Cell, Vol . 9, 333-345, October 1976, Copyright J 1976 by MIT
Small Circular DNA in Drosophila melanogaster
Sharon Stanfield and Donald R . Helinski Department of Biology University of California, San Diego La Jolla, California 92093
Covalently closed small circular DNA isolated from Drosophila melanogaster is described . The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2 . It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2cells ranges from 0 .09-7 .3µm, with a mean contour length of 1 .1 µm . This DNA has a buoyant density of 1 .703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells . The restriction enzyme EcoRl cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes . Both logarithmic and stationary phase cells contain approximately 3-40 average sized small circular DNA molecules per cell, representing a maximum of 0 .03% of the total cellular DNA . Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0 .3 µm . The drug-amplified DNA has a buoyant density in the range of 1 .698-1 .703 g/cc. No amplification was seen in cells treated with either inhibitor for 3 .5 hr . Ethidium bromide, cytosine arabinoside, 8-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA .
Vinograd, 1972), or unlike that of any other DNA component in the cell (Nass and Ben-Shaul, 1972) . Similarly, the intracellular locations of these DNAs, where determined, were found to vary with the organism, with both cytoplasmic and mitochondrial locations having been reported . The most detailed analysis to date of small circular DNAs in higher organisms was performed by Smith and Vinograd (1972) on HeLa cells . They found that the small polydisperse circular DNAs (spcDNA) of HeLa cells were present in the cytoplasm although they were of the same buoyant density as the nuclear DNA . They also showed that the number of spcDNA molecules per cell could be greatly increased by permitting the cells to sit in the stationary phase for 2-5 days or by treating the cells with cycloheximide . The small circular DNA amplified in this way was indistinguishable in size and buoyant density from nonamplified circles . Pulse-chase experiments with cycloheximideamplified circles suggested that they arose from preexisting nuclear DNA . Because none of the higher eucaryotes in which small circular DNA has been found have been particularly well characterized genetically, we decided to look for small circular DNAs in Drosophila melanogaster, an organism that has been extensively characterized both genetically and biochemically. We report here our discovery of heterogeneous small circular DNAs both in eggs and in cultured cells of D . melanogaster . The size distribution, intracellular location, buoyant density, number of copies per cell, and effect of some hormones and inhibitors were investigated for the circular DNA from tissue culture . In addition, the complexity of the circular DNA was investigated using the restriction enzyme EcoRl .
Introduction
Results
Small circular DNAs of unknown function have been described in a variety of eucaryotes, including Neurospora (Agsteribbe, Kroon, and Van Bruggen, 1972) ; Euglena (Nass and Ben-Shaul, 1972) ; three species of trypanosomes (Ono et al ., 1971) ; Saccharomyces (Billheimer and Avers, 1969) ; tobacco (Wong and Wildman, 1972) ; Xenopus (BuongiornoNardelli, Amaldi, and Lava-Sanchez, 1976) ; boar sperm (Hotta and Bassel, 1965) ; and a number of mammalian cell culture lines including those from monkey, mouse, and man (Smith and Vinograd, 1972 ; Rush, 1973) . In the majority of these cases, the circles were found to be heterogeneous in size and, depending upon the organism from which they were isolated, had buoyant densities either like that of the nuclear and/or mitochondrial DNA (Billheimer and Avers, 1969 ; Ono et al ., 1971 ; Smith and
Small Circular DNA Molecules from Drosophila Eggs Small circular DNA molecules were found in the supercoiled DNA region of CsCI/ethidium bromide gradients of homogenates of D . melanogaster eggs at the blastema stage of development . Figure 1 shows the size distribution of 78 light-nicked, small circular DNA molecules from eggs . These DNAs range in size from 0 .13-2 .03 µm, with a mean length of 0 .6 µm . Greater than 90% of the molecules were 1 pm in contour length . Size determinations were based on a contour length of 6 .0 pm for Drosophila egg mitochondrial DNA (Peacock et al ., 1973) . The small circular molecules were judged to be covalently closed on the basis of their supercoiled appearance in the electron microscope and by their location in the CsCI/ethidium bromide gradient .
Summary
Cell 334
and usually a single chromosome IV where these can be seen . The three telecentrics consist of two long chromosomes, approximately equal in length, and a single shorter chromosome . The shorter chromosome and one of the long chromosomes may correspond to the unequal X chromosomes seen in the,parental line, while the third telocentric resembles an X chromosome of the larger type . The karyotype may therefore best be described as 2N + X and probably haplo IV with a variable number of polyploids (10-30%) . The predominant polyploid was due to a single complete duplication of the basic karyotype .
Karyotypes of the Tissue Culture Lines Two established lines of Drosophila cells were used in this study . One of these was line 2, originally described by Schneider in 1972 . The other was a cloned subline of line 2, hereafter referred to as line BD6C . About 80% of log-phase line 2 cells are diploid female, with the two X chromosomes being of unequal lengths . The remainder of the cells are polyploid . Comparisons of the lengths of the arms of the X chromosomes relative to the lengths of the large autosomes were consistent with the idea that the shorter X was normal in length and the longer X contained an extra piece of chromosome roughly one third the size of the normal X . The predominant karyotype of the subline BD6C consists of two pairs of metacentrics (chromosomes II and III), three telocentric chromosomes,
Small Circular DNA Molecules from Cultured Drosophila Cells Figure 2 shows the size distributions of small circu-
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Figure 1 . Contour Length Distribution of Small Circular DNA from Eggs Eggs were collected as described in Experimental Procedures and allowed to develop at 26°C, 100% relative humidity, until the blastema state of development was reached (about 2 hr) . The small circular DNA was purified from the eggs as described in Experimental Procedures . Drosophila egg mitochondrial DNA (6 .0 µm) served as an internal length standard .
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LENGTH IN MICRONS Figure 2 . Contour Length Distributions of Small Circular DNA from Schneider's Line 2 Cells in the Logarithmic and Stationary Phases of Growth (a) 210 small circular DNA molecules from log-phase cells ; (b) 440 small circular DNA molecules from stationary phase cells . Molecules from stationary phase cells were obtained from the region of nuclear DNA density of the analytical CsCI gradient described in Figure 5a . Circular molecules from log-phase cells were obtained from the closed circular DNA region of the second of two successive CsCl/ethidium bromide gradients .
Small Circular DNA in D . melanogaster 335
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LENGTH IN MICRONS Figure 3 . Contour Length Distributions of Small Circular DNA from the Cloned Line BD6C during the Logarithmic Phase of Growth Circular DNA was obtained from the supercoiled DNA peak of the second of two successive CsCl/ethidium bromide gradients . Drosophila mitochondrial DNA from line BD6C (6 .0 µm) was used as an internal length standard . (a) and (b) small circular DNA from two different preparations of log-phase cells .
lar DNA molecules purified from log-phase and stationary phase cells of line 2 . Drosophila mitochondria) DNA from line 2 cells and antibiotic resistance plasmid R6K DNA (12 .04 µm) were used as internal length standards for both sets of measurements . Mitochondria) DNA from cultured cells has been determined to have a contour length of 6 .0 µm, based on the length of R6K DNA (S . Stanfield, unpublished results) . This value differs from the 5 .5 µm value found by Bultman and Laird (1973) for mitochondria) DNA from embryos, but agrees with that found by Peacock et al . (1973) . The majority of the small circular DNAs from cells in both the log and stationary phases ranged from about 0 .1-3 .0 µm, with each distribution having one very large circular molecule outside of this range . The mean sizes did not differ significantly, being 1 .09 µm for circles from log-phase cells and 1 .14 µm for circles from stationary phase cells . Chisquare analysis of the size distributions using five arbitrary size groupings containing at least twenty molecules each indicated a probability of 31% that the two distributions came from the same population of molecules . Based on a variation of 10% in the contour lengths of the internal DNA standards, it was estimated that the small circular DNA size distributions are compatible with at least eleven distinct size classes . Since Schneider's line 2 consists of a very heterogeneous population of cell types, it was believed that different cell types within this population might possess distinct subpopulations of circular molecules . For this reason, we investigated the circular DNAs of a cloned subline of line 2 . The size distribution from log-phase cells was determined twice, separated by a period of 5 weeks or approximately ten cell transfers . As shown in Figures 3a and 3b, the circles ranged in size from 0 .08-3 .88 um . The means of the two determinations did not differ significantly, being 1 .37 µm for the first determination
(Figure 3a) and 1 .33 ttm for the second (Figure 3b) . However, the contour length profiles did show some differences . The most obvious of these was that the major peak at 1 .76 µm, seen in the first distribution, was present only as a minor class in the second distribution . Chi-square analysis of the two size distribution profiles of BD6C indicated a probability of 28% that they could have been due to a random sampling of the same population . However, the probability that either of these distributions came from the same population of molecules as did those from line 2 was
Small Circular DNA in Drosophila melanogaster
Sharon Stanfield and Donald R . Helinski Department of Biology University of California, San Diego La Jolla, California 92093
Covalently closed small circular DNA isolated from Drosophila melanogaster is described . The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2 . It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2cells ranges from 0 .09-7 .3µm, with a mean contour length of 1 .1 µm . This DNA has a buoyant density of 1 .703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells . The restriction enzyme EcoRl cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes . Both logarithmic and stationary phase cells contain approximately 3-40 average sized small circular DNA molecules per cell, representing a maximum of 0 .03% of the total cellular DNA . Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0 .3 µm . The drug-amplified DNA has a buoyant density in the range of 1 .698-1 .703 g/cc. No amplification was seen in cells treated with either inhibitor for 3 .5 hr . Ethidium bromide, cytosine arabinoside, 8-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA .
Vinograd, 1972), or unlike that of any other DNA component in the cell (Nass and Ben-Shaul, 1972) . Similarly, the intracellular locations of these DNAs, where determined, were found to vary with the organism, with both cytoplasmic and mitochondrial locations having been reported . The most detailed analysis to date of small circular DNAs in higher organisms was performed by Smith and Vinograd (1972) on HeLa cells . They found that the small polydisperse circular DNAs (spcDNA) of HeLa cells were present in the cytoplasm although they were of the same buoyant density as the nuclear DNA . They also showed that the number of spcDNA molecules per cell could be greatly increased by permitting the cells to sit in the stationary phase for 2-5 days or by treating the cells with cycloheximide . The small circular DNA amplified in this way was indistinguishable in size and buoyant density from nonamplified circles . Pulse-chase experiments with cycloheximideamplified circles suggested that they arose from preexisting nuclear DNA . Because none of the higher eucaryotes in which small circular DNA has been found have been particularly well characterized genetically, we decided to look for small circular DNAs in Drosophila melanogaster, an organism that has been extensively characterized both genetically and biochemically. We report here our discovery of heterogeneous small circular DNAs both in eggs and in cultured cells of D . melanogaster . The size distribution, intracellular location, buoyant density, number of copies per cell, and effect of some hormones and inhibitors were investigated for the circular DNA from tissue culture . In addition, the complexity of the circular DNA was investigated using the restriction enzyme EcoRl .
Introduction
Results
Small circular DNAs of unknown function have been described in a variety of eucaryotes, including Neurospora (Agsteribbe, Kroon, and Van Bruggen, 1972) ; Euglena (Nass and Ben-Shaul, 1972) ; three species of trypanosomes (Ono et al ., 1971) ; Saccharomyces (Billheimer and Avers, 1969) ; tobacco (Wong and Wildman, 1972) ; Xenopus (BuongiornoNardelli, Amaldi, and Lava-Sanchez, 1976) ; boar sperm (Hotta and Bassel, 1965) ; and a number of mammalian cell culture lines including those from monkey, mouse, and man (Smith and Vinograd, 1972 ; Rush, 1973) . In the majority of these cases, the circles were found to be heterogeneous in size and, depending upon the organism from which they were isolated, had buoyant densities either like that of the nuclear and/or mitochondrial DNA (Billheimer and Avers, 1969 ; Ono et al ., 1971 ; Smith and
Small Circular DNA Molecules from Drosophila Eggs Small circular DNA molecules were found in the supercoiled DNA region of CsCI/ethidium bromide gradients of homogenates of D . melanogaster eggs at the blastema stage of development . Figure 1 shows the size distribution of 78 light-nicked, small circular DNA molecules from eggs . These DNAs range in size from 0 .13-2 .03 µm, with a mean length of 0 .6 µm . Greater than 90% of the molecules were 1 pm in contour length . Size determinations were based on a contour length of 6 .0 pm for Drosophila egg mitochondrial DNA (Peacock et al ., 1973) . The small circular molecules were judged to be covalently closed on the basis of their supercoiled appearance in the electron microscope and by their location in the CsCI/ethidium bromide gradient .
Summary
Cell 334
and usually a single chromosome IV where these can be seen . The three telecentrics consist of two long chromosomes, approximately equal in length, and a single shorter chromosome . The shorter chromosome and one of the long chromosomes may correspond to the unequal X chromosomes seen in the,parental line, while the third telocentric resembles an X chromosome of the larger type . The karyotype may therefore best be described as 2N + X and probably haplo IV with a variable number of polyploids (10-30%) . The predominant polyploid was due to a single complete duplication of the basic karyotype .
Karyotypes of the Tissue Culture Lines Two established lines of Drosophila cells were used in this study . One of these was line 2, originally described by Schneider in 1972 . The other was a cloned subline of line 2, hereafter referred to as line BD6C . About 80% of log-phase line 2 cells are diploid female, with the two X chromosomes being of unequal lengths . The remainder of the cells are polyploid . Comparisons of the lengths of the arms of the X chromosomes relative to the lengths of the large autosomes were consistent with the idea that the shorter X was normal in length and the longer X contained an extra piece of chromosome roughly one third the size of the normal X . The predominant karyotype of the subline BD6C consists of two pairs of metacentrics (chromosomes II and III), three telocentric chromosomes,
Small Circular DNA Molecules from Cultured Drosophila Cells Figure 2 shows the size distributions of small circu-
AVG,
4
1
2 O I ~PI111~ 0 0 .5
11
111 I
I
I,O
2 .0
1,5
LENGTH
2 .5
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Figure 1 . Contour Length Distribution of Small Circular DNA from Eggs Eggs were collected as described in Experimental Procedures and allowed to develop at 26°C, 100% relative humidity, until the blastema state of development was reached (about 2 hr) . The small circular DNA was purified from the eggs as described in Experimental Procedures . Drosophila egg mitochondrial DNA (6 .0 µm) served as an internal length standard .
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LENGTH IN MICRONS Figure 2 . Contour Length Distributions of Small Circular DNA from Schneider's Line 2 Cells in the Logarithmic and Stationary Phases of Growth (a) 210 small circular DNA molecules from log-phase cells ; (b) 440 small circular DNA molecules from stationary phase cells . Molecules from stationary phase cells were obtained from the region of nuclear DNA density of the analytical CsCI gradient described in Figure 5a . Circular molecules from log-phase cells were obtained from the closed circular DNA region of the second of two successive CsCl/ethidium bromide gradients .
Small Circular DNA in D . melanogaster 335
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LENGTH IN MICRONS Figure 3 . Contour Length Distributions of Small Circular DNA from the Cloned Line BD6C during the Logarithmic Phase of Growth Circular DNA was obtained from the supercoiled DNA peak of the second of two successive CsCl/ethidium bromide gradients . Drosophila mitochondrial DNA from line BD6C (6 .0 µm) was used as an internal length standard . (a) and (b) small circular DNA from two different preparations of log-phase cells .
lar DNA molecules purified from log-phase and stationary phase cells of line 2 . Drosophila mitochondria) DNA from line 2 cells and antibiotic resistance plasmid R6K DNA (12 .04 µm) were used as internal length standards for both sets of measurements . Mitochondria) DNA from cultured cells has been determined to have a contour length of 6 .0 µm, based on the length of R6K DNA (S . Stanfield, unpublished results) . This value differs from the 5 .5 µm value found by Bultman and Laird (1973) for mitochondria) DNA from embryos, but agrees with that found by Peacock et al . (1973) . The majority of the small circular DNAs from cells in both the log and stationary phases ranged from about 0 .1-3 .0 µm, with each distribution having one very large circular molecule outside of this range . The mean sizes did not differ significantly, being 1 .09 µm for circles from log-phase cells and 1 .14 µm for circles from stationary phase cells . Chisquare analysis of the size distributions using five arbitrary size groupings containing at least twenty molecules each indicated a probability of 31% that the two distributions came from the same population of molecules . Based on a variation of 10% in the contour lengths of the internal DNA standards, it was estimated that the small circular DNA size distributions are compatible with at least eleven distinct size classes . Since Schneider's line 2 consists of a very heterogeneous population of cell types, it was believed that different cell types within this population might possess distinct subpopulations of circular molecules . For this reason, we investigated the circular DNAs of a cloned subline of line 2 . The size distribution from log-phase cells was determined twice, separated by a period of 5 weeks or approximately ten cell transfers . As shown in Figures 3a and 3b, the circles ranged in size from 0 .08-3 .88 um . The means of the two determinations did not differ significantly, being 1 .37 µm for the first determination
(Figure 3a) and 1 .33 ttm for the second (Figure 3b) . However, the contour length profiles did show some differences . The most obvious of these was that the major peak at 1 .76 µm, seen in the first distribution, was present only as a minor class in the second distribution . Chi-square analysis of the two size distribution profiles of BD6C indicated a probability of 28% that they could have been due to a random sampling of the same population . However, the probability that either of these distributions came from the same population of molecules as did those from line 2 was
