Current Genetics
Current Genetics (1982) 5:187 -189
© Springer-Verlag 1982
Two Extramitochondrial Circular DNA Species in the Petite Negative Yeast Schizosaccharomycespombe: Relative Abundance and Size Determination by Electron Microscopy F. Manna 1 , L. Del Giudice a , W. H. Schrell ~, and K. Wolf2 1 Istituto Internazionale di Genetica e Biofisica, Consiglio Nazionale delle Ricerche, Via G. Marconi 10, 80125 Napoli, Italy 2 Insfitut ftir Genetik und Mikrobiologieder Universit5t Miinchen, MariaWard Strage la, 8000 Mtinchen 19, Federal Republic of Germany
Summary. In this paper we present the electron microscopic analysis of two distinct extramitochondrial circular DNA species in the fission yeast Schizosaccharomyces pombe (S. pombe). Both DNA species can be isolated from mitochondrial fractions, but disappear after DNase treatment of mitochondria, demonstrating their extramitochondrial location. The size of these molecular species is 3.08 + 0.18 lain and 2.00 + 0.09 tim (standard deviation). They are present in a ratio of approximately 9 : 1 in the DNA preparations analyzed.
tion of DNA are described by Del Gindice et al. (1978). For electron microscopy the protein monolayer method of Kleinschmidt (1968) was used. DNA was stained with uranyl acetate and shadowed with carbon platinum. Electron micrographs were taken with a Siemens Elmiscop 102. Molecules were photographed at a magnification of 10,000 and 20-fold magnified projections were measured with a map ruler. DNA of phage lambda with a molecular weight of 30.8 x 106 (Davidson and Szybalski 1971) was used as internal standard.
Results and Discussion Key words: Extramitochondrial DNA - S. pombe
Introduction
In Saccharomyces cerevisiae (S. cerevisiae) and other lower eukaryotes, several extrachromosomal genetic elements have been described. Some of them, like the 2 micron "ribosomal" DNA (Clark-Walker and Azad 1980) have been physically characterized; the biological role of these molecules is still a matter of speculation. Other genetic elements like psi (McCready and McLaughfin 1977) and URE (Aigle and Lacroute 1975) have only been identified genetically. In this paper we describe the electron microscopic analysis of 2 micron and 3 micron circular molecules in the fission yeast S. pombe. Materials and Methods Strain ade7-5Oh- (U. Leupold, Berne) was used. Preparation of protoplasts, purification of mitochondrial fractions, and isola-
Offprint requests to: L. Del Giudiee Editorial correspondence to: K. Wolf
Cells of strain ade7-5Oh- were converted into protoplasts and mitochondrial fractions were isolated by differential centrifugation. Mitochondria were opened by osmotic shock and DNA species inside and associated with mitochondrial structures prepared for electron microscopic analysis. Physical characterization of mitochondrial DNA from DNase treated mitochondrial fractions has recently been described by Manna et al. (1982). In addition, two distinct molecular species of approximately 2 and 3 micron circumference were detected in preparations without DNase treatment (Fig. 1). The absence of these two species in DNase-treated mitochondrial fractions clearly demonstrates their extramitochondrial location. Evaluation of 87 small circular molecules from four independent preparations exhibited similar ratios of 2 micron versus 3 micron molecules of approximately 1 : 9/ We have measured the size of 10 molecules of the 2 micron species and determined an average length of 2.00 -+ 0.09 pm (standard deviation). One of these molecules with a circumference of 1.91 pm is shown in Fig. 1C. Del Giudice et al. (1979) have discussed the possibility that the 2 micron DNA is homologous to the 2 micron DNA is homologous to the 2 micron DNA in S. cerevisiae. P. Philippsen (personal communication) could demonstrate that radioactively labelled 2 micron DNA O172-8083/82/0005/0187/$ O1.00
F. Manna et al.: Circular DNA in S. pombe
188
from S. cerevisiae did not hybrizide with total DNA of S. pombe. This rules out that these two molecular species are homologous. Work is in progress to clone and characterize the 2 micron DNA of S. pombe. The other type of molecule with approximately 3 micron circumference represents the major class of small circular molecules in mitochondrial fractions. Sixteen molecules of this type were measured yielding an average size of 3.08 +-0.18 pro. Using Student's t-test it could be shown that the size difference between the molecular species is highly significant. Figures 1A and 1B show molecules with a circumference of 2.92 and 3.03/Jan, respectively. Evidence for the existence of a 3 micron extramitochondrial DNA species was provided also by P. Q. Anziano, P. S. Perlman, B. F. Lang, and K. Wolf (personal communication) by restriction enzyme analysis o f mitochondrial DNA preparations. In HindIII digest of mitochondrial DNA from mitochondrial fractions not treated with DNase, they observed a weak band corresponding to a molecular weight of about 12 kilobase pairs. Using DNase-treated mitochondria they failed to observe this band. Independent electron microscopic analysis revealed that this 3 micron circular species was at least as abundant as mitochondrial DNA in these preparations. Fournier et al. (1982) have cloned this molecule in the plasmid pBR322. By restriction enzyme analysis they determined the size of this DNA species to be 10.8 kilobase pairs with a unique HindIII-site. They also could show that the 3 micron species hybridized with ribosomal DNA from S. cerevisiae. Thus this 3 micron DNA is very likely homologous to molecules of the same size found in S. cerevisiae (Clark-Walker and Azad 1980; Larinov et al. 1980) and Torulopsis glabrata (Clark-Walker and Azad 1980). The biological role of both 2 and 3 micron DNA is S. cerevisiae, Torulopsis glabrata, and S. pombe is uncertain. There are strains of S. cerevisiae which lack one or the other molecular species, indicating that neither 2 nor 3 micron DNA is essential for this organism. A systematic analysis of different S. pombe strains will reveal if these molecular species are always present or not.
Acknowledgement. The technical assistance of Mr. G. De Simone is acknowledged.
References
Fig. 1 A-C. Electron microgIaphs of small circular extramitochondrial DNA molecules. A contour length 2.92 t~m.B contour length 3.03 ~m. C contour length 1.91 t~m. The bar represents 1 ;zrn
Aigle M, Lacroute F (1975) Mol Gen Genet 136:327-335 Broach JR (1982) Cell 28 : 203-204 Clark-Walker GD, Azad AA (1980) Nucleic Acids Res 8 : 10091022 Davidson N, Szybalski W (...) In: Hershey AD (ed) The Bacteriophage Lambda. Cold Spring Harbor Laboratories, New York, p 45
F. Manna et al.: Circular DNA in S. pombe Del Giudice L, Wolf K, Sassone-Corsi P, Alvino C (1978) Mol Gen Genet 164:289-293 Del Giudice L, Wolf K, Sassone-Corsi P, Mazza A (1979) Mol Gen Genet 172 : 165-169 Fournier P, Gaillardin C. de Louvencourt L, Heslot H, Lang B, Kaudewitz F (1982) Curt Genet Kleinschmidt AK (1968) In: Kaplan NO, Colwick SP (eds) Methods in Enzymology XIIB. Academic Press, New York, p 361 Larinov VL, Gristfin AV, Smirnov MN (1980) Gene 12:41-49 Manna F, Del Giudice L, Schreil NH, Wolf K (1982) Mol Gen Genet 184:469-470
189 McCready SJ, McLaughlin CS (1977) Biocl-dm Biophys Acta 479:119 121 Wolf K, Lang B, Del Giudice L, Anziano PQ, Perlman PS (1982) Cold Spring Harbor Symposium Quant Biol (in press)
Communicated b y C. W. Birky, Jr. Received April 14 / May 20, 1982
Current Genetics (1982) 5:187 -189
© Springer-Verlag 1982
Two Extramitochondrial Circular DNA Species in the Petite Negative Yeast Schizosaccharomycespombe: Relative Abundance and Size Determination by Electron Microscopy F. Manna 1 , L. Del Giudice a , W. H. Schrell ~, and K. Wolf2 1 Istituto Internazionale di Genetica e Biofisica, Consiglio Nazionale delle Ricerche, Via G. Marconi 10, 80125 Napoli, Italy 2 Insfitut ftir Genetik und Mikrobiologieder Universit5t Miinchen, MariaWard Strage la, 8000 Mtinchen 19, Federal Republic of Germany
Summary. In this paper we present the electron microscopic analysis of two distinct extramitochondrial circular DNA species in the fission yeast Schizosaccharomyces pombe (S. pombe). Both DNA species can be isolated from mitochondrial fractions, but disappear after DNase treatment of mitochondria, demonstrating their extramitochondrial location. The size of these molecular species is 3.08 + 0.18 lain and 2.00 + 0.09 tim (standard deviation). They are present in a ratio of approximately 9 : 1 in the DNA preparations analyzed.
tion of DNA are described by Del Gindice et al. (1978). For electron microscopy the protein monolayer method of Kleinschmidt (1968) was used. DNA was stained with uranyl acetate and shadowed with carbon platinum. Electron micrographs were taken with a Siemens Elmiscop 102. Molecules were photographed at a magnification of 10,000 and 20-fold magnified projections were measured with a map ruler. DNA of phage lambda with a molecular weight of 30.8 x 106 (Davidson and Szybalski 1971) was used as internal standard.
Results and Discussion Key words: Extramitochondrial DNA - S. pombe
Introduction
In Saccharomyces cerevisiae (S. cerevisiae) and other lower eukaryotes, several extrachromosomal genetic elements have been described. Some of them, like the 2 micron "ribosomal" DNA (Clark-Walker and Azad 1980) have been physically characterized; the biological role of these molecules is still a matter of speculation. Other genetic elements like psi (McCready and McLaughfin 1977) and URE (Aigle and Lacroute 1975) have only been identified genetically. In this paper we describe the electron microscopic analysis of 2 micron and 3 micron circular molecules in the fission yeast S. pombe. Materials and Methods Strain ade7-5Oh- (U. Leupold, Berne) was used. Preparation of protoplasts, purification of mitochondrial fractions, and isola-
Offprint requests to: L. Del Giudiee Editorial correspondence to: K. Wolf
Cells of strain ade7-5Oh- were converted into protoplasts and mitochondrial fractions were isolated by differential centrifugation. Mitochondria were opened by osmotic shock and DNA species inside and associated with mitochondrial structures prepared for electron microscopic analysis. Physical characterization of mitochondrial DNA from DNase treated mitochondrial fractions has recently been described by Manna et al. (1982). In addition, two distinct molecular species of approximately 2 and 3 micron circumference were detected in preparations without DNase treatment (Fig. 1). The absence of these two species in DNase-treated mitochondrial fractions clearly demonstrates their extramitochondrial location. Evaluation of 87 small circular molecules from four independent preparations exhibited similar ratios of 2 micron versus 3 micron molecules of approximately 1 : 9/ We have measured the size of 10 molecules of the 2 micron species and determined an average length of 2.00 -+ 0.09 pm (standard deviation). One of these molecules with a circumference of 1.91 pm is shown in Fig. 1C. Del Giudice et al. (1979) have discussed the possibility that the 2 micron DNA is homologous to the 2 micron DNA is homologous to the 2 micron DNA in S. cerevisiae. P. Philippsen (personal communication) could demonstrate that radioactively labelled 2 micron DNA O172-8083/82/0005/0187/$ O1.00
F. Manna et al.: Circular DNA in S. pombe
188
from S. cerevisiae did not hybrizide with total DNA of S. pombe. This rules out that these two molecular species are homologous. Work is in progress to clone and characterize the 2 micron DNA of S. pombe. The other type of molecule with approximately 3 micron circumference represents the major class of small circular molecules in mitochondrial fractions. Sixteen molecules of this type were measured yielding an average size of 3.08 +-0.18 pro. Using Student's t-test it could be shown that the size difference between the molecular species is highly significant. Figures 1A and 1B show molecules with a circumference of 2.92 and 3.03/Jan, respectively. Evidence for the existence of a 3 micron extramitochondrial DNA species was provided also by P. Q. Anziano, P. S. Perlman, B. F. Lang, and K. Wolf (personal communication) by restriction enzyme analysis o f mitochondrial DNA preparations. In HindIII digest of mitochondrial DNA from mitochondrial fractions not treated with DNase, they observed a weak band corresponding to a molecular weight of about 12 kilobase pairs. Using DNase-treated mitochondria they failed to observe this band. Independent electron microscopic analysis revealed that this 3 micron circular species was at least as abundant as mitochondrial DNA in these preparations. Fournier et al. (1982) have cloned this molecule in the plasmid pBR322. By restriction enzyme analysis they determined the size of this DNA species to be 10.8 kilobase pairs with a unique HindIII-site. They also could show that the 3 micron species hybridized with ribosomal DNA from S. cerevisiae. Thus this 3 micron DNA is very likely homologous to molecules of the same size found in S. cerevisiae (Clark-Walker and Azad 1980; Larinov et al. 1980) and Torulopsis glabrata (Clark-Walker and Azad 1980). The biological role of both 2 and 3 micron DNA is S. cerevisiae, Torulopsis glabrata, and S. pombe is uncertain. There are strains of S. cerevisiae which lack one or the other molecular species, indicating that neither 2 nor 3 micron DNA is essential for this organism. A systematic analysis of different S. pombe strains will reveal if these molecular species are always present or not.
Acknowledgement. The technical assistance of Mr. G. De Simone is acknowledged.
References
Fig. 1 A-C. Electron microgIaphs of small circular extramitochondrial DNA molecules. A contour length 2.92 t~m.B contour length 3.03 ~m. C contour length 1.91 t~m. The bar represents 1 ;zrn
Aigle M, Lacroute F (1975) Mol Gen Genet 136:327-335 Broach JR (1982) Cell 28 : 203-204 Clark-Walker GD, Azad AA (1980) Nucleic Acids Res 8 : 10091022 Davidson N, Szybalski W (...) In: Hershey AD (ed) The Bacteriophage Lambda. Cold Spring Harbor Laboratories, New York, p 45
F. Manna et al.: Circular DNA in S. pombe Del Giudice L, Wolf K, Sassone-Corsi P, Alvino C (1978) Mol Gen Genet 164:289-293 Del Giudice L, Wolf K, Sassone-Corsi P, Mazza A (1979) Mol Gen Genet 172 : 165-169 Fournier P, Gaillardin C. de Louvencourt L, Heslot H, Lang B, Kaudewitz F (1982) Curt Genet Kleinschmidt AK (1968) In: Kaplan NO, Colwick SP (eds) Methods in Enzymology XIIB. Academic Press, New York, p 361 Larinov VL, Gristfin AV, Smirnov MN (1980) Gene 12:41-49 Manna F, Del Giudice L, Schreil NH, Wolf K (1982) Mol Gen Genet 184:469-470
189 McCready SJ, McLaughlin CS (1977) Biocl-dm Biophys Acta 479:119 121 Wolf K, Lang B, Del Giudice L, Anziano PQ, Perlman PS (1982) Cold Spring Harbor Symposium Quant Biol (in press)
Communicated b y C. W. Birky, Jr. Received April 14 / May 20, 1982
