DEVELOPMENTAL GENETICS 13:97-102 (1992)
Bacteriophage X DNA Fragments Replicate in the Paramecium Macronucleus: Absence of Active Copy Number Control CHUNG SOOK KIM, JOHN R. PREER, JR., AND BARRY POLISKY Program in Molecular, Cellular and Developmental Biology, Department of Biology, Indiana University, Bloomington, Indiana
ABSTRACT We show that bacteriophage A DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. o 1992 WiIev-Liss, Inc.
Key words: DNA replication control, ciliated protozoa, microinjection
INTRODUCTION The macronucleus of many holotrichous ciliated protozoans contains DNA arranged in a n unusual manner compared to that of higher eukaryotic nuclei. DNA is highly polyploid and present as linear forms ranging from 1 kb to 5 kb in Oxytricha nova to 200-600 kb in Paramecium [Klobutcher and Prescott, 1986; Preer and Preer, 19791. Cytological and genetic evidence indicates that macronuclear DNA of Tetrahymena lack centromeres; no mitotic apparatus is apparent in the macronucleus during cell division. Rather, the macronucleus pinches into two roughly equal parts, and the macronuclear DNA chromosomes are often distributed to the daughter cells unequally [Cleffman, 19801. The phenomenon of phenotypic assortment observed in Tetrahymena, in which clonal cell lines descended from a heterozygous micronucleus express only one of two alleles, is thought to result from unequal partitioning in amitotic macronuclear division [Bruns, 19861. In Paramecium, the macronucleus contains about 1,0001,500 copies of each gene derived from the diploid copies present in the micronucleus [Soldo and Godoy, 19721.
0 1992 WILEY-LISS, INC.
DNA injected into most eukaryotic nuclei studied previously is not capable of autonomous replication. In higher cells, injected DNA may integrate into the host genome with or without concatamer formation [Perucho et al., 1980; Folger et al., 19821, or it may be lost during subsequent cell divisions without integration. An exception is the Xenopus oocyte, in which i t has been shown that injected supercoiled DNA regardless of sequence was capable of replication as supercoiled DNA [Mechali and Kearsey, 19841. In the yeast, Saccharomyces cerevisiae, the ability to confer autonomous replication on a marker DNA fragment is not a general property of yeast DNA and has been used to selectively isolate DNA fragments that are candidates for replication origins in vivo [Struhl et al., 19791. Recently, a DNA transformation system has been developed for Paramecium. Cloned DNA encoding the serotype A surface antigen was microinjected into the macronucleus of A- mutant Paramecium and shown to confer the ability to synthesize the A surface antigen [Godiska et al., 19871. The expression of the A surface antigen gene carried on the injected DNA was subject to the same type of transcriptional regulation as were the A-genes present in wild-type macronuclear chromosomes [Godiska et al., 19871. Analysis of the DNA injected showed that it replicated predominately as unit-length linear molecules, although integration of injected molecules has been detected [F. Caron, personal communication]. Paramecium-like telomeres were appended to the termini, presumably by a n endogenous telomerase activity [Gilley et al., 1988; Bourgain and Katinka, 19911. The replication behavior of the injected DNA was unusual and raised two questions. First, what, if any, were the sequence requirements for autonomous DNA replication in Parame-
Received for publication October 1, 1991; accepted October 25, 1991. Address reprint requests to Dr. Barry Polisky, Program in Molecular, Cellular and Developmental Biology, Department of Biology, Indiana University, Bloomington, IN 47405.
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cium? Second, what factors might be involved in determining or modulating the copy number of the iniected DNA?
MATERIALS AND METHODS Paramecia Wild-type cells were P. tetraurelia, stock 51.s. The Mendelian d12 mutation, in which the macronucleus is deleted for the A surface antigen-encoding gene, has been described [Epstein and Forney, 19841. Paramecia were cultured either in Cerophyl (Cerophyl Co., Kansas City, MO) or in baked lettuce medium inoculated with Klebsiella pneumoniae. Cloned DNA The plasmid pSA14SB has been previously described [Godiska et al., 19881. It contains a 14-kb fragment of Paramecium DNA containing the A serotype gene and flanking sequences cloned into the expression vector pT7/T3-18 (Bethesda Research Labs Inc., Gaithersburg, MD). Microinjection Microinjection into the macronucleus has been described previously [Godiska et al., 19871. About 1-5 pl of DNA at various concentrations was injected into each macronucleus under continuous hydrostatic pressure. DNA concentrations ranged from 1to 3 mg/ml in various experiments. DNA Analysis and Hybridization DNA was isolated from cells after microinjection as previously described [Gilley et al., 19881. DNA blot hybridization was carried out as described by Maniatis et al. [1980]. DNA samples were applied to a Schleicher and Schuell slot blot apparatus a s described by the manufacturer. Hybridization was done at 50°C in 5 x SSC (1x SSC is 0.15 M NaC1, 0.015M sodium citrate), 20 mM sodium phosphate (pH 7.0), l o x Denhardt's solution, 5% sodium dodecyl sulfate (SDS), 100 pg/ml denatured salmon sperm DNA, and 10% dextran sulfate. Washes were 55°C for 1 hr in 2~ SSC, 10 mM sodium phosphate, followed by a 30-min wash in 0.1 x SSC, 10 mM sodium phosphate. Probes for hybridization were prepared by nick-translation. DNA Electrophoresis Field inversion gel electrophoresis (FIGE) in 0.8% or 1% agarose gels was carried out as previously described [Godiska et al., 19871. RESULTS AND DISCUSSION We have previously shown that a prokaryotic plasmid derived from the pUC replicon can replicate autonomously after injection into Paramecium [Gilley et al., 19881. To extend our studies of sequence requirements for replication in the macronucleus, we injected bacteriophage A DNA, which had been cleaved with
HindIII. This cleavage generates a set of eight DNA fragments of the following sizes: 23 kb, 9.4 kb, 6.5 kb, 4.3 kb, 2.3 kb, 2.0 kb, 564 bp, and 125 bp. The A DNA fragments were co-injected into Paramecium macronuclei with a linearized form of plasmid pSA14, which carries the Paramecium tetraurelia stock 51 A surface antigen gene [Godiska et al., 19871. This plasmid is known to transform the A- mutant recipient line d12 to A with high efficiency. A t transformants were obtained by screening for expression of the A surface antigen at 34"C, a growth temperature that induces A expression in wild-type cells. Total DNA from four transformed cell lines that had not undergone autogamy after injection was prepared 25 fissions after injection, and fractionated by field inversion gel electrophoresis (FIGE). At this point, the input DNA had been diluted by a factor of 3 x lo7. DNA blots were prepared and probed with either radioactive pSA14 DNA or A DNA. As previously reported [Godiska et al., 19871, pSA14 DNA replicated primarily as a unit length linear species whose electrophoretic mobility was slightly less than that of the linear input DNA because of the addition of telomeres to the termini of the injected DNA (data not shown). Hybridization with a A-specific probe revealed that the transformants also contained six of the injected A Hind11 fragments, which also migrated slightly slower than their uninjected counterparts (Fig. 1).The detectable fragments are the 23-, 9.4-, 6 . 5 , and 4.3-kb species. The 2.3- and 2.0-kb species are also faintly visible in this exposure and are slightly displaced in mobility from their uninjected counterparts. Their slower mobility corresponds to that expected if 100-200 bp were added to them. As expected, the A-specific probe does not hybridize with DNA extracted from uninjected d12 cells (Fig. 1).In addition to apparently intact A-DNA fragments, a smear below and above these fragments is detectable. The larger material may correspond to end-to-end fusion of fragments t h a t has been detected when nontelomerized DNA is injected into Paramecium [Bourgain and Katinka, 19911. The smaller material is likely a consequence of some degradation of DNA after microinjection. The relative stoichiometry of the A DNA fragment intensities in lines derived from injected cells varies somewhat among the transformants. The relative amounts of the 23-kb and the 4.3-kb species differ in different transformants, possibly as a consequence of A DNA sticky-end joining prior to or after microinjection. Note that in transformant C10, the 4.3-kb band is absent, and a band migrating more slowly than the 23-kb band is apparent. However, for transformant C1, densitometric-scans of the hybridization signals of the three largest A fragments (23, 9.4, and 6.5 kb) showed that their relative stoichiometry was similar to that of the input A DNA, indicating that each of these fragments was capable of replication in Paramecium with approximately equal efficiency. The detected frag+
REPLICATION OF MICROINJECTED DNA IN PARAMECIUM
Fig. 1. Field inversion gel electrophoresis analysis of lysates from cells microinjected with A Hind11 DNA fragments. DNA was isolated from cell populations 25 fissions after microinjection. DNA was isolated, electrophoresed, transferred to nitrocellulose, and probed with labeled A HindII DNA fragments. M, A HindII fragment marker DNA, U, uninjected samples. Letters at top of the lanes designate the line name. Numbers a t right are molecular weight sizes in kb. Exposure was 12 hr.
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ments could not be those originally injected because of their high copy number per cell and the number of fissions occurring between injection and DNA preparation. Thus it is concluded that A DNA fragments can replicate autonomously in Paramecium. The A DNA bands present in transformants, but not their uninjected counterparts, were shown to hybridize with a labeled oligonucleotide complementary to Paramecium telomeres, indicating that telomeric sequences are added to the injected DNA (data not shown). We believe that telomere addition is responsible for the altered mobility of the 2.0- and 2.3-kb fragments; however DNA sequencing is required to prove this. These results extend those reported previously with injected pUC DNA [Gilley et al., 19881. The ability of prokaryotic DNA to replicate extensively in Paramecium raises questions about how this replication is controlled. The first fact that must be addressed about copy number of DNAs microinjected into Paramecia is that different transformed lines can have widely different copy numbers. For lines microinjected with pSA14 DNA, we have observed copy number levels ranging from 1,000 to 2,000 per cell up to 200,000 per cell (Polisky and Preer, unpublished data). We believe t h a t the wide variation is attributable to the different amounts of DNA introduced initially into each macronucleus during the injection process. I n general, we have observed that copy number is maintained at a relatively constant level until autogamy. However, this is not universally true. Some lines initially established with high copy numbers do undergo loss of copies progressively before autogamy (Polisky and Preer, unpublished data). The basis for this loss is not known. An additional consideration is cell to cell differences in copy number. Our copy number determinations are made on populations; analysis of lines derived from subclones has not been carried out because autogamy usually occurs before such subclones reach appreciable population size. We wanted to distinguish between two possible modes of control of the steady-state copy number of the A DNA fragments after injection. In one model, designated the active mode, replication of the fragments would be controlled by the level of some positive or negative acting replication factor(s), analogous to those known to regulate replication of other extrachromosoma1 elements, such as bacterial plasmids. In the active model, steady-state copy number depends on the concentration of the regulators and the affinity of the regulators for their targets. In this model, injected DNA would be expected to be capable of amplification; i.e., more than one replication per cell generation at low input concentrations. In the alternative model, designated the passive mode, steady state copy number would not be subject to control by replicon-specific elements, but rather would simply reflect the input copy number, faithfully duplicated once each generation. Injected DNA would not be capable of amplification re-
gardless of input levels. In the passive model, copy number would not be actively controlled, but re-replication in a single generation might be precluded by a general negative control mechanism governing replication of all macronuclear DNA. We carried out a simple experiment t h a t distinguished between these two models. We co-injected different ratios of HindIII-cleaved A DNA and pSA14 DNA into macronuclei and compared the steady-state copy number of the two types of DNAs to the input copy number ratio. If copy number of the A DNA fragments were actively regulated, their total steady-state copy number should be independent of the concentration of the input DNA, and might be expected to be approximately equal regardless of input. Alternatively, if copy number were passively controlled, we expect that steady-state copy number ratios would strictly reflect the input ratio. A DNA Hind11 fragments were mixed with linear pSA14 DNA in the following molar ratios: 1:3,1:6, 1:12, 1124,and 1:48. The total concentration of DNA during the microinjection process was constant in the different mixtures (1mg/ml). Transformants capable of expressing the A surface antigen were screened and grown into large populations. Autogamy was suppressed by providing sufficient culture medium to keep cells in logarithmic growth. DNA lysates were prepared from populations that had grown for 25 fissions after injection. Lysates were prepared from three separate transformants in each group. The amounts of pSA14 and A DNA per cell in transformant DNA lysates were determined by slot blot analysis using labeled pSA14 DNA or A DNA as probes. Known amounts of A DNA and pSA14 DNA were used to calibrate the hybridization signal from the lysates. Typical slot-blot data are shown in Figure 2. The copy number of A DNA and pSA14 DNA molecules in each of the transformants from the five DNA mixtures used for microinjection is shown in Table 1.The ratio of the two DNAs in transformants is plotted as a function of the input ratio in Figure 3. The results show a close relationship between the ratio of DNAs microinjected and the ratio present in the transformed cells after establishment of the injected DNAs. In each case, the original injected ratio is maintained for at least 25 fissions. These results are not compatible with a n active regulatory model, but are expected if no fragment-specific regulatory factors are involved. The data are consistent with the idea that all DNA molecules introduced by microinjection, regardless of sequence, receive telomeres and acquire the ability to replicate autonomously in a linear form. The results suggest that the macronuclear replication machinery replicates each molecule only once per cell generation, implying the existence of a marking system to prevent re-replication. If this is the case, the injected DNA does not amplify in copy number, but rather is maintained at the introduced copy number. We do not know whether sim-
REPLICATION OF MICROINJECTED DNA IN PARAMECIUM
1 probe
101
pSA14 probe
A1 L
82
P Y
c
E"s
c3
r
cn
E
0
D7
E7 d12 d12
A
E
F '
G
Fig. 2. DNA slot blot of transformant DNA preparations. DNA was prepared from 15 transformant lines after microinjection with mixtures of pSA14 and lambda DNAs. Approximately 250 ng of DNA was loaded into each slot. The designations A l , A3, etc., refer to the transformant line. A, B, etc., refer to lines obtained after microinjection with DNA mixture A, B, etc. In the upper part of the blot, identical samples were probed with either labeled A DNA (left) or labeled pSA14 DNA (right). The input DNA samples used for microinjection were also spotted onto the filter and probed (middle section). In the lower sets of slots, F-K, purified A DNA (F, G, H) or pSA14 DNA (I, J, K) was spotted in increasing amounts to permit calibration. These amounts were 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 4.8, and 6.4 ng from left to right.
ilar features apply to the replication of macronuclear chromosomes. There are indications that the copy number of these chromosomes might be under more active
control, which would enable them to adjust replication initiation frequencies to copy number fluctuations resulting from unequal segregation [Berger, 1979; Preer
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TABLE 1. Copy Number of X DNA and pSA14 DNA Molecules in Transformants From Five DNA Mixtures Used for Microinjection*
Input ratio 3 3 3
Xkell 3.3 3.5 5.8
pSA14kell 9.1 17 22.1
B2 B5 B6
6 6 6
3.4 3.7 6.3
19.6 36.2 43.4
c3 C6 c7
12 12 12
1.6 .68 .90
32 9.6 11.9
D7 D11 D14
24 24 24
.35 .45 1.4
13.5 16.5 25.5
E7 El2 El3
48 48 48
.50 .21 .48
38 13.5 32.9
Transformant A1 A3 A9
pSA14A 2.76 4.86 3.81 (3.81) 5.8 9.8 6.9 (7.50) 11.4 14.1 13.2 (12.9) 38.6 36.7 18.2 (31.2) 76 64 68 (69.5)
*The data were obtained from analysis of the slot-blot shown in Fig. 2. Bands were excised from the slot blot and counted in a scintillation counter. Input ratio is the ratio of pSA14 molecules to A DNA molecules in the microinjection mixture. A/cell and pSAl4kell data are molecules per cell x lo-”. They were calculated from the radioactivity in the individual slots and the reconstruction slots. Numbers in parentheses are the averages for the three transformants of a particular class. 80 0
70
o
m
o
-
-
m
N
o
N
m
m
o
m
m -
t
o
*
m
m
o
Input Ratio Fig. 3. Graph of data from Table 1.The input ratio of pSA14IA DNA molecules was plotted against the average of the observed ratio of these molecules 25 fissions after microinjection. The “expected output” is equivalent to the input. The observed output was derived from the slot-blot analysis.
and Preer, 19791. Paramecium discard their macronuclei periodically and produce a new one derived from the micronucleus following autogamy or conjugation
with another cell of appropriate mating type. Thus, unusual DNA in the macronucleus poses no long-term genetic burden for these cells. A major question concerns when and how the copy number of macronuclear chromosomes is determined. It is possible that during or shortly after the DNA processing steps generating macronuclear DNA from micronuclear DNA, DNA amplification events occur that set the copy number to a characteristic level, after which it is maintained during vegetative growth by a passive regulatory mechanism similar to that described here for microinjected DNA.
ACKNOWLEDGMENT This work was supported by NIH grant GM 31745. REFERENCES Bourgain FM, Katinka MD (1991): Telomeres inhibit end to end fusion and enhance maintenance of linear DNA molecules injected into the Paramecium primaurelia macronucleus. Nucl Acids Res 19:1541-1547. Bruns PJ (1986): Genetic organization of Tetrahymena. In Gall J G (ed): “The Molecular Biology of Ciliated Protozoa.” San Diego: Academic Press, pp 27-44. Cleffman G (1980): Chromatin elimination and the genetic organization of the macronucleus in Tetrahymena thermophila. Chromosoma 78:313-325. Epstein LM, Forney J D (1984): Mendelian and nowMendelian mutations affecting surface antigen expression in Paramecium tetraurelia. Mol Cell Biol 4:1583-1590. Folger KR, Wong EA, Wahl G, Capecchi MR (1982): Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol 2:1372-1387. Gilley D, Preer J R J r , Aufderheide KJ, Polisky B (1988): Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei. Mol Cell Biol 8:4765-4772. Godiska R, Aufderheide KJ, Gilley D, Hendrie P, Fitzwater T, Preer LB, Polisky B, Preer J R Jr (1987): Transformation of Paramecium by microinjection of a cloned serotype gene. Proc Natl Acad Sci USA 84:7590-7594. Karrer K (1986): The nuclear DNAs of holotrichous ciliates. In Gall J G (ed): “The Molecular Biology of Ciliated Protozoa.” San Diego: Academic Press, pp 85-105. Klobutcher LA, Prescott DM (1986): The special case of the hypotrichs. In Gall J G (ed): “The Molecular Biology of Ciliated Protozoa.” San Diego: Academic Press, pp 111-154. Maniatis T, Fritsch EF, Sambrook J (1982): “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Mechali M, Kearsey S (1984): Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high specificity in yeast. Cell 38:55-64. Perucho M, Hanahan D, Wigler M (1980):Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22:309-317. Preer JR Jr, Preer LB (1979): The size of macronuclear DNA and its relationship to models for maintaining genic balance. J Protozool 26:14-18. Soldo AT, Godoy GA (1972): The kinetic complexity of Paramecium macronuclear deoxyribonucleic acid. J Protozool 19573-678. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979): High freauency transformation of yeast: Autonomous reulication of hybrid ‘ DNA molecules. Proc Nati Acad Sci USA 76:1035-1039.
Bacteriophage X DNA Fragments Replicate in the Paramecium Macronucleus: Absence of Active Copy Number Control CHUNG SOOK KIM, JOHN R. PREER, JR., AND BARRY POLISKY Program in Molecular, Cellular and Developmental Biology, Department of Biology, Indiana University, Bloomington, Indiana
ABSTRACT We show that bacteriophage A DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. o 1992 WiIev-Liss, Inc.
Key words: DNA replication control, ciliated protozoa, microinjection
INTRODUCTION The macronucleus of many holotrichous ciliated protozoans contains DNA arranged in a n unusual manner compared to that of higher eukaryotic nuclei. DNA is highly polyploid and present as linear forms ranging from 1 kb to 5 kb in Oxytricha nova to 200-600 kb in Paramecium [Klobutcher and Prescott, 1986; Preer and Preer, 19791. Cytological and genetic evidence indicates that macronuclear DNA of Tetrahymena lack centromeres; no mitotic apparatus is apparent in the macronucleus during cell division. Rather, the macronucleus pinches into two roughly equal parts, and the macronuclear DNA chromosomes are often distributed to the daughter cells unequally [Cleffman, 19801. The phenomenon of phenotypic assortment observed in Tetrahymena, in which clonal cell lines descended from a heterozygous micronucleus express only one of two alleles, is thought to result from unequal partitioning in amitotic macronuclear division [Bruns, 19861. In Paramecium, the macronucleus contains about 1,0001,500 copies of each gene derived from the diploid copies present in the micronucleus [Soldo and Godoy, 19721.
0 1992 WILEY-LISS, INC.
DNA injected into most eukaryotic nuclei studied previously is not capable of autonomous replication. In higher cells, injected DNA may integrate into the host genome with or without concatamer formation [Perucho et al., 1980; Folger et al., 19821, or it may be lost during subsequent cell divisions without integration. An exception is the Xenopus oocyte, in which i t has been shown that injected supercoiled DNA regardless of sequence was capable of replication as supercoiled DNA [Mechali and Kearsey, 19841. In the yeast, Saccharomyces cerevisiae, the ability to confer autonomous replication on a marker DNA fragment is not a general property of yeast DNA and has been used to selectively isolate DNA fragments that are candidates for replication origins in vivo [Struhl et al., 19791. Recently, a DNA transformation system has been developed for Paramecium. Cloned DNA encoding the serotype A surface antigen was microinjected into the macronucleus of A- mutant Paramecium and shown to confer the ability to synthesize the A surface antigen [Godiska et al., 19871. The expression of the A surface antigen gene carried on the injected DNA was subject to the same type of transcriptional regulation as were the A-genes present in wild-type macronuclear chromosomes [Godiska et al., 19871. Analysis of the DNA injected showed that it replicated predominately as unit-length linear molecules, although integration of injected molecules has been detected [F. Caron, personal communication]. Paramecium-like telomeres were appended to the termini, presumably by a n endogenous telomerase activity [Gilley et al., 1988; Bourgain and Katinka, 19911. The replication behavior of the injected DNA was unusual and raised two questions. First, what, if any, were the sequence requirements for autonomous DNA replication in Parame-
Received for publication October 1, 1991; accepted October 25, 1991. Address reprint requests to Dr. Barry Polisky, Program in Molecular, Cellular and Developmental Biology, Department of Biology, Indiana University, Bloomington, IN 47405.
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cium? Second, what factors might be involved in determining or modulating the copy number of the iniected DNA?
MATERIALS AND METHODS Paramecia Wild-type cells were P. tetraurelia, stock 51.s. The Mendelian d12 mutation, in which the macronucleus is deleted for the A surface antigen-encoding gene, has been described [Epstein and Forney, 19841. Paramecia were cultured either in Cerophyl (Cerophyl Co., Kansas City, MO) or in baked lettuce medium inoculated with Klebsiella pneumoniae. Cloned DNA The plasmid pSA14SB has been previously described [Godiska et al., 19881. It contains a 14-kb fragment of Paramecium DNA containing the A serotype gene and flanking sequences cloned into the expression vector pT7/T3-18 (Bethesda Research Labs Inc., Gaithersburg, MD). Microinjection Microinjection into the macronucleus has been described previously [Godiska et al., 19871. About 1-5 pl of DNA at various concentrations was injected into each macronucleus under continuous hydrostatic pressure. DNA concentrations ranged from 1to 3 mg/ml in various experiments. DNA Analysis and Hybridization DNA was isolated from cells after microinjection as previously described [Gilley et al., 19881. DNA blot hybridization was carried out as described by Maniatis et al. [1980]. DNA samples were applied to a Schleicher and Schuell slot blot apparatus a s described by the manufacturer. Hybridization was done at 50°C in 5 x SSC (1x SSC is 0.15 M NaC1, 0.015M sodium citrate), 20 mM sodium phosphate (pH 7.0), l o x Denhardt's solution, 5% sodium dodecyl sulfate (SDS), 100 pg/ml denatured salmon sperm DNA, and 10% dextran sulfate. Washes were 55°C for 1 hr in 2~ SSC, 10 mM sodium phosphate, followed by a 30-min wash in 0.1 x SSC, 10 mM sodium phosphate. Probes for hybridization were prepared by nick-translation. DNA Electrophoresis Field inversion gel electrophoresis (FIGE) in 0.8% or 1% agarose gels was carried out as previously described [Godiska et al., 19871. RESULTS AND DISCUSSION We have previously shown that a prokaryotic plasmid derived from the pUC replicon can replicate autonomously after injection into Paramecium [Gilley et al., 19881. To extend our studies of sequence requirements for replication in the macronucleus, we injected bacteriophage A DNA, which had been cleaved with
HindIII. This cleavage generates a set of eight DNA fragments of the following sizes: 23 kb, 9.4 kb, 6.5 kb, 4.3 kb, 2.3 kb, 2.0 kb, 564 bp, and 125 bp. The A DNA fragments were co-injected into Paramecium macronuclei with a linearized form of plasmid pSA14, which carries the Paramecium tetraurelia stock 51 A surface antigen gene [Godiska et al., 19871. This plasmid is known to transform the A- mutant recipient line d12 to A with high efficiency. A t transformants were obtained by screening for expression of the A surface antigen at 34"C, a growth temperature that induces A expression in wild-type cells. Total DNA from four transformed cell lines that had not undergone autogamy after injection was prepared 25 fissions after injection, and fractionated by field inversion gel electrophoresis (FIGE). At this point, the input DNA had been diluted by a factor of 3 x lo7. DNA blots were prepared and probed with either radioactive pSA14 DNA or A DNA. As previously reported [Godiska et al., 19871, pSA14 DNA replicated primarily as a unit length linear species whose electrophoretic mobility was slightly less than that of the linear input DNA because of the addition of telomeres to the termini of the injected DNA (data not shown). Hybridization with a A-specific probe revealed that the transformants also contained six of the injected A Hind11 fragments, which also migrated slightly slower than their uninjected counterparts (Fig. 1).The detectable fragments are the 23-, 9.4-, 6 . 5 , and 4.3-kb species. The 2.3- and 2.0-kb species are also faintly visible in this exposure and are slightly displaced in mobility from their uninjected counterparts. Their slower mobility corresponds to that expected if 100-200 bp were added to them. As expected, the A-specific probe does not hybridize with DNA extracted from uninjected d12 cells (Fig. 1).In addition to apparently intact A-DNA fragments, a smear below and above these fragments is detectable. The larger material may correspond to end-to-end fusion of fragments t h a t has been detected when nontelomerized DNA is injected into Paramecium [Bourgain and Katinka, 19911. The smaller material is likely a consequence of some degradation of DNA after microinjection. The relative stoichiometry of the A DNA fragment intensities in lines derived from injected cells varies somewhat among the transformants. The relative amounts of the 23-kb and the 4.3-kb species differ in different transformants, possibly as a consequence of A DNA sticky-end joining prior to or after microinjection. Note that in transformant C10, the 4.3-kb band is absent, and a band migrating more slowly than the 23-kb band is apparent. However, for transformant C1, densitometric-scans of the hybridization signals of the three largest A fragments (23, 9.4, and 6.5 kb) showed that their relative stoichiometry was similar to that of the input A DNA, indicating that each of these fragments was capable of replication in Paramecium with approximately equal efficiency. The detected frag+
REPLICATION OF MICROINJECTED DNA IN PARAMECIUM
Fig. 1. Field inversion gel electrophoresis analysis of lysates from cells microinjected with A Hind11 DNA fragments. DNA was isolated from cell populations 25 fissions after microinjection. DNA was isolated, electrophoresed, transferred to nitrocellulose, and probed with labeled A HindII DNA fragments. M, A HindII fragment marker DNA, U, uninjected samples. Letters at top of the lanes designate the line name. Numbers a t right are molecular weight sizes in kb. Exposure was 12 hr.
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ments could not be those originally injected because of their high copy number per cell and the number of fissions occurring between injection and DNA preparation. Thus it is concluded that A DNA fragments can replicate autonomously in Paramecium. The A DNA bands present in transformants, but not their uninjected counterparts, were shown to hybridize with a labeled oligonucleotide complementary to Paramecium telomeres, indicating that telomeric sequences are added to the injected DNA (data not shown). We believe that telomere addition is responsible for the altered mobility of the 2.0- and 2.3-kb fragments; however DNA sequencing is required to prove this. These results extend those reported previously with injected pUC DNA [Gilley et al., 19881. The ability of prokaryotic DNA to replicate extensively in Paramecium raises questions about how this replication is controlled. The first fact that must be addressed about copy number of DNAs microinjected into Paramecia is that different transformed lines can have widely different copy numbers. For lines microinjected with pSA14 DNA, we have observed copy number levels ranging from 1,000 to 2,000 per cell up to 200,000 per cell (Polisky and Preer, unpublished data). We believe t h a t the wide variation is attributable to the different amounts of DNA introduced initially into each macronucleus during the injection process. I n general, we have observed that copy number is maintained at a relatively constant level until autogamy. However, this is not universally true. Some lines initially established with high copy numbers do undergo loss of copies progressively before autogamy (Polisky and Preer, unpublished data). The basis for this loss is not known. An additional consideration is cell to cell differences in copy number. Our copy number determinations are made on populations; analysis of lines derived from subclones has not been carried out because autogamy usually occurs before such subclones reach appreciable population size. We wanted to distinguish between two possible modes of control of the steady-state copy number of the A DNA fragments after injection. In one model, designated the active mode, replication of the fragments would be controlled by the level of some positive or negative acting replication factor(s), analogous to those known to regulate replication of other extrachromosoma1 elements, such as bacterial plasmids. In the active model, steady-state copy number depends on the concentration of the regulators and the affinity of the regulators for their targets. In this model, injected DNA would be expected to be capable of amplification; i.e., more than one replication per cell generation at low input concentrations. In the alternative model, designated the passive mode, steady state copy number would not be subject to control by replicon-specific elements, but rather would simply reflect the input copy number, faithfully duplicated once each generation. Injected DNA would not be capable of amplification re-
gardless of input levels. In the passive model, copy number would not be actively controlled, but re-replication in a single generation might be precluded by a general negative control mechanism governing replication of all macronuclear DNA. We carried out a simple experiment t h a t distinguished between these two models. We co-injected different ratios of HindIII-cleaved A DNA and pSA14 DNA into macronuclei and compared the steady-state copy number of the two types of DNAs to the input copy number ratio. If copy number of the A DNA fragments were actively regulated, their total steady-state copy number should be independent of the concentration of the input DNA, and might be expected to be approximately equal regardless of input. Alternatively, if copy number were passively controlled, we expect that steady-state copy number ratios would strictly reflect the input ratio. A DNA Hind11 fragments were mixed with linear pSA14 DNA in the following molar ratios: 1:3,1:6, 1:12, 1124,and 1:48. The total concentration of DNA during the microinjection process was constant in the different mixtures (1mg/ml). Transformants capable of expressing the A surface antigen were screened and grown into large populations. Autogamy was suppressed by providing sufficient culture medium to keep cells in logarithmic growth. DNA lysates were prepared from populations that had grown for 25 fissions after injection. Lysates were prepared from three separate transformants in each group. The amounts of pSA14 and A DNA per cell in transformant DNA lysates were determined by slot blot analysis using labeled pSA14 DNA or A DNA as probes. Known amounts of A DNA and pSA14 DNA were used to calibrate the hybridization signal from the lysates. Typical slot-blot data are shown in Figure 2. The copy number of A DNA and pSA14 DNA molecules in each of the transformants from the five DNA mixtures used for microinjection is shown in Table 1.The ratio of the two DNAs in transformants is plotted as a function of the input ratio in Figure 3. The results show a close relationship between the ratio of DNAs microinjected and the ratio present in the transformed cells after establishment of the injected DNAs. In each case, the original injected ratio is maintained for at least 25 fissions. These results are not compatible with a n active regulatory model, but are expected if no fragment-specific regulatory factors are involved. The data are consistent with the idea that all DNA molecules introduced by microinjection, regardless of sequence, receive telomeres and acquire the ability to replicate autonomously in a linear form. The results suggest that the macronuclear replication machinery replicates each molecule only once per cell generation, implying the existence of a marking system to prevent re-replication. If this is the case, the injected DNA does not amplify in copy number, but rather is maintained at the introduced copy number. We do not know whether sim-
REPLICATION OF MICROINJECTED DNA IN PARAMECIUM
1 probe
101
pSA14 probe
A1 L
82
P Y
c
E"s
c3
r
cn
E
0
D7
E7 d12 d12
A
E
F '
G
Fig. 2. DNA slot blot of transformant DNA preparations. DNA was prepared from 15 transformant lines after microinjection with mixtures of pSA14 and lambda DNAs. Approximately 250 ng of DNA was loaded into each slot. The designations A l , A3, etc., refer to the transformant line. A, B, etc., refer to lines obtained after microinjection with DNA mixture A, B, etc. In the upper part of the blot, identical samples were probed with either labeled A DNA (left) or labeled pSA14 DNA (right). The input DNA samples used for microinjection were also spotted onto the filter and probed (middle section). In the lower sets of slots, F-K, purified A DNA (F, G, H) or pSA14 DNA (I, J, K) was spotted in increasing amounts to permit calibration. These amounts were 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 4.8, and 6.4 ng from left to right.
ilar features apply to the replication of macronuclear chromosomes. There are indications that the copy number of these chromosomes might be under more active
control, which would enable them to adjust replication initiation frequencies to copy number fluctuations resulting from unequal segregation [Berger, 1979; Preer
102
KIMETAL.
TABLE 1. Copy Number of X DNA and pSA14 DNA Molecules in Transformants From Five DNA Mixtures Used for Microinjection*
Input ratio 3 3 3
Xkell 3.3 3.5 5.8
pSA14kell 9.1 17 22.1
B2 B5 B6
6 6 6
3.4 3.7 6.3
19.6 36.2 43.4
c3 C6 c7
12 12 12
1.6 .68 .90
32 9.6 11.9
D7 D11 D14
24 24 24
.35 .45 1.4
13.5 16.5 25.5
E7 El2 El3
48 48 48
.50 .21 .48
38 13.5 32.9
Transformant A1 A3 A9
pSA14A 2.76 4.86 3.81 (3.81) 5.8 9.8 6.9 (7.50) 11.4 14.1 13.2 (12.9) 38.6 36.7 18.2 (31.2) 76 64 68 (69.5)
*The data were obtained from analysis of the slot-blot shown in Fig. 2. Bands were excised from the slot blot and counted in a scintillation counter. Input ratio is the ratio of pSA14 molecules to A DNA molecules in the microinjection mixture. A/cell and pSAl4kell data are molecules per cell x lo-”. They were calculated from the radioactivity in the individual slots and the reconstruction slots. Numbers in parentheses are the averages for the three transformants of a particular class. 80 0
70
o
m
o
-
-
m
N
o
N
m
m
o
m
m -
t
o
*
m
m
o
Input Ratio Fig. 3. Graph of data from Table 1.The input ratio of pSA14IA DNA molecules was plotted against the average of the observed ratio of these molecules 25 fissions after microinjection. The “expected output” is equivalent to the input. The observed output was derived from the slot-blot analysis.
and Preer, 19791. Paramecium discard their macronuclei periodically and produce a new one derived from the micronucleus following autogamy or conjugation
with another cell of appropriate mating type. Thus, unusual DNA in the macronucleus poses no long-term genetic burden for these cells. A major question concerns when and how the copy number of macronuclear chromosomes is determined. It is possible that during or shortly after the DNA processing steps generating macronuclear DNA from micronuclear DNA, DNA amplification events occur that set the copy number to a characteristic level, after which it is maintained during vegetative growth by a passive regulatory mechanism similar to that described here for microinjected DNA.
ACKNOWLEDGMENT This work was supported by NIH grant GM 31745. REFERENCES Bourgain FM, Katinka MD (1991): Telomeres inhibit end to end fusion and enhance maintenance of linear DNA molecules injected into the Paramecium primaurelia macronucleus. Nucl Acids Res 19:1541-1547. Bruns PJ (1986): Genetic organization of Tetrahymena. In Gall J G (ed): “The Molecular Biology of Ciliated Protozoa.” San Diego: Academic Press, pp 27-44. Cleffman G (1980): Chromatin elimination and the genetic organization of the macronucleus in Tetrahymena thermophila. Chromosoma 78:313-325. Epstein LM, Forney J D (1984): Mendelian and nowMendelian mutations affecting surface antigen expression in Paramecium tetraurelia. Mol Cell Biol 4:1583-1590. Folger KR, Wong EA, Wahl G, Capecchi MR (1982): Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol 2:1372-1387. Gilley D, Preer J R J r , Aufderheide KJ, Polisky B (1988): Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei. Mol Cell Biol 8:4765-4772. Godiska R, Aufderheide KJ, Gilley D, Hendrie P, Fitzwater T, Preer LB, Polisky B, Preer J R Jr (1987): Transformation of Paramecium by microinjection of a cloned serotype gene. Proc Natl Acad Sci USA 84:7590-7594. Karrer K (1986): The nuclear DNAs of holotrichous ciliates. In Gall J G (ed): “The Molecular Biology of Ciliated Protozoa.” San Diego: Academic Press, pp 85-105. Klobutcher LA, Prescott DM (1986): The special case of the hypotrichs. In Gall J G (ed): “The Molecular Biology of Ciliated Protozoa.” San Diego: Academic Press, pp 111-154. Maniatis T, Fritsch EF, Sambrook J (1982): “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Mechali M, Kearsey S (1984): Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high specificity in yeast. Cell 38:55-64. Perucho M, Hanahan D, Wigler M (1980):Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22:309-317. Preer JR Jr, Preer LB (1979): The size of macronuclear DNA and its relationship to models for maintaining genic balance. J Protozool 26:14-18. Soldo AT, Godoy GA (1972): The kinetic complexity of Paramecium macronuclear deoxyribonucleic acid. J Protozool 19573-678. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979): High freauency transformation of yeast: Autonomous reulication of hybrid ‘ DNA molecules. Proc Nati Acad Sci USA 76:1035-1039.
