Vol. 169, No. 12

Yeast Plasmids Resembling 2pLm DNA: Regional Similarities andDiversities at the Molecular Level

IKUYO UTATSU, SUSUMU SAKAMOTO, TOSHIAKI IMURA, AND AKIO TOH-E*Department of Fermentation Technology, Hiroshima University, Shitami, Saijo, Higashihiroshima, Hiroshima 724, Japan

Received 13 April 1987/Accepted 31 August 1987

The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated fromZygosaccharomyces bailii, and pSMl (5,416 base pairs), isolated from Zygosaccharomycesfermentati Naganishi,was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids thatresemble 2,um DNA indicated regional sequence similarities among FLP proteins. Greater similarities were

seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSRl, and pSM1) than other combinations. A putativerecognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especiallyin the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putativegenes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling2,Lm DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSRl are most closely related, since not onlywere the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of theputative P gene of pSRl showed clear homology with that of open reading frame B of pSB2.

Since the discovery of 2,um DNA in Saccharomycesspecies (21; see reference 6 for a review), several yeastplasmids resembling the 2Rm plasmid were isolated andcharacterized. We call these plasmids 2,um DNA-like plas-mids. For unknown reasons, 2ptm DNA-like plasmids werefound frequently in Zygosaccharomyces species. We havefound five plasmids in Zygosaccharomyces yeasts: pSB1 andpSB2 from Z. bailii (23), pSR1 and pSB3 from Z. rouxii (23,24), and pSM1 from Z. fermentati (unpublished data). Re-cently, another 2,um DNA-like plasmid, pKD1, was discov-ered in Kluyveromyces drosophilarum (8). Of these, thenucleotide sequences of 2,um DNA (13), pSR1 (2), pSB3(25), and pKD1 (8) have been published. These plasmids,along with 2,um DNA from Saccharomyces cerevisiae, haveseveral structural features in common: (i) they are closedcircular DNA plasmids whose size is ca. 6 kilobases (kb), (ii)they have a pair of inverted repeats (IRs) consisting ofseveral hundred bases, (iii) they are cryptic and exist in highcopy number, and (iv) intramolecular recombination resultsin two isomeric forms. In spite of resemblances in theirglobal structure, homology at the nucleotide sequence levelis poor (23).Plasmid-encoded functions have been most extensively

studied in 2,um DNA. The prominent features of genes on2,um DNA are briefly summarized as follows. It has onereplication origin, which is activated once in the cell cycle bythe same replication mechanism as that for replication ofchromosomes (16, 28). It has four open reading frames(ORFs) or genes, A, B, C, and D. The A gene (or FLP gene)encodes a recombination enzyme which functions as asite-specific recombinase (7). The B and C genes producetrans-acting factors that are needed for the stable mainte-nance of this plasmid. These two genes, along with thecis-acting locus STB, participate in partitioning of plasmidmolecules during cell division (14, 15). Recently, the func-tion of the D gene was analyzed (A. M. Cashmore, M.Albury, and P. A. Meacock, personal communication), and

* Corresponding author.

the D gene was shown to positively regulate expression ofthe C gene. Other plasmids have a structural organizationsimilar to that of 2,um DNA; they have an FLP gene andthree to four large ORFs. In the case of pSB3, two ORFsother than the one encoding FLP enzyme were shown toparticipate in the stable maintenance of this plasmid (25).Our main interest was the evolution of these plasmids. It

was necessary to carry out comparative studies of thefunctional genes of each plasmid as well as of the nucleotidesequences of many yeast plasmids resembling 2pLm DNA. Inthis communication, we describe the structural analyses oftwo plasmids, pSB2 and pSM1. Comparative studies of themand other plasmids whose nucleotide sequences have beenpublished reveal that there are limited homologies in nucle-otide sequences of the FLP genes and proteins encoded bythem and that pSB2 is most closely related to pSR1.

MATERIALS AND METHODS

Strains and plasmids. Zygosaccharomyces bailii IFO1047(pSB2) and Zygosaccharomyces fermentati NaganishiIFO 0021(pSM1) were from the Institute for Fermentation,Osaka. Escherichia coli JA221 (F- leuB6 AtrpE5 hsdR recAllac I) (9) was used for construction and amplification ofplasmids. JM103 [A(lac pro) supE thi strA endA sbcBJ5hsdR4(F'traD36 proABlaclqZ AM15)] (17) was a host toclone DNA segments into M13-based cloning vectors. YIp5(Apr Tcr URA3) and YIp32 (Apr LEU2) were described byBotstein et al. (5) and were used as starting plasmids toconstruct various recombinant plasmids containing the se-quences of pSB2 or pSM1, which in turn were used as thestarting materials for nucleotide sequencing.

Isolation of plasmids. Yeast plasmids were isolated as

described previously (24) with partial modifications. Pro-toplasts were lysed in a buffer containing 5% guanidinethiocyanate. By this method, degradation of DNA duringextraction can be avoided. A minipreparation of plasmidDNA from E. coli was done by the method described byBirnboim and Doly (4), and the DNA was subjected to

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TCTAGAGCCAAAAGCGG;ATCGCATCGTCCCTTACATGGGAAAAAATAGATAAACCTTTCAATGCTAGTCTTTGTCTCTGGAArAAAGGCCCCsCAATrATAC 100AGATCTCGGTTTT,c(;ccGrAGCGTAGCAGGGAATG1'ACCCTT'ITTTATCTATTTGGAAAGTTACGATCAGAAACAGAGAcc,rTGT1'ITCCGG(;CG'11'A'ATrIGACCAAGGTGAGGATCTGCGAI'GATTTGAAAAGTGCTGGGGTCGGCGTTTTTCAAATCATTAGCCCTACAGCAGI'TCATGAAI'GTGAGTTGsAAGCAGAAA 200TGGTTCCACTCCTAGACGCTACTAAACTTTTCACGACCCCAGCCGCAAAAAGTTTAGTAATCGGGATGTCGTCAAGTACTFTACACTCAAC¶'TCGTCTTT

CCTATACGCAACTc,rTGTCGTGGGCTGGCGGGTGCGGGCTTCTATCAGATCCATTGTTc,rCTCGATAAAAC'rCCAAGTAGAATCTTTCGTCTCA(;CCAGT 300GGATATrGCG;TTGAGAACAGCACCCGACCGCCCACGCCCGAAGATAGTCTAGGTAACAAGAGAGCTATTTTGAGGTTCATCTTAGAAAGCA(;AGTCGGTCA

TTAGAGATCTCGGCGCCAACATTGTTGCTGAGAATGCTTCTGTGGCCCGCTTTCCTCTCATGCACGCTTGAGAGCTGTTTCTGCCAATCAATTGGCTTCT 400

TGTGCACCTTTACCACGAAATTGTAGGGCGCAATGGCTTGCTCGAGGCCCTCCTGTAGATCTTGTGTATTTTTGAGATGGTATTCGAAGAATACTGTCTT 500

GCTrTGACATATCATAATACAATGACTTCGCAACATTTCTACGATATTTAGTAAAGGTGCTAAGCTTGATTGGAGAGCTCTTCCTCTTTTTTGACATGTTG 600

ACCGTTAGGATCACCATAGTTAGTAGAGAAGCTAACCTTTGTAAAGGTATAGGGTCGCCGCGACTCAAAATACGCTTTATTTCTGCA ACCTGGTCTAATG 700

GGAGAATTCTGACAAGCTCGCTAAACTCGGACATAGGATACTAAACCTTTGATTAATTCAGTTTGGCGTCCTCATGTCGTAATCCTTGTTCGCTCGATTT 800CCTCTTAAGACTGTTCGAGCGATTTGAGCCTGTATCCTATGAT'TTGGAAACTAATTAAGTCAAACCGCAGGAGTACAGCATTAGGAACAAGCGAGCTAAA

--ATTTTTCTGCGAACCCGTGCGCAACGCTTTTTCATTCCGTACAGGTTATTTTCCGAAACAAAAAAAAAAGACCAAACTGTGCTCAAACCCATCACTGCCCT 900AAAAAGACGCTTGGGCACGCGTTGCGAAAAAGTAAGGCATGTCCAATAAAAGGCTTTGTTTTTTTTTTCTGGTTTGACACGAGTTTGGGTAGTGACGGGA

C--o3CTTATCGACGTATTGAGAGAGACACTACGACAGGGATAGTTCAGCGAGAXTACCGGTTTMGA(GAT'GAT'GTrTC 1000GAATAGCTGCATAACTCTCTCTGTGATGCTGTCCCTATCAAGTCGCTCTTACTTGAGTCTCAAGAGAGAACGCATGCCTTTACACCTGAGACTAATACGA

CTCGATiCTGCI'TGAACGGCTTGATTCCAACTGGAAGGGCACCGAGCTCTTTACTCATATrACGCGAAACCTTTCAAAT'TG(;ccTT,GGCAATGTT,ATCATAG 1100GAGCTAGACGAACTTGCCGAACT'AAGGTTGACCTTCCCGTGGCTCGAGAAATGAGTATATGCGCTTTGGAAAGTTTAACCGGAACCGTTACAATAGTATC

TGTCAGAACAGAGTGAAAGCCTTAGAATACCCCCTTCACTACTTGGTAGCAGTAGTCCAGCAGATAGCGACAATAGTCCTCCAGGAACACCTACTAATGA 1200ACAGTCTTGTCTCACTTT1CGGAATCTTATGGGGGAAGTGATGAACCATCGTCATCAGGTCGTCTATCGCTGTTATCAGGAGGTCCTTGTGGATGATTACT

AGCGCAACCCTGGT'TTATTTCTGAAGATCTCTCGAAAGGCCCTTTCACGGAAGCCCAGT'CAACTCAATCATCTATTGAGACACTlCGAAGG'l'GAGCACCAT 1300TCGCGTTGGGACCAAATAAAGACTTCTAGAGAGCTTTCCGGGAAAGTGCCTTCGGGTCAGTTGAGTTAGTAGATAACTCTGTGAGCTTCCACTCGTGGTA

GCTGTGTCTTCTCT'GCACCT'GAAGCTAAATGGCCTCTCCTGT'ATTGGACGTGCTGTATGGCGGGCTACTCGCAAAATGGATACGAGAACAGAGGTGGACG 1400CGACACAGAAGAGACGTGGACTTCGATTTACCGGAGAGGACATAACCTGCACGACATACCGCCCGATGAGCGTTTTACCTATGCTCTTGTCTCCACCTGC

ACATATTAAACTCAATAACAGAACCCAGAAGACTCACATI'ACCCGGTATCAACAAGATG.CGTCAATGCAT'TGTGCCGTCTATTGCTT'CTCGTACCGATCCA 1 500TGTATAATTTGAGTTATTGTCTTGGGTCTTCTGAGTGTAATGGGCCATAGTTGTTCTACGCAGTTACGTAACACGCAGATAACGAAGAGCATGGCTAGGT

AGTACGAGAAGAGATCCTTTcl",rTCGCCATAGCTTCGGGGATACCCTCAGAAACAATAGAAGATATTCGATCTTCAACAAATATTrTCAGCTGTTGATACC 1600TCATGCTCTTCTCTAGGAAAGAAAGCGGTATCGAAGCCCCTATGGGAGTCTTTGTTATCTTCTATAAGCTAGAAGTTGTTTATAAAGTCGACAACTATGG

4G100

TTACCGTCTCCGTATCGTGTATTAAGGTTTTTCGCCAGTAATCGCGGTTGTGTTCTAAGTGCGTTAAATGCGGCAGCTTAGTCCCCTGTATGGGTTATCT

ATCAATCATTTAGTGTGGCAGGAGTAATTTATATCTATTGGT hATGTATGAGGGATGTAGAGGTAGGCATATAGGATAGAAT'TAATGAGATATGAGATAA 1800TAGTTAGTAAATCACACCGTCCTCATTAAATATAGATAACCA rTACATACTCCCTACATCTCCATCCGTATATCCTATCTTAATTACTCTATACTCTATT

TGGTTCATAGA GGGG('AG('TCCTACGGAAGATATI'CGGAGTCATAGGGACGGTAI'TGAArTAGTTATTr1900ACTCTACACGCTACTCCATACCGCCCGGTCCCAGACGCGCAGTCGCCCTTTCTATAAGAccc'rCAGTGATCCCTGCTCAAAGTAATCCCTTAATCAATAA

CCTTAATGAAACTCTCCTAAAATGGAAATGCACTACCCGGACTCTTTCTGCCGCCCAAATGCTGCCTGCCATACTCTCCAGCACCTTACAATTCTTTGAT 2000GGAATTACTTTGAGAGGATTTTACCTTTACGTGATGGGCCTGAGAAAGACGGCGGGTTTACGACGGACGGTATGAGAGGTrCGTGGAATGTTAAGAAACTA

5100

ACCTATAAAACCCGAGGAGACGGTTCGGGACACAAACGTACGTAAAAACATAATACATAATACGTGATGGCACGAGAAACGGGACAAATACGGCCGCCCi

TCCGTAGAAGTGTTACTGTGCTTGCTAAACGTGAAATAGCCAACATAATATACAACATACAAATrl'CAACACTCTCTGAAACTCCTTTGGCATTTCAATTG 2200AGGCATCTTCACAATGACACGAACGATTTGCACTTTATCGGTTGTATTATATGTTGTATGTTTAAGTTGTGAGAGACTTl'GAGGAAACCGTAAAGTTAAC

CCGCTCTCATATGGCTC pTCATAGTTAAATTTCACATATTTCCTAACATTACCTATTAATACTTGAACGCCTAAAATACAATATATACAAAATACAAAA 2300GGCGAGAGTATACCGAG sAGTATCAATTTAAAGTGTATAAAGGATTGTAATGGATAATTATGAACTTGCGGATTTTATGTTATATATGTTTTATGTTTT

GGGATCTTTTCTGTTTTATCCCAGTCCACCAGAGGAACCAGCCTTGGCACCAAAATATTTTTTCTTCATCCGACAAATCTACTGGCTGACTTCGGTCTCA 2400CCCTAGAAAAGACAAAATAGGGTCAGGTGGTCTCCTTGGTCGGAACCGTGGTTTTATAAAAAAGAAGTAGGCTGTTTAGATGACCGACTGAAGCCAGAGT

GAGCCTGCTTCCTTGTCGCTCTCCTTCTCTCGAACGTCACACGCCAGGTCTAACAAAATATCGTCCAGAATG,rTATCTTTTGAGGTCAACTCTATGTTAA 2500CTCGGACGAAGGAACAGCGAGAGGAAGAGAGCTTGCAGTGTGCGGTCCAGATTGTTTTATAGCAGGTCTTACAATAGAAAACTCCAGTTrGAGATACAATT

CCCAATACGACGAAATATGGCGGGCAGTTTTTAATTTTGAAGCTCACCTTTAATTGCCATTTTTCTTGCATCTGATGcc,rCTGTATATTCTAAATTCCAG 2600GGGTTATGCTrGCTTTATACCGCCCGTCAAAAAT-TAAAACTTCAGTGG^AAATTAACGGThAAAAAGAACGTAGACTACGGAGACATATAAGATT'rAArGsTC

CTGGTTCCCAGACACTCTTTGAGATTTATATAAAAAAGAGCGCGGGCAGTATCCTTTTGCTTTGTAAATGTTTGGAGTATTTTTT'TATG.GCCCAGTAAAA 2700GACCAAGGGTCTGTGAGAAACTCTAAATATAnTTTTTCCGCGCCCGTCATAGGAAAACGAAACATTTACAAACCTCA'rhAAA^AAAA'ACCGGGTCATnTT

FIG. 1. Complete nucleotide sequence of pSB2. Noncoding strands of the large ORFs, A, B, and C, are shown. The largest ORF, A,encodes the FLP enzyme. IRs are boxed. The putative recombination sites are shown as inverted arrows within the IRs.

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VOL. 169, 1987 PHYLOGENETIC RELATIONSHIPS AMONG YEAST PLASMIDS 5539

AAACTGCCGCTTTCCTGCACTCT1TCTAACTGTG,r,rG('AGAr,GTGCGTAGCTCA,r1'A,rGGAGCGCCCT'GATAGCTI'CC('AAAA'tr.ArTCCTCC(;TrTCTAT 2800TTTrGACGGCGAAAGGACG'I'GAGAAGATTGACACAACCTiCTCCAcGr,ATCGAG1'AAI'ACCTC(; CGGGACTATCGAAGG.GTTrqTACTAAGGAGGSCAAAGwATA

GGCATTAATTTGAGTATTCAGAG(CGTCCAAACCATTrGGATGTSGGerATCCAGAGCCTCTGATTrCCTCTCGGGCTGTCGTAGTTTGTTCG'CCCGATGCATTA 2900CCGTAATTAAACTCATAACTCI'CGCAGGsTTTGGTAACCTACACCATAGGTCTCGGAGACTAAGGAGrAGCCCCGACAGCA,rcAAACAAGCGG(;(:rA(-GsTAATCGCTCTI'CTI'CCTCGAGATCATTAnrCGTCAAT-,rGCATTTTTATTGG^AAACCATGCCGCACACTATCTCCCCTATCCAGAc,rTTrACGAACAITTGGTSsGCCA 3000

TAAACTGGAGTTCTTSGGGrGTGTCCI'CCGAACAAAGCTI'TACACG;ATATGAGCG'ITTTGACCTTGCACAAGCCACC(1ICCAAATCTCCGG1'TAAT(;TACC 31 00ATTTGACCTCAAGAACCCA('ACAGG-A(,GGe,rTTTTCeGAAATGI'GCTATACTCGCAAAAcTrGGAACGTGTrTCGGI'GGGA(GGITTTAGAG(;CCAATTAC.ATGG.r

ATCCTGAAGTTTTCTGAAGTrACTCATCCAATTCTTTTCTCGCA-r,rA'GTGGCATCAATAAAGGTCTCCTCAATCTCTTTTGTAACATTI'TTCGTcrTlTTTT 3200TAGGACT'rCAAA-AGACrrCATGAGTAGGTTAAGAAAAGAGcGTrAATCACCGTAGTT'ArlTTCCAGAGGAGTTAGAGAAAACAT'TG-rAAAAAGCAGAAAAAA

GGCTGCCCTAATCGTTCATTCTTCTTAGACTCAGGAATCTTAAI'ACCCGAGGCAATTGCATTTTGAAGTCTTTCAACGTAGACATCAGCATTTTTGTTTG 3300,CCGZAC-GGGATiTAGCAAGTAAGwAAGAATCTGAGTCCTTAGAATTATGGGCTCCGTTAACGTAAAAC'I'TCAGAAAG,TTGCAT%CTG.TAGTCGTAAAAACAAAC

CAAATAGTTTATGGTCCTGGAAACGCTTAGTGATGGTATTCACCACTACATAGGGTTCCGGCCAGTTCACGGTGCTTGGAAGAAGATCGTACGGATTTTT 3400GTTTATCAAATACCAGGACCTTTGCGAATrCACTACCATAAGT(;GTIGA,rGTATCCCAAGGCCGGTrCAAGTGCCACGAACCTTCTTCTAGCATGCCTA AAAA

CTTCATTAAATATTTGACGTAGAAGATGGCCAATATGGGATAATTTTGATAGTTTTCAGGTATCTCAACTGTGCCTTTT'GGTGI'GATAAGTATATTCTTG 3500

GAAGGAACATGTACCGCGAAGTAAGGAAATGTCATAAGCACATCAAAGATCATCTTCATCTCTTTAGTGGGCCTGGAGGCCCTAACCTCTTCCCTGGAGA 3600CTTCCTTGTACATGGCGCTICATTCCTTTACAGTATTCGTGTAGTTTCTAGTAGAAGTAGAGAAATCACCCGGACCTCCGGGATTGGA(;AAGGGACC.TCTACATAATTGCTCAGCAAAACCATCTTGAGGACTAAACAGACGCACTTCTATCGTTGAAGATTTGGGAAATTTGTcTrGATCGGTATTTATACGCTTAAGCA 3700,TTAACGAGTCGTTTTGGTAGAACTCCTGATTl'GTCTGCGTGAAGATAGCAACTTrC'l'AAACCCTTTAAACAGACTAGCCATAAATATGCGAATTCGTB

CATCCTGAGGTGAAAAAACATTCTTGTGAGCGAAAAAAAACAAAAAGTTTTAGCAGAGAGCAAAAAAAATAAAAAATAGTTTTGTTAAAGCACACAAAAA 3800GTAGGACTCCACTTTTTTGTAAGAACACTCGCTTTTTTTTGTTTTTCAAAATCGTCTCTCGTTTTTTTTATTTTTTATCAAAACAATTTCGTG1'GTTTTT

TTACAGAAAAACGCCAAAAGCATACAACCAATTAAATATTTTTACGTCAAGCGCAAATTATATTTATAATTTAGGAACGCGCACAAGCATATTATTAAAA 3900AATGTCTTTTTGCGCTI'TTCGTATGTTGGTTAATTTATAAAAATGCAGTTCGCGI'TTAA'rATAAATATTrAAATCCTTGCGCG'rGTTCGTATAA'rAATTTT

AGTAACAGTrAA^GCACACCACTTAATACAAAAAGTTAGAGTAAGCATAGCAAAAAATGCTGCATGTGCGCATTAAGCATGGTAAAAAtATAAAACTATTTA 4 000TCATTOTCATTTCGTGTCGTGAATTATGT,TITTrCAATCT'CATTCGTATCGTTTTTTACGACGTACACGCGTAATTCGTACCATTTTTTATTTTGATAAAT

TAAAGTTTATTCTGTAAGTACATAAATATACCATACGAAAG,TTGGTAGAAAGCTTTTATTTTTAAGCACTATTrTGTAGGTAGAA,rATTTTTT1AAGCACAT 4100ATTTCAAATAAGACATTCATGTATTTATATGGTATGC'TTTCAACCATCTTTCGAAAATAAAAATTCGTGATAAACATCCATCTTATAAAAAATT'CGTGTA

CTTTAAATCTTAAATTTATTCTGAGCACTTTAATCCATGAGCATTT,rTTGTAGAGCAGGGTCTCCTATCATTTATTTTCTGG.ATCATTGGCCGGGTAAGT 4200GAAATTTAGAATTTAAATAAGACTCGTGAAATTrACG'TACTCG'rAAAAAACATCTCGTCCCAGAGGATAG'rAAATAAAAGACCTAGTAACCGGCCCAT'TCA

IRATAATCATGATCGCGGTtAAAG ,GAPCcAlATGAGAGCGGCAATTGAAATrGCCAAAGGAGTTTCAGAGAG rG ll GAATTI'TATz G l GTA rATTrATGTT 4 300TATTACTACTAGCGCCAATTTC -----GTTACTCTCGCCGTTAACTTTACGGTTTCC.TCAAAGTCTCTCACAACTTAAACATACAACATATAATACAA

2800

CCGATAAAGTGCAAATCGTTCGTGTCArrGTGAAGATGCCTCGGGCGGCCGTATFTTGTCCCGTTTCTCGTGCCATCAC(;'rATTATGTA,iwrAA1%GTTTTTAC

35000

GTACGTTTGTGTCCGhCCGTCTCCI'CGGGTTTTATAGG'rTAGTTTCTTAACATTCCACGACCTCTCATACCGTCCGTCGTAAACCCGCCGTrC'rTTrCTC

TCCGGGTAGTGCATrlTCCATTTTAGGAGAGTiT,TCA'rTAAGGAXTAACTAATTCCCTAA'GAAACI'CG,rcccrAGTGACTU'CCAGAA'I'ATcTiT,TCCCGCTG 4600

ACGCGCAGACCCTGGCCCGCCATACCTCATCGCACATCTC(AT,rA'I'CTCATATCTCATTAA'I'TCTATCCTATA1sGcc,rACC'1%CTACATCCCsTCATACAT 44700

AGCAAATTCGTGTATTT'rATGTA(;,CTACTCTTCTCCAGGTGTCATTAGACCAGTGGAGAG,rGTCTTTGGAGCAGTCCAGGTCAAGGGTGTCGATGAGCCT 4800TCGTTTAAGCACATAAAATACATCGATGAGAAGA(;(;'rcCACAGTAATCT.GGTCACCTCTCACAGAAAcc'rCGTCA(;GTCCAGTT'CCCACAGCl'ACTCGGA

GTCGATTGAGGAGTTrAAGAAGGGAI'GCGCTGGTGATTCACTACTGAATAGCCTATTTTGCTCATTGGGAI'CGGCCAATrTGTTTTrCGCTTCTGCTGCGSCAT 4900

ACGTGTAGAGATACAGCAGTGCG('TCrTTI'TG.TATCACTTTCGCATTTTTCCCGTACCGCCC.ACAGAT('AATTTCTCGTI'C.TTGTrTATTIGrGAAGTCGTTTT 5000

GTCGTAATCCAGGGGATTATACAGTGTTTCAGCCAGCACGTACTCGCCTTGGTTGGACTTT,rTGTAGTAGCCGGATAAAAATGCA AA'TAGATAI'GAAGGT s 10o

GGACTTTTTTtAACCGTGTC.CATC,tAT4C(;GCTGTCAGCCATTTTGCTGACGCCCTCTTGCCTTTCCACAGACCAGtTGCCGTAGAGTGTGGCCTC(;CTCT 5200

TGAGACTGTTGTTTCCAAGGTATGATGCCATTAGATGCCGeCCCCAAATCAGCTTTGGGCCCATTAGGTATTTTGAAAATATTTTCCTTTGATTCT,rTGG.C 5 30 0

GATAAACCTGTCGTrACGA AAT('AAGAGAGTATCT,rG.AAGAAGCTGGTAATCATATTGAGTCTCTTCATCGGTAGTr,C.AGATTTTGC,GCA(-,CTGGGCCAA CC 54 0o

CACAGGAGATAGGAAGTGTCCTCTATCC"rT

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Xba I

SacH1in ' BamH I

Bg1 II

,Xho I

bpaa

FIG. 2. Model of pSB2 and pSM1. Central constrictions indicate IRs. Dashed lines indicate the putative site of FLP reaction. Open boxesshow ORFs, with the tail indicated by the arrowed end. Length (in nucleotide pairs) and the number of amino acid residues (aa) deduced fromthe nucleotide sequences are also shown. *, Consensus sequence for ARS of S. cerevisiae.

preliminary structural analysis. Plasmid DNA was purifiedby CsCl-ethidium bromide banding (10) as necessary.Media and growth conditions. YEPD contained 20 g of

Polypeptone (Daigo-Eiyo), 20 g of glucose, and 10 g of yeastextract (Oriental) per liter. Yeasts were grown at 30°C withshaking. L-broth contained 10 g of Tryptone (Difco), 5 g ofNaCl, and 5 g of yeast extract per liter. TY mediumcontained 8 g of Bacto-tryptone (Difco), 2.5 g of NaCl, and5 g of yeast extract per liter. TY medium was used forgrowing M13 phage and its derivatives. E. coli cells weregrown at 37°C. Ampicillin (50 ,ug/ml) or tetracycline (10,ug/ml) was added as appropriate. Agar (2%) was added toprepare solid medium. E. coli transformation was done asdescribed previously (11).

Southern hybridization. 32P-labeled probes were preparedby nick translation (18). Purified yeast plasmids (24im DNA,pSB1, pSB2, pSB3, pSM1, and pSR1) were electrophoresedon a 1% agarose gel and transferred to a Biodyne membrane(Pall Ultrafine Filtration Corp., New York) by the proce-dures described by Southern (22). Then filter hybridizationwas carried out with a 32P-labeled probe.

Determination of nucleotide sequences. Nucleotide se-quences were determined by the M13 chain terminationmethod (19). DNA segments were subcloned into single-stranded DNA vectors, either M13mp8 or M13mp9 (17).Chemicals and enzymes. Restriction endonucleases and

other DNA-modifying enzymes were purchased from TakaraShuzo (Kyoto, Japan), Toyobo Biochemicals (Kyoto, Ja-pan), or Nippon Gene (Niigata, Japan) and were usedaccording to instructions provided by the manufacturers.

[ox-32P]dCTP was from Amersham Corp. M13 sequencingkits were purchased from Takara Shuzo.

RESULTS

Homology test by Southern analysis. Isolation and prelim-inary characterization of pSB2 were described previously(23). pSM1 was purified from a 1-liter culture of Z.fermentati Naganishi IFO 0021 (formerly Saccharomycesmontanus) by CsCl-ethidium bromide banding. When[32P]pSB2 or [32P]pSM1 was used as a probe in Southernhybridization experiments under intermediately stringentconditions (65°C for 18 h) in a cocktail containing 3x SSC(1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.02%Ficoll 400, 0.02% bovine serum albumin, 0.02% polyvinyl-pyrrolidone, 50 [Lg of denatured sonicated calf thymus DNAper ml, and 0.1% sodium dodecyl sulfate, neither probeshowed cross-hybridization with other plasmid DNAs(pKD1 was not tested) (data not shown). This result, laterconfirmed by sequencing, indicates that both pSB2 andpSM1 are unique plasmids.

Characterization of pSB2. Nucleotide sequences of bothstrands of pSB2 were determined by the M13 chain termi-nation method (Fig. 1). A structural model of pSB2 is shownin Fig. 2. pSB2 contained 5,415 base pairs (bp). Inspection ofthe sequence revealed that it had a pair of IRs, eachconsisting of 477 bp and three large ORFs: A (1,422 bp), B(1,071 bp), and C (474 bp). As shown previously (23), pAT63or pAT65, in which the plasmid was interrupted at the EcoRIsite, did not undergo intramolecular recombination in an S.

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cerevisiae host. However, intramolecular recombination didoccur when pAT63 was present with pSB2 (data not shown).Since the EcoRI site was located in ORF A, we infer that theA gene is the structural gene for FLP of pSB2. The functionsof ORFs B and C are not known.

Characterization of pSMl. The nucleotide sequences ofboth strands of pSM1 were determined as described above(Fig. 3), and its model was shown in Fig. 2. pSM1 was 5,416bp long and contained a pair of IRs consisting of 352 bp andfour large ORFs, FLP (1,116 bp), A (780 bp), B (672 bp), andC (600 bp). The largest ORF, FLP, encoded the FLP enzyme

of pSM1, since disruption of this frame resulted in defectiveFLP function in this plasmid (data not shown).

Search for similarities among yeast plasmids. When nucle-otide sequences of 2,um DNA-like plasmids were comparedpairwise in all possible combinations, partial similarity was

always found in the ORFs encoding the FLP enzymes. Nosimilarity (more than 60%) was found outside the FLP gene.One exception was homology between the B gene of pSB2and P gene of pSR1. Then, the amino acid sequence wasdeduced from the nucleotide sequence of each ORF of theplasmids, and dot-matrix analyses were carried out betweenORFs in all possible combinations. Limited similarity wasalways found between FLP gene products (data not shown).When amino acid alignment of the FLP proteins was per-

formed (Fig. 4), the length of the N-terminal and C-terminalregions varied from one plasmid to another; FLP of pSM1had the shortest N terminus and FLP of 2,um DNA had theshortest C terminus. In the case of pSB3, whose FLP proteinhad the longest C terminus, 102 residues from the C terminuscould be deleted without affecting FLP function (unpub-lished data). Two clusters of homologous regions were seen,

from residues 206 to 224 and from residues 317 to 335 (Fig.4). These two regions were detected as homologous regionsby searching for homology at the nucleotide sequence levelas mentioned above. The number of residues matched ineach comparison between residues 59 and 475 of Fig. 4 isshown in Table 1. These figures show that pSB2 and pSR1are the most closely related of the plasmids tested.There were a pair of short inverted repeats in the IRs of

each plasmid, which are most probably a recognition site forthe FLP enzyme. In the case of 2,um DNA, this sequencehas been proved to be the recombination site by the FLPsystem (1). When these sites were compared (Fig. 5), thoseof the Zygosaccharomyces plasmids seemed to be moreclosely related; the sequence of 4 bases flanking the spacerwas conserved.Other than the combinations between FLP genes, homol-

ogy was detected only between gene B (encoding 357 aminoacid residues) of pSB2 and gene P (encoding 410 amino acidresidues) of pSR1 by dot matrix analysis. When the aminoacid sequences of these two proteins deduced from thenucleotide sequences were aligned, 102 amino acid residuesdirectly matched were concentrated in the N-terminal two-thirds of the B protein.

DISCUSSION

Two new plasmids, pSB2 from Z. bailii and pSM1 from Z.fermentati, were sequenced. Sequence comparisons amongyeast plasmids confirm that they have a similar globalstructure; each plasmid has a pair of complete IRs and threeto four ORFs, one of which encodes the FLP protein.Furthermore, the nucleotide sequences suggest that putativetranscriptional orientation of the ORFs is always towards theIRs.

The FLP reaction of 2,um DNA was studied extensively(1, 20). FLP enzyme of 2,um DNA requires an 8-bp spacerand 13-bp IRs flanking the spacer and produces staggeredstrand breaks at the junctions of spacer and IRs. However,the enzyme does not recognize the nucleotide sequence ofthe spacer. The enzyme also catalyzes a rejoining reaction.As for the biological meaning of the FLP reaction, Futcher(12) proposed that the FLP enzyme of 2,um DNA functionsto recover high copy number when 2,um DNA copy numberis reduced by chance. Since replication of 2Rm DNA isactivated only once in each cell cycle, it is necessary to havea special mechanism to recover the copy number. Futcher'smodel predicts that recombination mediated by the FLPprotein allows the replication fork of 2p.m DNA to proceedmore than one round in a division cycle; in this way, areduced copy number of 2,um DNA can be restored. Volkertand Broach (27) devised a method to construct a cellcontaining one copy of 2,um DNA by excising Flp- 2,umDNA flanked by direct repeats of the recombination sitesthrough the action of the FLP enzyme whose production hadbeen placed under control of the GAL1O promoter. By usingthis system, they proved Futcher's model.The site-specific recombinases in phage X, +80, P22, P2,

186, P4, and P1 catalyze a similar reaction. Although theserecombinase proteins show little global sequence homology,Argos et al. (3) found a conserved region in the C-terminalhalves. They also found that the region is also homologous toa region near the C terminus of the 2pim DNA FLP proteinand that three positions, His-396, Arg-399, and Tyr-433 (intheir coordinates) are perfectly conserved in the above eightcases. These positions are also conserved in 2pRm DNA-likeplasmids; His-327, Arg-330, and Tyr-368 in the coordinatesshown in Fig. 4.Comparisons of nucleotide sequences and putative pro-

teins encoded by six yeast plasmids indicate that there arealways local homologies in the FLP proteins. The extent ofhomology is variable, and in general, the FLP proteins ofZygosaccharomyces plasmids are more closely related toeach other than to those from other sources. Judging fromthe homology between the FLP proteins, pSB2 is closest topSR1 (Table 1). The close relationship between these twoplasmids is supported by the homology between the B geneof pSB2 and the P gene of pSR1. Putative recognition sitesfor the FLP enzymes of Zygosaccharomyces plasmids alsoshow some sequence similarity. The homologous regions arenot scattered evenly along the yeast plasmid sequences.Instead, such regions are restricted to the FLP gene. Even inthe comparison between pSB2 and pSR1, which seem to bethe most closely related, other parts of plasmids than theFLPs and B (pSB2) and P (pSR1) do not show similarity.Although this suggests that yeast plasmids had divergedevolutionarily before they established their present config-

TABLE 1. Number of mnatching residuesa

No. of matching residuesPlasmid

pSR1 pSB3 pSM1 2,um, pKD1

pSB2 266 161 164 147 128pSRl 165 167 152 128pSB3 164 142 109pSMl 139 1252p.m 119

aComparisons were made between amino acid residues 59 to 475 in thecoordinates shown in Fig. 4.

5541VOL. 169, 1987

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AAGCTTGTGCTCGCTATTTAGCATCTCTTTGAGCTCATTGACT^AGAGATGTTATGTTTCGCG^A ACTAGCTCTTrCAAATAATGATATTCr,G(;GCG(';CATT 100TTCGAACACGAGCGATAAATCGTAGAGAAACTCGAGTAACTGATCTCTACAATACAAAGCGCsTCTGA'TCGAGAAGTTTAT'IACTATAAGCCCCGCCGTAA

TTATTGCCCTCTGCCCAAATTCTGCAACTCCTCTTAACTGTTACAGAGCCTGAAGACAAGAAACGCCATTTGTTCCCGTGAAAAATGCATTTTAA ATTGC 200AATAACGGGAGACGGGTTTAAGACGTTGAGGAGAATTGACAATGTCI'CGGACTTCTGTTCTTTGCGGTAAACAAGGGCACrTTTTTACG'AAAATTTrAACG

ACTTrCTGGGTAAAAAATACAATACAAGGTrGCGCAAGACGTAAACTGAAAGCGTTCCAAAGGGACCAAAGAGACCGAATACTACGCAAAATGGATTGGAAA 300TGAAGACCCATTTTTTATGTTATGTTCCACGCGTTCTGCATTwTGACTTTCGCAAGGTTTCCCTGGTTTCTCTGGCTTATGATGCGTTTTACCTAACCTTT

ATTGATACATCAGACCTCACAGCTACTAGATATTATGCTTATTCGGCATCTGATGCCTTAGTTGTGCTACTCAGAAGAATATGI'TCCCCATATGC(;ACAA 400TAACTATGTAGTCTGGAGTGTCGATGATCTATAATACGAATAAGCCGTAGACTACGGAATCAACACGATGAGTCTTCTTATACAAGQGGGTATACGCTGTT

CGAGGTTTGACAGCTCGAGCTGTAACAAGATGTCTCAGACCGACTATCCCCAATATGAGATTAAGACTrGGAACGGATGTG;TCACAC.AATAGAGACTTGTC SooCtTCCAAACTGTCGAG0CTCGACATTGTTCTACAGAGTCTGGCTGATAGGGGTTATACTCTAATTCTGACCTTGCCTACACAGTGTCTTATCTCTGAACAGTTTTGATTACATGAAAAAGCCCACGAAGTCCACTGCAAAAGTACTCAGAGGAGTAGGTGTGATTGATGA ATGGTrTCGCAAAACGCCTCCT'rGGACCTCGA 600AAAACTAATGTACTTTTTCGGGTGCTTCAGGTGACGTTTTCATGAGTCTCCTCATCCACACTAACTACTTACCAAGCGTTT'TGCGGAGGAACCTGGAGCT

GGATCTAATCCGGATCTTCCATTTGCTGCTCTTCTGTGTCGAGATCAACTGTACAGGGAGTGGATAACACTAATTCGTATGCI'TACACTAGTGTTGAAAC 700CCTAGATTAGGCCTAGAAGGTAAACGACGAGAAGACACAGCTCTACTTGACATGTCCCTCACCTATTGTGATTAAGCATACGAATGTGATCACAACTTTG

ATG'TGCCAAAGGCAAAAGTGAAGGAAGCACATGATATGATCCTTGAATTATATCCCAACTACAGAAGAAGGAATATc'M',CATI'CCGGAAATAAAG(;AAAG 800

TACACGGTTTCCGTTTTCACTTCCTTCGTGTACTATACTAGGAACTTAATATAGGGTTGATGTCTTCTTCCTTATAGAAGTAAGGCCTTTATTTCCTTTC

TGATATACATGATATCAAGAATGCCTTGCGCTCGCTAGTACGTCAGcTrTTCAAGCAGAACAAATATCCTAGAATGGCCCGAAGCCGCTAAATATCCCACT 900ACTATATGTACTATAGTTCTTACGGAACGCGAGCGATCATGCAGTCGAAAGTTCGTCTTGTTTATAGGATCTTACCGGGCTTCGGCGATTTATAGGGTGA

TT'ATTTGAGGAATTAGTAAGGGAAAAG.AGGTCACAAGAGACACAAAAGACCTCTAGTACGAAAAATrCTTCTCAATCGT'CGAAATGCCAGAAAAAGAAGGC 1000AATAAACTCCTTAATCATTCCCTTTTCTCCAGTGTTCTCTGTGTTTTCTGGAGATCATGCTTTTTAGAAGAGTTAGCAGCTTTACGGTCTTTTTCTTCCG

GGTTGTCCAGCGACATGGCGGCTGAACGGCCTGCTGGCAAATGGAAGAAccGccTrTCGGAGCACGCAATAG3CTATTTTGTTAGTTGGCTAAG AGTAT 1100CCAACAGGTCGCTGTACCGCCGACTTGCCGGACGACCGTTTACCTTCTTGGCGGAAGCCTCGTGCGTTATCGATAAAACAATCAACCGATTC GT

GGAACGTGGACACAGGTGGTGGGGCAAGATGTGCTGGAGTATGTACACTCATACGCGATGGGTAAGCTAGGCATAAGGAAATAAATGTATGTTCAGTAG'r 1200

GCAAGCGTTGGAAGTGATTGATTTGGAAAGGAACGAATCTGTAACCGCTGACGTGAAAAGCGAGAGAAATGGAAAGGAATGGTTCAT1'CCTTTCCATTTC 1300CGTsTCGCAACCTTCACTAACTAAACCTTTCCTTGcTTrAGACATTGG,CGACTGCACTTTTCGCTCTCTTTACCTTTCCTTACCAAGTAAGG.AAAGGTAAAG

GCTCATCTTTGACGGGTTTCATrCTTTACCGCCAATGGGTTATATATATGATTATACTI'AGCATGG,rACTCATAGTGTTGCAA,rGGcCAT,rTACTGTAGTAC 1400

18~~~~~~~~~~~~~~~~~0

CCAACCATCTTCTGCCTTTTCTrGATCCTGTTCAGGCCTTACGCA1 TGAAAAATAATCGTAATTGTrCCATAATAAAGCAGATGTGCACATATATGTCTGT 1500FaGTTG',ACGG CCGGAA ~~ACTTTTTATTAGCATTAACAGGTATTATTTCGTCTACACGTGTATATACAGACA

GAACTGCCACTACAGGTTGTTACCTAATGTACGACTCTATGCTTATTGGACCGCCCTCTTCTACTTCCGTTGCTGCTGAAAGTACTTAGAAGAGGATATT 1600CTTGACGGTGATGTCCAACAATGATCTCGGTCATACGCGAAGTAGCAGCAT'AGA'TCCTTA

TCTTCAGAGCCTGACGAGCGGATTTTTGTACATTAGTATTCI'TTATGGCATCCTCAATCTTCTTTCTCAGCTGCGTTAGCTc'rTCTGTTGTTAACTCTAC 1700AGAAGTCTCGGACTGCTCgCCTAAAAACATGTAATCATAAGAAATACCGTAGGAGTTAGAAGAAAGAGTCGACGCAATCGAGAAGACAACAATTGAGATG

CATGAACGGCTTGTTTGCGAACCAGGGCATTAGCACGTCATCGATTCGTTTCTGATCTGCGGGGTCTTCTACCAAATTTATCAGGTCGTTTAAATATGAT 1800GTACTTGCCGAACAAACGCI'TGGTCCCGTAATCGTGCAGTAGCTAAGCAAAGACTArGACGCCCCAGAAGATGGTTTAAATAG'rCCAGCAAAT'rTATACTrA

CTGGTTTCAAGCTCCCACTGACGCTCTTGCATTGAACGATAATGTAGTAATCGCAGTAGTTGATTTGCTGATAATTCTAGATTAG'l'Tl'GTCTGACGAAGC 1900

GTTCAGCAGATTCCGCCAATCTGACAGCAGTGTCAACATTCGTGATGCCCTCTGAGACATCGAAGTCCTCCTTAGACCAATCAGGAACTTCATATCTTGT 2000CAAGTCGTCTAAGGCGGTTAGACTGTCGTCACAGTTGTAAGCACTACGGGAGACTCTGTAGCTTCAGGAGGAATCTGGTTAGTCCTTGAAGTATAGAACA

TTCTAATGCCTCCCAATGCGCGCGAATAGTTTCAGATTCTTCATGCGCCTCTTGAGCGTACTTGTCATCAAGATGTTCCGATGCGGAGCGCATAACATTA 2100AAGATTACGGAGGG'l'TACGCGCGCTTATCAAAGTCTAAGAAGTACGCGGAGAACTCGCATGAACAGTAGTTCTACAAGGCTACGCCTCGCGTATT(;TAAT

GAAAAAGTTCTGTAAACATACGCCTGTAATGGAATTGCCAGTAACCTTGTTTCAGTCATGTCAGTAGCATCGTAATCTTCATTGTAAAGGGCTCCCATAA 2200CTTTTTCAAGACATTTGTATGCGGACATTACCTTAACGGTCATTGGAACAAAGTCAGTACAGTCATCGTAGCATTAGAAGTAACATTTrCCCGAGGGTATT

GGAAGGATATGCGCTCAGCCTTGATTATAGGGATTATTCAAAAATCATACAATTGCATCTACCCCCAAtAAAACATATCGAAGTCGCAAThAAAAACATTTT 2300CCTTCCTATACGCGAGTCGGAACTAATATCCCTAATAAGTTTTTAGTATGTTAACGTAGATGGGGGTTTTTTGTATAGCTTCAGCGTTATTTTTGTAAAA

TTACAATATGCGTCTTTTTTT,TTATGGCCAAAAGAGAAAAAA^GCTGCACTTTCTACTCATTTTTTAATTTTTATTGCAATI'TTI'ACATAAAAAAtCAATTT 2400AATGTTATACGCAGAAAAAAAAATACCGGTTTTCTCTTTTTTCGACGTGAAAGATGAGTAAAAAATTAAAAATAACGTTAAAAATGTATTTTTTGTTAAA

TTATTATCCATGTACAATTATGTACG,rAATAATTAACTTTTTTTAACAGCTTGCAGTACACATTAATTTTTTTCAAAATTATTTTTT,rATAATTGCTTTT 2500AATAATAGGTACATGTTAATACATGCATTATTAATTGAAAAAAATTGTCGAACGTCATGTGTAATTAAAAAAAGTTTTAATAAAAAAATATTAACGAAAA

AAAACTTTTTCAGTTGTTTTTTAAGATACAGAGTGCTTTTTCTATTGTTATT,TTTGACACACAAATTGCGCTTTTGCTGCTCATACACGAGAtAAACATCT 2600TTTTG^AAAAAGTCAACAAAAAATTCTATGTCTCACGAAxAAAGATAACAATAAAAACTGTGTGTTTAACGCGAAAACGACGACTATGTGC'rCT'rTl'GTAGA

TTTCATTGTTTTGAAAGATAAAGTGCAGTAACCTTCACCCCAGATATTGAAATTAGGAAACCAAGAACACTCTTAAGATGGAAGATGATAGTGATAAACC 2700AAAGTAACAAAACTTTCTATTTCACGTCATTGGAAGTGGGGTCTATAACTTTAATCCTTTGGTTCTTGTGAGAATTCTACCTTCTACTATCACTATTTGG

FIG. 3. Complete nucleotide sequence of pSM1. Noncoding strands of the four large ORFs (FLP, A, B, and C) are shown. The sequencesof IRs are boxed. The putative target sites of the FLP enzyme are shown by inverted arrows within the IRs.

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VOL. 169, 1987 PHYLOGENETIC RELATIONSHIPS AMONG YEAST PLASMIDS 5543

GTTTCI'TAGTGACCCAGAAGGTAI-TTTATTATGTCTTAGGGAATGCTI'TATGATAGGATCCCCGGATGsTTGTCATTGTACCTGATCTTGr,AATCATGCAA 2BOOCAAAGAATCACTGGGTCTTCCATAAAATAATACAGAATcCCC'TACGAAATACTATCCTAGGGGCCTACAACAGTAACATrGGACTAGAACCTTAGTACGTT

TCACCTTCGGATCAGTTTCGCTGCCAGCAAAGTACCAGCAATATCCCCAGTTGGCACTGTTTTACTI'CAGTGCTGTTCTCAATCAAAATGGACTCATTCC 2900AGTGGAAGCCTAGTCAAAGCGACGGTCGTTTCATGGTCGTTATAGGGGTCAACCGTGACAAAATGAAGTCACGACAAGAGTTAGTTTTACCTGAGTAAGG

AGAAGATCTTCACCTAAATTTTGAATGGCCTGAGCCTTATGTGGTACTAAATACTTTGATzCAAACGCATAGAGCAACACAGATTTGTCACAAAAAGcG,GC 3000TCTTCTAGAAGTGGATTTAAAACTTACCGGACTCGGAATACACCATGATTTATGAAACTAGTTTGCGTATCTCGTTGTGTCTAAACAGTGTTTTTCGCCG

TATAAAGGATTCCCCAGCTTCATAAAAGAGCAAATTGCACCTCATCTCGAGTGTCCTGATGATGAACTGGTCCGTGGTGGTAATATCCTTGATTTAGATC 3100ATATTTCCTAAGGGGTCGAAGTATTTTCTCGTTTAACGTGGAGTAGAGCTCACAGGACTACTACTTGACCAGGCACCACCATTATAGGAACTAAATCTAG

CCGGCACGAAACAACTTGTTAAGGTACGTTTCGAGAATAAAGACTCAATTGTGGAAGATGTCAATCAGTTTTTTGCAAGAATTACTGACCTACCGACAGT 3200GGCCGTGCTTTGTTGAACAATTCCATGCAAAGCTCTTATTTCTGAGTTAACACCTTCTACAGTTAGTCAAAAAACGTTCTTAATGACTGGATGGCTGTCA

C _-oACTTTCTTCATTTCAGCATCATACGAAGTTAGATGTCAAAATATCTTGTCGGCAATGGATGACATCGTAC'GAGTTGAGC 3300TGAAAGAAGTAAAGTCGTAGTATGCTTCAATCTACAGTTTTATAGAACAGCCGTTACCTACCTTCGGTTAGGACCACTTGACCTTGCAAAACCTTTCCGA

ATTAGAAAGATATGGCTCTATGAAACTATCATGCTGGGAGCTGAAAAAATTGCCAAAGGTAACTTAAGTGAGGAAAGTACCTCTACATCAGAATCGGAAA 3400TAATCTTTCTATACCGAGATACTTTGATAGTACGACCCTCGACTTTTTTAACGGTTTCCATTGAATTCACTCCTTTCATGGAGATGTAGTCTTAGCCTTT

TCCCAAACACCTCTAGCCAGCAGGATGTCATCTaCTTCTTCAGAACATrG,rAAGCGATrTCCTTGAAGTTTTGGGAGGTATTAAACTTc'r,rAGAAGTATCGTG 3500AGGGTTTGTGGAGATCGGTCGTCCTACAGTAGAGAAGAAGTCTTGTACATTCGCTAAGGAACTTCAAAACCCTCCATAATTTGAAGAATCTTCATAGCAC

GGTACCACATAGTGGCGAGGATGGTGTTCAACACAAGTTATCTCAGTATGTGGAAAAGGATCCTI'CTACTTTTGAAGAGCTCAAAAAGCGAAGACTCGCG 3600CCATGGTGTATCACCGCTCCTACCACAAGTTGTGTTCAATAGAGTCATACACCTTTTCCTAGGAAGATGAAAACTTCTCGAGTTTTTCGCTTCTGAGCGC

GCAAAAAAI'GATGTATATGGCTACGTTAGGCAAATCATGTATTCCAAGGGACAAGTAAGGATTTACAGAGGI'GTTAGATGCAAACCAAATGGTGTTCTCA 3700CGTTTTTTACTACATATACCGATGCAATCCGTTTAGTACATAAGGTTCCCTGTTCATTCCTAAATGTCTCCACAATCTACGTTTGGTTTACCACAAGAGT

AAGTGAGTTTGTTCTATGGACCTTGCAAATGGGTAACATCAATGTGGGTTACGTTAGTGCCCATCACATTAAGAGAGAATGGc,rCGC.CAGAAGACATTGA 3800TTCACTCAAACAAGATACCTGGAACGTTTACCCATTGTAGTTACACCCAATGCAATCACGGGTAGTGTAATTCTCTCTTACCGAGCGATCTTCTGTAACT

ATTTACTT.,'CAATTGCTCACTAAATCTCAAGAGCTCCAAGGCTATTTCGGCGGGTATGTAATAATTCATGTAATTTGAACAAATAATAATGGTAATGAAA 3900TAAATGAAAGTTAACGAGTGATTTAGAGTTCTCGAGGTTCCGATAAAGCCGCCCATACATTATTAAGTACATTAAACTTGTTTATTATTACCATTACTTT

TTAAAGAGAGAAGTATTTATTTAGACGACTTAAATCGATAATCAGAATAAACGTGAAzAAATATTAAAATCATTCTGTTTAATACTAGATCAAAAAC*ATG 4000AATTTCTCTCTTCATAAATAAATCTGCTGAATTTAGCTATTAGTCTTATTTGCACTTTTTATAATrTTTAGTAAGACAAATTATGATCTAGTTTTTGa A

CGTAAGGCCTGAACAGGATCAGAAAAGGCAGAAGATGGTTGGGTACTACAGTAAATGCCATTGCAACACTATGAGTACCATGCTAAGTATAATCATATAT 4100GCAl'TCCGGACTTGTCCTAGTCTTTTCCGTCTTC,rTACCAACCCATGATGTCATTTACGGTAACGTTGTGATACTCATGGTACGATTCATATATTT

ATAACCCATTGGCGGTAAAGATGAAACCCGTCAAATA-TGAGCGAAATGGAAAGGAATGAACCATlI'CCI'TTCCATTTCTCTCGCTTTTCACGTCAGCGGTT 4200TATTGGGTAACCGCCATTTCTACTTTGGGCAGTTTCTACTCGCTTTACCTTTCCTTACTTGGTAAGGAAAGGTAAAGAGAGCGAAAAgTGCAGTCGCCAA

-ACAGATTCGTTCCTTTCCAAATCAATCACTTCCAACGCTTGCACTACTGAACATACATTTATTTCCTTATGCCTAGCTTACCCATCGCGTATGAGTGTAC 4300TGTCTAAGCAAGGAAAGGTTTAGTTAGTGAAGGTTGCGAACGTGATGACTTGTATGTAAATAAGGAATCGGATgAATGG AGCCATACCACAT

ATACTCCAGCACATCTTGCCCCACCACCTGTGTCCACGTTCCATAGCT W,TGGAAATATCCTCCTGCGTTGAAAGGTTGTTGATAAAGGTAGTGGGCTGC 4400IAGGTGGAACGGGTGCCGTC& TCAAACCTTTATAGGAGGACGCAACTTTCCAACAACTATTTCCATCACCCGACG=TCACTACTGTCAATGAGTTCGAAATCTCCAGATGGTGTTTGAATATAATATTGGGATAGAAACGCAAACAATTCATCTGGAATATCAGCTTGGATGTGTA 4500AGTGATGACAGTTACTCAAGCTTTAGAGGTCTACCACAAACTTATATTATAACCCTATCTTTGCGTTTGTTAAGTAGACCTTATAGTCGAACCTACACAT

CGTACTTGGCTCTAGCCACGTTACTTTCCACCGTATCCTTACCTGCACTCCAATTACCAAAAGGGCTTACCCACTGACCATGATTAGTGTGGCTTAAGTA 4600GCATGAACCGAGATCGGTGCAATGAAAGGTGGCATAGGAATGGACGTGAGGTTAATGGTTTTCCCGAATGGGTGACTGGTACTAATCACACCGAATTCAT

CGAAGACATCAAGTGCCGCCCCAAATGGGACTTGGGGCCATGCTTGATACCAAATACAGCTTGCTTAGCGTGCGTGGCTATAAATCTGTCATAATTGGTG 4700GCTTCTGTAGTTCACGGCGGGGTTTACCCTGAACCCCGGTACGAACTATGGTTTATGTCGAACGAATCGCACGCACCGATATTTAGACAGTATTAACCAC

AGCAGAGAATCACGCAGCATCTGCCATTCTTGATCTGTTTTCTGATGAGATGCTCTGCTTTTGGGTACTGGCTCAGCTTCCGAGAAAAGATCATGGAGAG 4800TCGTCTCTTAGTGCGTCGTAGACGGTAAGAACTAGACAAAAGACTACTCTACGAGACGAAAACCCATGACCGAGTCGAAGGCTCTTTl'CTAGTACCTCTC

CTATTAAAGGATCTGTTTTCTTATTGACGGGGAAAAAGTATATGTATCGTGGCTTTCGGGTTTTAGTCTCGCACACTAGAACCCTTAATACACGATTCAA 4900GATAATTTCCTAGACAAAAGAATAACTGCCCCTTTTTCATATACATAGCACCGAAAGCCCAAAATCAGAGCGTGTGATCITgGGAzATTATgTGCTAAGTT

ATACCTGTTCTTCACCAATTCGAATTTCGTTGGATCTGCATTTTTCAAATCACTGTGTCTGCTGCAGTTAAAAAAAGATGTTATCAACACATATTTTAAT 5000TATGGACAAGAAGTGGTTAAGCTTAAAGCAACCTAGACGTAAAAAGTTTAGTGACACAGACGACGTCAATTTTTTTrCTACAATAGTTGTGTATAAAATTA

TCAGCTCTGAGAGACCGTTTAAACGCACGTTTTTGAATCGTCACTTCTACACCTGACAGTAGAGTCCAGATATCATCTGCTCTTACAATTTTGGTGATCT S100AGTCGAGACTCTCTGGCAAATTTGCGTGCAAAAACTTAGCAGTGAAGATGTGGACTGTrATCTCAGGTCTATATGnA AATC.1TI tCATG

CCTCCGCGACTGCGTTGTGCCCGCCTTTACCTTCTACTTTGTCCATTTTAGCCGCCAATTCAACCATTGACTCAACTTTTTTGGTTGACGATACTTCGAA 5200GGAGGCGCTGACGCAACACGGGCGGAAATGGAAGATGAAACAGGTAAAATCGGCGGTTAAGTTGGTAACTGAGTTGAAAAAACCAACTGCTATGAAGCTT

TACTATTGGCGCAAATGTCTTATCCAAGACATCTTTTAGCTCATGTGGTCGCTTCAGATGATAATTGAATTTTACTATTTGCGCTTCTCTGTCATATTGT 5300ATGATAACCGCGTTTACAGAATAGGTTCTGTAGAAAATCGAGTACACCAGCGAAGTCTACTATTAACTTAAAATGATAAACGCGAAGAGACAGTATAACA

ACTGATTGTCTTATCTCCCGCGAGTACTTGATGAATGTGCTACGCTTTCTTTCACTCAATTTACTGAAGGTAGCCATTATCACGTATGCTAGCAGAGACG 5400TGACTAACAGAATAGAGGGCGCTCATGAACTACTTACACGATGCGAAAGAAAGTGAGTTAAATGACTTCCATCGGTAkATAGTGCATACGATCGTCTCTGCCGAACTTCTCACTGGGF

GCTTGAAGAGTGACCC~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~ 20

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PHYLOGENETIC RELATIONSHIPS AMONG YEAST PLASMIDS

_ _ -I r I

pSB2 G A G T T T C A T T A A GIG A A TIA A G T AIA T T CIC C T A A T G A A A C T C

pS83 G G T T G C T T A AIG A A TIA A G T AIA T T CiT T A A G C A A C C

pSM1 G A A A T G G A A A G1G A A T G G T T CIA T T CIC T T T C C A T T T C

pSRI T T G A T G A A A,G A A TA C G T TA T TCTT T T C A T C A A

pKDI ... C A T T T G T C T G ATlA ATIGAAGCIATTIATCAGACAAATG ...

2,um G A A G T T C C T A T T C T C T A G A A A G T A T A G G A A C T T C

FIG. 5. Sequence comparison of putative recombination sites.Part of the IRs of pKD1 is shown. The block of nucleotides boxed bythe dashed lines shows a conserved sequence seen in all plasmidsexcept 2p.m. This block is tentatively placed at the border of the IRs.

uration, a circular molecule carrying a pair of IRs, morework is required to reach this conclusion.

ACKNOWLEDGMENTS

We thank R. Wickner for his critical reading of the manuscript.Part of this study was supported by a Grant-in-Aid for Scientific

Research from the Ministry of Education, Science, and Culture,Japan.

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