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Rapid identification of Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii
international Journal of Food Microbiology, 16 (i 992) 63-67 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1605/92/$05.00
63
FOOD 00512
Rapid identification of Saccharomyces
cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii B.M. Pearson and R.A. McKee AFRC Institute of Ftual Research. Norwich Laboratory, Norwich Research Park, Colne~: Norwich, England, UK (Received 5 August 1991: accepted 17 April 1992)
Most strains of Saccharomyces cerel'isiae. Zygosaccharomyces bailii and Zygosaccharomyces rm~xii have been found to contain plasmid DNA. The sequences of the plasmids from these three yeasts are known to be different. We have used two primers within the plasmid from each yeast species in a multiplex polymerase chain reaction (PCR) to discriminate between these three yeasts. The primers were designed to give easily distinguishable fragment sizes when run on a simple agarose gel. Due to the sensitivity of PCR, crude cells can be used with no need to isolate DNA. The method is rapid when compared with current methods. Key words: Yeast: Saccharomyces; Zygosaccharomyces: Identification: Polymerase chain reaction
Introduction Yeasts of the Zygosaccharomyces genus are well known for their spoilage of food products. T h e i r ability to grow in low water activity environments, often in the presence of preservatives, makes these yeasts a problem. Both Z. bailii and Z. rouxii were shown to contain plasmids (Painting and Kirsop, 1984; T o h e - e et al., 1982; T o h c - e et al., 1984) which are similar in structure to the 2 / ~ m circle of S. cerevisiae. All these plasmids have inverted repeats, exist in two isomeric forms through intramolecular recombination and are approximately 6000 bp in length (Araki et al., 1985; Utatsu et al., 1987). Generally these yeasts are difficult to separate and any differences are based on the assimilation of a relatively small n u m b e r of sugars. Despite this, low D N A homologies are found between the various Zygosaccharomyces species (Kurtzman, 1990). A m e t h o d which could be rapidly deployed to specifically identify these yeasts would be of benefit.
Correspondence address: B.M. Pearson, AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, England, UK. Telephone, 0603 56122.
With the development of the polymerase chain reaction (PCR) (Mullis and Faloona, 1987; Saiki ct el., 1988) it is now possible to detect very low quantities of DNA. The amplification of target sequences is made possible by the thermostable Taq polymerase and oligonucleotide primers specific to the ends of the target DNA sequence. We have designed and made primers to regions within the plasmids of S. ceret'isiae, Z. bailii and Z. rouxii. These primers can be used together in a multiplex PCR to amplify up specific lengths of target sequence. In our system we have arranged the primers so that they give rise to fragments which correspond in length with a standard DNA which is run alongside on each agarose gel as a marker. Materials and Methods Oligonucleotide primers were synthesized using phosphoramidite chemistry on an Applied Biosystems 381A DNA synthesizer. These were resuspended to a concentration of 1000 p.g/ml. The primer sequences were chosen so that their annealing temperatures were similar, their 3' base was the same, and they amplified a sequence which did not contain any of the plasmid inverted repeats. In addition, primers were checked for secondary structure and cross complementarity so that the mix of six primers could be used in the same multiplex PCR reaction. The primers chosen are shown in Table I. Primers 1 and 2 will hybridize with the 2 p.m plasmid of S. ceret'isiae and give rise to a 310 bp fragment. Primers 3 and 4 will hybridize with the pSB2 plasmid of Z. bailii and give rise to a 603 bp fragment. Primers 5 and 6 will hybridize with the pSRI plasmid of Z. rouxii and give rise to a 872 bp fragment. Following growth on YEPD plates (containing per litre: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar) a small sample of colony was removed. Typical samples contained approximately 100000 cells. The sample was then resuspended in 10 pA of water on ice. Ninety p.I of PCR mix was then added and the reaction mix covered with 60 /.tl of sterile paraffin oil overlay. The final reaction mix contains: 10 mM Tris-HCI, pH 9.0, 50 mM KCI, 1.5 mM MgCI2, 0.1% gelatin (w/v), 0.1% Triton X-100, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTI'P and 200 ng of cach of the six primers. One unit of Promega Taq polymerase is used per reaction. The polymerase chain reaction was carried out in a Hybaid TABLE 1 Primers used in multiplex PCR Primer I Primer 2 Primer 3 Primer 4 Primer 5 Primer 6
5'GGAGCACCTAATAACATI'CT3' 5'ACAGTVFATATCGGTI'GCATI"3' 5' CATI"GTI'GCTGAGAATGC'IT 3' 5'TCI'CTCTCAATACGTCGAT 3' 5'AGTi'CTGTACTTCCACAGAACT3' 5' TYCTCACTCTGTCATATGCT 3'
TABLE !! Strains used fi)r analysis
Saccharomyccs cercl'isiae
NCYC 234. NCYC 241. NCY(" IIX)2, NCYC 1027. NCYC 161)5. NCYC 16(18. NCYC 128. NCYC 417, NCYC 563, NCYC 580, NCYC 14iX). NCYC 1427. NCYC 152(I, NCYC 1521. NCYC 1553. NCYC 1554, NCYC 1555, NCYC 1556, NCYC 1557, NCYC 1558, NCYC 1766. NCYC 1495. NCYC 381. NCYC 561. NCYC 564, NCYC 568. NCYC .569. NCYC 579, NCYC 581, NCYC 582, NCYC 583. NCYC 72(I, NCYC 1508.
Zygosaccharomyccs bailii
Zygosaccharomycc.s" bi,~poru,~ Z):gosaccharomyccs rotL~'ii
thermocycler. The cycling parameters were as follows: 92°C for 2 min, 55°C for 3 rain, 72°C for 2 min and repeated for 30 cycles. Following the polymerase chain reaction, 10 p.I of sample was removed mixed with 2 /zl loading dye containing 40% sucrose, 0.2% bromophenol blue. The sample was then run on a 1.5% agarose gel at 100 V for 2 h, stained with 1 ~.g/ml ethidium bromide and photographed under UV (220 nm) light illumination.
1
2
3
4
5
6
lO~I~
~-
,
6mi~
3~1~11~ l~Ibp
Fig. I. Different PCR fragments visualised under U V light. Lanes 1 and 6, ~ X 1 7 4 H a e l i l D N A markers; lane 2, Zygosaccharomyces bispon~s NCYC 1495: lane 3, Succhuromyces ceret'isiae NCYC 1027: lane 4, Zygosaccharom)ces bailii NCYC 1427: lane 5, Zygosaccharomyces rowdi NCYC 568.
66 Yeast (Table
strains were
all obtained
from
the l~Iational Collection
of Yeast
Cultures
il).
Results a n d D i s c u s s i o n All
strains
produced
were
were
analysed
all strong
and
using
the
sharp
without
desc:ibed
PCR
any fragment
technique. length
The
T A B L E !!1 T h e results after P C R fragments were run on a 1.5% agarose gel (Fig. 1)
Present classification
Saccharomyces ceret'isiae N C Y C Saccharomyces ceret'isiae N C Y C Saccharomyces cerel'isiae N C Y C Saccharomyces ceret'isiae NCYC Saccharomyces ceret'isiae N C Y C Saccharomyces cerel'isiae N C Y C
Fragments observed 234 241 1002 1027 1605 1608
Zygosaccharomyces bailii N C Y C 128 Zygosaccharomyces bailii N C Y C 417 Zygosaccharomyces bailii N C Y C 563 Zygosaccharomyces bailii N C Y C 580 Zygosaccharomyces bailii N C Y C 1400 Zygosaccharomyces bailii N C Y C 1427 Zygosaccharomyces bailii N C Y C 1520 Zygosaccharomyces bailii N C Y C 1521" Zygosaccharomyces bailii N C Y C 1553 Zygosaccharomyces bailii N C Y C 1554 Zygosaccharomyces bailii N C Y C 1555" Zygosaccharomyces bailii N C Y C 1556 Zygosaccharomyces bailii N C Y C 1557 Zygosaccharomyces bailii N C Y C 1558 Zygosaccharomyces bailii N C Y C 1766 Zygosaccharomyces bisponts N C Y C 1495 Zygosaccharomyces rouxii N C Y C 381 Zygosaccharomyces rouxii N C Y C 561" Zygosaccharomyces rouxii N C Y C 564 Zygosaccharomyces rowcii N C Y C 568 Zygosaccharomyces rouxii N C Y C 569 Zygosaccharomyces rouxii N C Y C 579* Zygosaccharomyces rouxii N C Y C 58 I Zygosaccharomyces rouxii N C Y C 582" Zygosaccharomyces rouxii N C Y C 583 Zygosaccharomyces rouxii N C Y C 720 Zygosaccharomyces rouxii N C Y C 1508
310 bp
603 bp
+ + + + + +
-
-
+ + + + + + + + + + + + +
bands
polymorphisms
872 bp
within the groups. Figure 1 illustrates the clear distinction between the different fragment sizes. it can be seen from Figure 1 that S. ceret'isiae N C Y C 1027 gives rise to a 310 bp fragment, Z. bailii N C Y C 1427 a 603 bp fragment and Z. roaxii N C Y C 568 a 872 bp fragment. A closely related Z. bispon~ N C Y C 1495 was included as a negative control. T h e results are summarized in Table !il. W h e r e the expected fragment was not observed, m a r k e d by * in Table !il, the isolates were examined further. It was clear from the sugar assimilation data available on these strains from NCYC, that the identification of different Zygosaccharornyces sp. is very problematical. However, we noted that both the strains N C Y C 1521 and N C Y C 1555 produced only 2 spores per ascus where the others tested produced 4 spores per a ~ u s . This suggested that these may be Z. bisporus. T h e Z. rouxii group was expected to be diverse as it had been subjected to a great deal of reclassification, the species now has 48 different synonyms (Barnett et al., 1990). T h e Z. rouxii isolates which tested negative had originally b e e n deposited under different names: N C Y C 561 Zygosaccharornyces t~ariabilis, N C Y C 579 Zygosaccharomyces barkeri, N C Y C 582 Zygosaccaromyces amaeboidetts, in the light of our findings we believe these isolates should be examined more closely, p e r h a p s looking at D N A homology. It is clear that the m e t h o d is very good at identifying these three yeasts. T h e s e plasmids are all confined to their own species so this method should be very reliable at positively identifying these yeasts. T h e only problem is the possible lack of plasmids in some strains. Since the isolates tested were all derived from different types of food products it should be possible to use this technique to rapidly classify yeasts found in a variety of food environments.
References
Araki, H., Jearnpipatkul, A., Tatsumi. T., Ushio, T.S.H., Muta, T. and Oshima, Y. (1985) Molecular and functional organization of yeast plasmid pSRI. J. Mol. Biol. 182, 191-203. Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeasts: characteristics and identification. Cambridge University Press, Cambridge, pp. 693-694. Kurtzman, C.P. (1990) DNA relatedne~ among species of the genus Zygosaccharomyces. Yeast 6, 213-219. Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol. 155, 335-350. Painting, K.A. and Kirsop, B. (1984) A note on the presence of novel DNA species in the spoilage yeasts Zygosaccharomycesbailii and Pichiamerabranefaciens. J. Appl. Bacteriol 56, 331-335. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-494. Tohe-e, A., Araki, H., Utatsu, I. and Oshima, Y. (1984) Plasmids resembling 2-micron DNA in the osmotolerant yeasts Saccharomyces bailii and Sacchar~myces bispon~s. J. Gen. Microbiol 130, 2527-2534. Tohe-e, A., Tada, S. and Oshima, Y. (1982) 2-micron DNA-like plasmids in the osmophilic haploid yeast Saccharomycesrouxii. J. Bacteriol. 151, 1380-1390. Utatsu, !., Sakamo',o, S., lmura, T. and Tohe-e, A. (1987) Yeast plasmids resembling 2-micron DNA: r~,gional similarities and diversities at the molect.lar level. J. Bacteriol. 169, 5537-5545.
63
FOOD 00512
Rapid identification of Saccharomyces
cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii B.M. Pearson and R.A. McKee AFRC Institute of Ftual Research. Norwich Laboratory, Norwich Research Park, Colne~: Norwich, England, UK (Received 5 August 1991: accepted 17 April 1992)
Most strains of Saccharomyces cerel'isiae. Zygosaccharomyces bailii and Zygosaccharomyces rm~xii have been found to contain plasmid DNA. The sequences of the plasmids from these three yeasts are known to be different. We have used two primers within the plasmid from each yeast species in a multiplex polymerase chain reaction (PCR) to discriminate between these three yeasts. The primers were designed to give easily distinguishable fragment sizes when run on a simple agarose gel. Due to the sensitivity of PCR, crude cells can be used with no need to isolate DNA. The method is rapid when compared with current methods. Key words: Yeast: Saccharomyces; Zygosaccharomyces: Identification: Polymerase chain reaction
Introduction Yeasts of the Zygosaccharomyces genus are well known for their spoilage of food products. T h e i r ability to grow in low water activity environments, often in the presence of preservatives, makes these yeasts a problem. Both Z. bailii and Z. rouxii were shown to contain plasmids (Painting and Kirsop, 1984; T o h e - e et al., 1982; T o h c - e et al., 1984) which are similar in structure to the 2 / ~ m circle of S. cerevisiae. All these plasmids have inverted repeats, exist in two isomeric forms through intramolecular recombination and are approximately 6000 bp in length (Araki et al., 1985; Utatsu et al., 1987). Generally these yeasts are difficult to separate and any differences are based on the assimilation of a relatively small n u m b e r of sugars. Despite this, low D N A homologies are found between the various Zygosaccharomyces species (Kurtzman, 1990). A m e t h o d which could be rapidly deployed to specifically identify these yeasts would be of benefit.
Correspondence address: B.M. Pearson, AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, England, UK. Telephone, 0603 56122.
With the development of the polymerase chain reaction (PCR) (Mullis and Faloona, 1987; Saiki ct el., 1988) it is now possible to detect very low quantities of DNA. The amplification of target sequences is made possible by the thermostable Taq polymerase and oligonucleotide primers specific to the ends of the target DNA sequence. We have designed and made primers to regions within the plasmids of S. ceret'isiae, Z. bailii and Z. rouxii. These primers can be used together in a multiplex PCR to amplify up specific lengths of target sequence. In our system we have arranged the primers so that they give rise to fragments which correspond in length with a standard DNA which is run alongside on each agarose gel as a marker. Materials and Methods Oligonucleotide primers were synthesized using phosphoramidite chemistry on an Applied Biosystems 381A DNA synthesizer. These were resuspended to a concentration of 1000 p.g/ml. The primer sequences were chosen so that their annealing temperatures were similar, their 3' base was the same, and they amplified a sequence which did not contain any of the plasmid inverted repeats. In addition, primers were checked for secondary structure and cross complementarity so that the mix of six primers could be used in the same multiplex PCR reaction. The primers chosen are shown in Table I. Primers 1 and 2 will hybridize with the 2 p.m plasmid of S. ceret'isiae and give rise to a 310 bp fragment. Primers 3 and 4 will hybridize with the pSB2 plasmid of Z. bailii and give rise to a 603 bp fragment. Primers 5 and 6 will hybridize with the pSRI plasmid of Z. rouxii and give rise to a 872 bp fragment. Following growth on YEPD plates (containing per litre: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar) a small sample of colony was removed. Typical samples contained approximately 100000 cells. The sample was then resuspended in 10 pA of water on ice. Ninety p.I of PCR mix was then added and the reaction mix covered with 60 /.tl of sterile paraffin oil overlay. The final reaction mix contains: 10 mM Tris-HCI, pH 9.0, 50 mM KCI, 1.5 mM MgCI2, 0.1% gelatin (w/v), 0.1% Triton X-100, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTI'P and 200 ng of cach of the six primers. One unit of Promega Taq polymerase is used per reaction. The polymerase chain reaction was carried out in a Hybaid TABLE 1 Primers used in multiplex PCR Primer I Primer 2 Primer 3 Primer 4 Primer 5 Primer 6
5'GGAGCACCTAATAACATI'CT3' 5'ACAGTVFATATCGGTI'GCATI"3' 5' CATI"GTI'GCTGAGAATGC'IT 3' 5'TCI'CTCTCAATACGTCGAT 3' 5'AGTi'CTGTACTTCCACAGAACT3' 5' TYCTCACTCTGTCATATGCT 3'
TABLE !! Strains used fi)r analysis
Saccharomyccs cercl'isiae
NCYC 234. NCYC 241. NCY(" IIX)2, NCYC 1027. NCYC 161)5. NCYC 16(18. NCYC 128. NCYC 417, NCYC 563, NCYC 580, NCYC 14iX). NCYC 1427. NCYC 152(I, NCYC 1521. NCYC 1553. NCYC 1554, NCYC 1555, NCYC 1556, NCYC 1557, NCYC 1558, NCYC 1766. NCYC 1495. NCYC 381. NCYC 561. NCYC 564, NCYC 568. NCYC .569. NCYC 579, NCYC 581, NCYC 582, NCYC 583. NCYC 72(I, NCYC 1508.
Zygosaccharomyccs bailii
Zygosaccharomycc.s" bi,~poru,~ Z):gosaccharomyccs rotL~'ii
thermocycler. The cycling parameters were as follows: 92°C for 2 min, 55°C for 3 rain, 72°C for 2 min and repeated for 30 cycles. Following the polymerase chain reaction, 10 p.I of sample was removed mixed with 2 /zl loading dye containing 40% sucrose, 0.2% bromophenol blue. The sample was then run on a 1.5% agarose gel at 100 V for 2 h, stained with 1 ~.g/ml ethidium bromide and photographed under UV (220 nm) light illumination.
1
2
3
4
5
6
lO~I~
~-
,
6mi~
3~1~11~ l~Ibp
Fig. I. Different PCR fragments visualised under U V light. Lanes 1 and 6, ~ X 1 7 4 H a e l i l D N A markers; lane 2, Zygosaccharomyces bispon~s NCYC 1495: lane 3, Succhuromyces ceret'isiae NCYC 1027: lane 4, Zygosaccharom)ces bailii NCYC 1427: lane 5, Zygosaccharomyces rowdi NCYC 568.
66 Yeast (Table
strains were
all obtained
from
the l~Iational Collection
of Yeast
Cultures
il).
Results a n d D i s c u s s i o n All
strains
produced
were
were
analysed
all strong
and
using
the
sharp
without
desc:ibed
PCR
any fragment
technique. length
The
T A B L E !!1 T h e results after P C R fragments were run on a 1.5% agarose gel (Fig. 1)
Present classification
Saccharomyces ceret'isiae N C Y C Saccharomyces ceret'isiae N C Y C Saccharomyces cerel'isiae N C Y C Saccharomyces ceret'isiae NCYC Saccharomyces ceret'isiae N C Y C Saccharomyces cerel'isiae N C Y C
Fragments observed 234 241 1002 1027 1605 1608
Zygosaccharomyces bailii N C Y C 128 Zygosaccharomyces bailii N C Y C 417 Zygosaccharomyces bailii N C Y C 563 Zygosaccharomyces bailii N C Y C 580 Zygosaccharomyces bailii N C Y C 1400 Zygosaccharomyces bailii N C Y C 1427 Zygosaccharomyces bailii N C Y C 1520 Zygosaccharomyces bailii N C Y C 1521" Zygosaccharomyces bailii N C Y C 1553 Zygosaccharomyces bailii N C Y C 1554 Zygosaccharomyces bailii N C Y C 1555" Zygosaccharomyces bailii N C Y C 1556 Zygosaccharomyces bailii N C Y C 1557 Zygosaccharomyces bailii N C Y C 1558 Zygosaccharomyces bailii N C Y C 1766 Zygosaccharomyces bisponts N C Y C 1495 Zygosaccharomyces rouxii N C Y C 381 Zygosaccharomyces rouxii N C Y C 561" Zygosaccharomyces rouxii N C Y C 564 Zygosaccharomyces rowcii N C Y C 568 Zygosaccharomyces rouxii N C Y C 569 Zygosaccharomyces rouxii N C Y C 579* Zygosaccharomyces rouxii N C Y C 58 I Zygosaccharomyces rouxii N C Y C 582" Zygosaccharomyces rouxii N C Y C 583 Zygosaccharomyces rouxii N C Y C 720 Zygosaccharomyces rouxii N C Y C 1508
310 bp
603 bp
+ + + + + +
-
-
+ + + + + + + + + + + + +
bands
polymorphisms
872 bp
within the groups. Figure 1 illustrates the clear distinction between the different fragment sizes. it can be seen from Figure 1 that S. ceret'isiae N C Y C 1027 gives rise to a 310 bp fragment, Z. bailii N C Y C 1427 a 603 bp fragment and Z. roaxii N C Y C 568 a 872 bp fragment. A closely related Z. bispon~ N C Y C 1495 was included as a negative control. T h e results are summarized in Table !il. W h e r e the expected fragment was not observed, m a r k e d by * in Table !il, the isolates were examined further. It was clear from the sugar assimilation data available on these strains from NCYC, that the identification of different Zygosaccharornyces sp. is very problematical. However, we noted that both the strains N C Y C 1521 and N C Y C 1555 produced only 2 spores per ascus where the others tested produced 4 spores per a ~ u s . This suggested that these may be Z. bisporus. T h e Z. rouxii group was expected to be diverse as it had been subjected to a great deal of reclassification, the species now has 48 different synonyms (Barnett et al., 1990). T h e Z. rouxii isolates which tested negative had originally b e e n deposited under different names: N C Y C 561 Zygosaccharornyces t~ariabilis, N C Y C 579 Zygosaccharomyces barkeri, N C Y C 582 Zygosaccaromyces amaeboidetts, in the light of our findings we believe these isolates should be examined more closely, p e r h a p s looking at D N A homology. It is clear that the m e t h o d is very good at identifying these three yeasts. T h e s e plasmids are all confined to their own species so this method should be very reliable at positively identifying these yeasts. T h e only problem is the possible lack of plasmids in some strains. Since the isolates tested were all derived from different types of food products it should be possible to use this technique to rapidly classify yeasts found in a variety of food environments.
References
Araki, H., Jearnpipatkul, A., Tatsumi. T., Ushio, T.S.H., Muta, T. and Oshima, Y. (1985) Molecular and functional organization of yeast plasmid pSRI. J. Mol. Biol. 182, 191-203. Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeasts: characteristics and identification. Cambridge University Press, Cambridge, pp. 693-694. Kurtzman, C.P. (1990) DNA relatedne~ among species of the genus Zygosaccharomyces. Yeast 6, 213-219. Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol. 155, 335-350. Painting, K.A. and Kirsop, B. (1984) A note on the presence of novel DNA species in the spoilage yeasts Zygosaccharomycesbailii and Pichiamerabranefaciens. J. Appl. Bacteriol 56, 331-335. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-494. Tohe-e, A., Araki, H., Utatsu, I. and Oshima, Y. (1984) Plasmids resembling 2-micron DNA in the osmotolerant yeasts Saccharomyces bailii and Sacchar~myces bispon~s. J. Gen. Microbiol 130, 2527-2534. Tohe-e, A., Tada, S. and Oshima, Y. (1982) 2-micron DNA-like plasmids in the osmophilic haploid yeast Saccharomycesrouxii. J. Bacteriol. 151, 1380-1390. Utatsu, !., Sakamo',o, S., lmura, T. and Tohe-e, A. (1987) Yeast plasmids resembling 2-micron DNA: r~,gional similarities and diversities at the molect.lar level. J. Bacteriol. 169, 5537-5545.