nuclear and mitochondrial DNAs.The 2-m DNA bands represent the open circular(OC) and closed circular (CC) forms. The bands belowthe 2-aum DNA (OC) on the B and C lanes are 2-pmDNA dimers (CC), and the faint bands at thepositionofpGK12 are the OC form of2-un DNA dimers (25).

ber of gels by the freeze and squeeze technique(33) for further study.Structure and size of pGKll and pGK12

plasmids. In electron microscope analysis (Fig.2), the pGKll and pGK12 plasmid fractions pur-ified from gels were found to consist of homo-geneous populations of linear DNA of 2.6 to 2.7,um (molecular weight, 5.4 x 106) and 4.1 to 4.2,um (molecular weight, 8.4 x 106) in length, re-spectively. No circular DNA molecules were de-tected, except for the marker pBR322. Becauseof the linear structure of the DNA molecules,the estimation of molecular weights based onthe mobility by electrophoresis was conductedby comparison with linear fragments fromHndIII digestion of A DNA. As a result, thepGKl1 and pGK12 DNAs were shown to havemolecular weights of 5.3 x 106 to 5.4 x 106 and8.3 x 106 to 8.4 x 106, respectively. The valuesare in agreement with the sizes estimated byelectron microscope, evidence ofthe linear struc-tures of the pGKI plasmids.

J. BACTERIOL.

Digestion with restriction endonuclea-ses. The pGKll and pGK12 plasmids weretreated with EcoRI, HindHI, BamHI, PstI, andHpaI, and the sizes of the resulting fragmentswere determined by coelectrophoresis withHindu digests of A DNA and HincH digests of46X174 DNA. The results of single and doubledigestions of pGKll (Table 1) led to the con-struction ofrestriction map (Fig. 3). No digestionwas observed with HpaI. pGK12 was digested byEcoRI into seven fragments (molecular weights,2.5 x 106, 2.3 x 106, 0.95 x 10", 0.85 x 10¶, 0.7 x106, 0.55 x 106, and 0.5 x 106) and digested byBamHI into two fragments (molecular weights,4.8 x 106 and 3.6 x 106). Double digestion byEcoRI and BamHI indicated that the EcoRIfragment (molecular weight, 0.85 x 10') con-tained the single BamHI site and was digestedby BamHI into fragments with molecularweights of 0.65 x 10" and 0.2 x 106. H dII,PstI, and HpaI did not cleave pGK12; therefore,the physical map of pGK12 was difficult to con-struct. The existence of several definite cleavagesites for restriction enzymes ruled out the pos-sibility that the linear DNAs may have comefrom random fragmentations of mitochondrialor chromosomal DNAs during DNA isolation.The top band DNAs, mainly consisting of chro-mosomal DNA, gave diffuse fragments whendigested by EcoRI or HindmI.Buoyant density. Buoyant densities of the

plasmid DNAs were determined in neutral CsClsolution at 40,000 rpm for 42 h at 16°C in aHitachi analytical centrifuge model 282 by usinginternal density markers of phage SPOl DNA(p = 1.742 g/cm3) and E. coli DNA (p = 1.710 g/cm3). Both pGKl1 and pGK12 plasmids showedan identical density of 1.687 g/cm3. The DNAsisolated from the top band fraction of gels were

g -

FIG. 2. Electron micrograph ofpGKl1 andpGK12plasmids: a, pGKl1; b, pGK12; c, open circular formofpBR322. The bar represents 1 um.

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VOL. 145, 1981 LINEAR DNA PLASMIDS IN KILLER YEAST 385

TABLE 1. Molecular weights of restriction fragments ofplasmidpGK1:Mol wt (106) of restriction fragments of the following enzymes:

Frag- EcoRI EcoRI HindHIment.- EcoRI HindIl BamHI PstI Plus Plus EcoRI lu HindflI Pstl plus

HindI BamHI plus PstI BarmHI plus PstI BamHIA 3.0 3.4 2.7 3.9 1.8 2.7 2.5 2.1 2.9 2.2

2.7

B 2.4 1.5 _b 1.0 1.6 2.4 1.4 1.4 1.0 1.7

C - 0.5 - 0.5 1.5 0.3 1.0 1.3 0.5 1.00.50.5

D - - - - 0.5 - 0.5 0.5 - 0.5

Total 5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.3 5.4 5.4

aFragments were designated A to D in order of increasing molecular weight.b_- No fragment.

co =

III I I I 1

0.5 0.5 1.4 0.3

I?

I 61.2 1.0 0.5 x 1 0 d.

FIG. 3. Restriction map of the pGKlI plasmid. The map was constructed on the basis of the data ofTable 1.

resolved into two peaks: a main peak of a densityof 1.699 g/cm3 due to nuclear DNA (20, 21, 28)and a faint peak of a density of 1.692 g/cm3 dueto mitochondrial DNA (20, 21, 28). Thus, thedensities of the plasmids are lower than those ofthe mitochondrial and nuclear DNAs, in con-trast to the density of the 2-um DNA of S.cerevisiae, which is the same as the density ofnuclear DNA (p = 1.700 g/cm3) and is higherthan that of mitochondrial DNA (p = 1.683 g/cm3).

Killing activity ofK lactis EFO 1267. Kill-ing activity of yeasts was examined as describedabove. S. cerevisiae killer strains B060, F38-4A,and B511-4C killed S. cerevisiae sensitive strainsG102D and M1-7C at a suitable pH (pH 5)(Table 2). The killer strains did not kill eachother, indicating that they were immune to toxicsubstances produced by themselves. The 12Kluyveromyces lactis strains listed above weretested for killing activity against S. cerevisiaestrains and among themselves. As a result, K.lactis IFO 1267 carrying the pGKll and pGK12plasmids was found to kill the S. cerevisiaesensitive and killer strains and K. lactis strainsIFO 1903, IFO 0433, L3a, W600B, L4, and L5. K.lactis strains K43, WM37, K51, IFO 0648, and

IFO 1090 were not killed. The 11 K. lactis strainslacking the pGKI plasmids did not kill any of thestrains of S. cerevisiae and K. lactis mentionedabove. All K. lactis strains were resistant to theS. cerevisiae killers.To further characterize the killer phenotypes

of K. lactis IFO 1267, the killing tests wereextended to strains of other yeasts: Saccharo-myces italicus, several Kluyveromyces species,and Schizosaccharomycespombe (Table 2). TheS. cerevisiae killers killed all of the S. italicusstrains tested, whereas K. lactis strain IFO 1267weakly killed S. italicus strains IFO 0253 andIFO 1049, but did not kill S. italicus IFO 0725.On the other hand, K. lactis IFO 1267 killed K.thermnotolerans IFO 0622 and IC. vanudenii IFO1673, although the S. cerevisiae killers and the11 K. lactis strains lacking the pGKl plasmidsdid not kill them. Schizosaccharomyces pombestrains were not killed by either group of yeasts.The effect of pH on the killing was tested by

using S. cerevisiae strains as testers. S. cerevi-siae killers were active in the range of pH 4.5 to5, but not at pH 6, in agreement with the pre-vious reports (23, 29) (Table 3). In contrast, thekilling of K. lactis IFO 1267 was expressed in therange of pH 5 to 7, but not at pH 4.5. Thus, the

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386 GUNGE ET AL.I

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LINEAR DNA PLASMIDS IN KILLER YEAST

TABLE 3. Killing action at various pH'sKillig of S. cerevisiae strains":

Streaked G102D B5114Cstrain

4.5 5 6 7 4.5 5 6 7

S. cerevisiaeG102D - - - - - - -

B060 + + -- -

B511-45C + + - - - - - -K. lactisW600B - - - - - - - NTIFO 1267 - + + + - + + NTTetrad8A - + + + - + + NTTetrad8B - + + + - + + NTTetrad8C - + + + - + + NTTetrad8D - + + + - + + NT

NT, Not tested; +, killing,-, nonkilling; ±, weak Ikilling.b pH value.

killer phenotype of K. lactis IFO 1267 was dis-tinguishable from that of S. cerevisiae with re-gard to the killing spectrum against variousyeasts and the pH range of killing.

Cells of K. lactis IFO 1267 were grown inYEPD medium which contained 20% glycerol tostabilize the toxin (22) and centrifuged. The cell-free solution showed the killing activity, but didnot when treated with papain.AssOciation of the killer character with

the pGKI plasmids. To understand the geneticrelationship of the killer character with thepGKl plsmids, K. lactis IFO 1267 (mating typea prototroph) was crossed with K. lactis W600B(a adel ade2 leu), one of the nonkiller K. lactisstrains lacking the pGKl plasmids, and the re-sulting hybrid was subjected to tetrad analysisafter sporulation. Out of 17 tetrads dissected, allgave 2:2 segregation for mating types and leumarker and irregular (2:2, 1:3, and 0:4) segrega-tion for ade marker, which was due to the in-dependent segregation of adel and ade2 muta-tions involved in the cross. In contrast, the killerphenotype of K. lactis IFO 1267 was segregatedin a 4:0 ratio (Table 4): all tetrads from the crosskilled S. cerevisiae sensitive and killer strains,the six nonkiller K. lactis strains (IFO 1093, IFO0433, L3a, W600B, L4, and L5), S. italicus IFO0253 and IFO 1049, K. therrnotolerans IFO 0622,and K. vanudenii IFO 1673, as did K. lactis IFO1267 (Table 2, tetrads 8A to 8D). Again, killingwas expressed in the range of pH 5 to 7, but notat pH 4.5, against S. cerevisiae strains (Table 3,tetrads 8A to 8D). From these 17 tetrads, 7 wereselected at random and examined for the exist-ence ofplasmids. All four spores from the tetradscontained both pGKll and pGK12 plasmids (Ta-ble 4). Thus, both the pGKll and pGK12 plas-mids exhibited an extrachromosomal inherit-ance and were associated with the killer char-

TABLE 4. Tetrad analysis of a cross between K.lactis IFO 1267 (a) and K. lactis W600B (a adel

ade2 ku)-Kill

Stantetrad MT ade leu ing pGIl1 pGKl2activ-ity

K. lactis IFO 1267 a + + - - -

K. lactis W600B

Tetrad1A1B1C1D

3A3B3C3D

5A5B5C5D

8A8B8C8D

14A14B14C14D

a

aaaa

_ +

+ + ++ + +

_ - +

a - - +a - + +a + + +a - - +

a - - +a + + +a - - +a - + +

+ ++ ++ ++ +

+ ++ ++ ++ +

+ ++ ++ ++ +

a + - + + +a + + + + +a - + + + +a _ - + + +

a - + + + +a - + + + +a - - + + +a + _ + + +

16A16B16C16D

18A18B18C18D

a - - +a + + +a - - +a + + +

a - - +a + - +a - + +a - + +

+ ++ ++ ++ +

+ ++ ++ ++ +

a Out of 17 tetrads dissected, 7 were selected for thedetection of plasmids and the results listed above. Therema.iing 10 tetrads gave 2:2 segregation for matingtypes (MT) and leu marker and irregular segregation(2:2 in 5, 1:3 in 4 and 0:4 in 1) for ade marker. Killingactivity segregated at a ratio of 4:0.

acter during meiosis.Examination of dsRNA plasmids. RNA

fractions prepared from protoplast lysate weresubjected to agarose gel electrophoresis. Asshown in Fig. 4 (lane L), two discrete RNAbands were detectd from a killer strain of S.cerevisiae: the molecular weights of the upper

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A B C D E F G H I J K l

FIG. 4. Electrophoresis of DNase-treated samplesof nucleic acid extract from protoplast lysates. A,cytoplasmicpolyhedrosis virus RNA: B, K. lactis IFO1267; C, K lactis W600B; D to G, tetrads 3A to 3D,respectively; H to K, tetrads 8A to 8D, respectively; L,S. cerevisiae killer strain F38-4A. Arrows 1 and 2 inlane L correspond to dsRNA's of molecular weights2.7 x 106 and 1.3 x 106, respectively. Cytoplasmicpolyhedrosis virus RNA consists of 10 segments hav-ing the following molecular weights (x 106): 2.55,2.42,2.32, 2.03, 1.82, 1.12, 0.84, 0.62, 0.56 and 0.35 (8).

and lower bands were estimated to be 2.7 x 106and 1.3 x 106, respectively, by using cytoplasmicpolyhedrosis virus RNA for internal molecularweight markers. The RNA bands were highlyresistant to digestion with T2 RNase at 270C for15 min as compared with E. coli tRNA (Sigma)(data not shown), indicating that they consistedofdsRNA (26). The RNA fraction obtained fromsensitive strains of S. cerevisiae gave only a

single band corresponding to dsRNA of 2.7 x106; the dsRNA with a molecular weight of 1.3x 106 was not detected (data not shown). Thesedata are in agreement with the results of killerand sensitive strains of S. cerevisiae reportedpreviously (2, 31, 35). When K. lactis strains IFO1267 and W600B and tetrads from the cross were

examined by the same procedure, no dsRNAbands could be detected (Fig. 4, lanes B to K).Similarly, all of the 11 nonkiller strains of K.lactis lacked both dsRNA's. Thus, the killercharacter of K. lactis strains could not be due tothe dsRNA.A chromosomal gene controlling the re-

sistance to theK lactis killer. K. lactis killerstrains (K. lactis IFO 1267 and tetrads from thecross with strain W600B carrying the pGKl plas-mids) were immune to each other (Table 2),implying that the plasmids encoded the resist-ance to the killer toxin. Certain strains of K.lactis (K43, WM37, K51, IFO 0648, and IFO1090), however, were also resistant to the K.lactis killers in spite of the lack of pGKl plas-mids. This may be explained by assuming the

J. BACrERIOL.

involvement of chromosomal gene(s) in the re-sistance. To prove this, a cross was made bymating a resistant strain K43 (a tip) with asensitive strain L3a (a met), and tetrad analysiswas conducted after sporulation. All of the 11tetrads analyzed gave 2:2 segregation for matingtypes and auxotrophic (met and trp) markers(Table 5). Sensitivity and resistance to K. lactiskillers (K. lactis IFO 1267 and tetrads 1A to 1D)also segregated in a ratio of 2:2, showing that theresistance of strain K43 to K. lactis killers wascontrolled by a single chromosomal gene, kkr(Kluyveromyces killer resistance). The hybridbetween strains K43 and L3a was killed by K.lactis killers; therefore, the kkr gene was reces-sive. Linkage analysis indicated that the kkrgene was closely linked to the trp gene (PD:NPD:T = 5:0:6).

DISCUSSIONWe searched for plasmids among 70 yeast

strains of 17 different genera and found twonovel DNA plasmids, pGKll (molecular weight,5.4 x 106) and pGK12 (molecular weight, 8.4 x106), in K. lactis IFO 1267. These plasmids werecharacterized by a linear structure unlike thecircular structure of bacterial plasmids and 2-,umDNA. The isolation of a linear plasmid-likeDNA from a strain of Streptomyces sp. has beenreported (13). Two linear DNA species associ-ated with mitochondria have been demonstratedin a eucaryote, Zea mays (24). The present pa-per, however, is the first to disclose the existenceof linear DNA plasmids in yeasts, although theintracellular localization remains obscure atpresent. The absence of the pGKl plasmidsamong the K. lactis strains tested, except K.lactis IFO 1267, indicates that the plasmids arenot necessary for proliferation of cells, in analogywith the 2-pm DNA in S. cerevisiae (19).

K. lactis IFO 1267 was found to kill bothsensitive and killer strains of S. cerevisiae at pH5 to 7, but not at pH 4.5, in contrast to S.cerevisiae killers with dsRNA, which showedthe killing at pH 4.5 to 5, but not at pH 7. Instudying the killing action of various yeasts be-longing to the genera Saccharomyces, Hansen-ula, Kluyveromyces, Pichia, Debaryomyces,Candida, and Torulopsis, Philliskirk and Young(23) have also demonstrated that a pH of 4.5 isoptimum or close to optimum for the killingfunction of most yeasts. Thus, it appears thatthe killing at pH 5 to 7 is rather unique to the K.lactis killer. All of the 11 tested strains of K.lactis lacking the pGKll and pGK12 plasmidswere nonkillers. Tetrad analysis indicated anextrachromosomal inheritance of the killer char-acter in association with the pGKl plasmids. In

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LINEAR DNA PLASMIDS IN KILLER YEAST 389

TABLE 5. Tetrad analy8i8 of the cross K43 (a tp) xL3a (a met)

Killing by:b

K. lactisStrain/tetrad MTa met trp R._Iacti Tet-radsIFO W600B lA-lD

1267

K. IactisK43 a + - - - -K. lactisLMa a - + + - +

TetradIA a +lB a - + + - +iC a - - + - +ID a + +

2A a - + + - +2B a +2C a +2D a - + + - +

3A a - - - - -

3B a

3C a + + + - +3D a + + + - +

4A a + - + - +4B a

4C a - + + - +4D a + +

5A a + - - - -

6B a + + + - +5C a - + + - +5D a

6A _C + +6B a - + + - +6C a6D a +

7A a - +7B a + - + - +7C a7D a + + + - +

8A a +8B a - + + - +8C a + +8D a - - + - +

9A a - +9B a - - + - +9C a + + + - +9D a +

1OA a + - + - +10B a - + + - +1OC a - +10D a +

11A a +11B alic a + + + - +

11D a - + + -

'MT, Mating type.+, Killing; -, nonkling ±, weak killing.

c_, Nonmating.

addition, electrophoretic analysis has revealedthat the killer character was not due to thepresence of dsRNA, which encodes the produc-tion of protein toxin in S. cerevisiae (3). Thus,it seems highly probable that the killer characterof K. lactis strains was under the control bf thepGKI plasmids. To further evidence this, simul-taneous curing of the killer character and thepGKI plasmids was attempted. The treatmentswith ethidium bromide and acriflavine were in-effective. Cycloheximide treatment (7) andgrowth at an elevated temperature (3800) (34)have been reported to eliminate the dsRNAkiller plasmid of S. cerevisiae, but they also gavenegative results so far. Hereupon, it will be in-teresting to study whether nonkiller K. lactis orS. cerevisiae strains are transformed into killerstrains with one or both of the pGKll and pGK12plasmids. Such transformation experiments arenow in progress.Young and Yagiu (36) have investigated var-

ious killer strains of different yeast genera andhave found that killer yeasts other than Saccha-romyces sp. do not carry dsRNA plasmids.Whether chromosomal gene(s) are responsiblefor the killings of such killer yeasts or whetherthe pGKl or other DNA plasmids are involvedremains to be studied.

ACKNOWLDGMENTSWe are grateful to J. Sugiyama for providing K. lactis IFO

1267, to L Del Giudice for K. lactis strains K43, L3a, WM37,and W600B, to A. M. Guerini for K. lactis strains L4, L5, andL51, to T. Yamazaki for the Saccharomycodes ludwigiistrins, and to C. Shimoda for the Schizosaccharomycespombe strains. We express our thanks to K. Miura for provid-ing the cytoplamic polyhedrosis virus RNA, to S. Irie for hisgenerous help in analytical centrifugation, and to T. Shiba, T.Tanaka, and N. Nagahari for their helpful discussions.

ADDENDUM IN PROOF

Since this paper was submitted for publication, wehave noticed that the pGKl plasmids and the killerphenotype were cured in the presence of ethidiumbromide or by heat shock at 650C. The details will bepublished elsewhere.

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