Current Genetics
Current Genetics (1982) 6:119-122
© Springer-Verlag 1982
MinicircularDNA Having Sequence Homologies with Chloroplast DNA in a Bleached Mutant of Euglena gracilis Philippe Heizmann, Patrick Ravel-Chapuis, and Victor Nigon Universit6 Claude Bernard Lyon I, D6partement de Biologie G6n6rale, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France
Summary. Chloroplast DNA was isolated from total celMar DNA of a bleached mutant of Euglena gracilis (Y3BUD) by enrichment of the light component (p = 1.686) by repeated CsC1equilibrium centrifugations. Electron microscope visualization of this DNA showed minicircular DNA molecules in addition to large circular molecules (42 #m) identical to wild type chloroplast DNA. They were heterogenous in size and their contour lengths ranged from 0.8 to 8.5 lma. Fractionnation by agarose gel electrophoresis gave several discrete bands. Some of them hybridized with pure chloroplast DNA and with several cloned chloroplast DNA fragments, particularly to ribosomal fragments, while others did not show homology with chloroplast DNA being probably of extrachloroplatic origin. Key words: Minicircular DNA - Chloroplast DNA Bleached mutant
Introduction It is generally accepted that chloroplast DNA constitute homogeneous populations of identical molecules as judged from the combined results of electron microscope visualization, restriction endonuclease mapping and renaturation kinetics analyzes (Manning and Richards 1972; Kopecka et al. 1977; Stutz and Vandrey 1971; Kolodner and Tewari 1975; Chu et al. 1981). Relative variations of chloroplast DNA sequences, presumed to be chloroplast ribosomal DNA on the basis of their buoyant density (p = 1.701-1.691) in CsC1 gradients, were suggested in the course of growth cycle in Euglena; minicircular DNA of various densities (p = Offprint requests to: P. Heizmann
1.700-1.686) have also been described (Gibson and Hershberger 1975; Nass and Ben-Shaul 1972; Manning and Richards 1972; Mielenz and Hershberger 1974). However Stutz et al. (1975) and Kopecka et al. (1977) did not confirm these results after rigourous identification and estimation of ribosomal DNA by hybridization with chloroplast ribosomal RNA and restriction digestion. The case of bleached mutants of Euglena offers a different situation where non-stoechiometry among various chloroplast DNA sequences has been shown to occur: - in the ~ - bleached mutants having low amounts of chloroplast DNA, ribosomal cistrons are amplified relative to non ribosomal sequences (preceding paper); - in the c p - bleached mutants having about as much chloroplast as wild type cells some sequences are more frequent than others. Moreover the restriction pattern of the ribosomal fragments of one of these mutants (Y3BUD) displays the characteristic modifications appearing during mutagenesis (Heizmann et al. 1982). The high content in mutated chloroplast DNA in the strain Y3BUD allowed its isolation for direct analysis. The present paper describes some features of this DNA.
Materials and Methods Strains and Cultures. The wild type reference strain was a Z type strain (1224-5/25 from the G6tfingen Institute Algensammlung); the Y3BUD mutant derived from a bacillaris strain was kindly provided by Prof. J. A. Schiff (Brandeis University). Cells were grown on an organotrophic medium and harvested in late logarithmic phase of growth (Freyssinet et al. 1970). DNA Extraeffon and Analysis. Reference chloroplast DNA from chloroplasts purified by flottation and total DNA preparation from Y3BUD were extracted by mild sarkosyl lysis and phenol/ chloroform treatment according to Richards and Manning (1975). Total DNA from Y3BUD were run on CsC1preparative gradients including 0.5 /~g/mlof Ethidium bromide for UV inspection of the gradients. The material banding above the main nuclear band
13172-8083/82/0006/0119/$ 01.00
120
P. Heizmann et al.: Minicircles Homologous with Chloroplast DNA
Figs 1 and 2. Spreading of chloroplast DNA prepared from Y3BUD by CsC1 centrifugations. Fig. 1: minicircular molecule of 2.0/~m. Fig. 2: minicircular molecules in OC and CCC configurations heterodisperse in size among linear molecules of sheared chloroplast DNA
at the position of wild type chloroplast DNA was pipetted out of the gradient and rebanded two additional times. Agarose gel electrophoresis, Southern transfers, in vitro DNA labeling by nicktranslation have been described or referenced in the preceding paper. The cloned restriction fragments of chloroplast DNA used as probes were excised from their plasmid vector (RSF 2124) in order to increase the label effectively incorporated in the probe and to avoid eventual cross-hybridization of the vector with sequences of DNA from Euglena. DNA samples were spread by the formamide method of Davis et al. (1971), stained with uranyl acetate, shadowed with Pt/Pd and observed with an Hitachi HU12 electron microscope. Lengths of molecules were determined with a map-measurer and replica gratings as standards.
Results Plastid remnants from bleached mutantslike Y3BUD have not been isolated and purified as plain chloroplast so far. We attempted thus to enrich their chloroplast DNA from total DNA preparations by repeated CsC1 preparative centrifugations on the basis of their buoyant density (p = 1.686) lower than that of nuclear DNA (p = 1.707). (Schiff and Epstein 1967). After three cycles o f banding the upper band o f chloroplast DNA represented the major part of the DNA and could be isolated with minimal contamination by nuclear DNA (probably less than 10%).
Electron microscope examination o f the DNA thus prepared showed a large majority (about 80% of the mass of total DNA) of linear molecules with lengths of 10 to 30/am. They could represent either broken chloroplast DNA or nuclear contaminants for a minor part. Some large molecules apparently similar with intact wild type chloroplast DNA (42 #m of contour length) were also visualized. At last numerous minicircular molecules of DNA were observed (Figs. 1 and 2). Both covalently closed and open circular molecules were found. Length measurements of 50 molecules indicated contour length ranging from 0.8 to 8.5/am with plurimodal distribution: there are at least four distinct classes with sizes averaging at 1/am, 2 - 4 / a m , 5 lain and 8/am. The second class itself is very broad and certainly constituted of several overlapping subclasses (Fig. 3). Upon fractionation by agarose gel electrophoresis, these DNA preparations showed discrete bands clearly separated from each other confirming the occurence of several definite classes o f molecule sizes (Fig. 4). At least 6 sharp bands (noted from 1 to 6 on Fig. 4) move faster than the major fluorescent band of unresolved high molecular weight DNA (MW > 15.106 daltons o f the EcoA reference fragment). A diffuse zone also appears between the bands 1 and 2 at the level o f reference fragments EcoF and EcoG.
P. Heizmann et al.: Minicircles Homologous with Chloroplast DNA
121 All the six bands were resistant to b o t h RNase A and to S 1 nuclease treatments b u t disappeared after DNasel digestion: they are thus made o f plain double stranded DNA. Hybridization with total nick-translated chloroplast DNA clearly labeled band 1 and 3, b u t also five other bands not detected under UV-Ethidium bromide fluorescence. On the opposite, bands numbered 2, 4, 5, 6 and the diffuse zone between bands 1 and 2 were not significantly labeled b y the chloroplast probe even after long exposures o f the X-ray films with the Southern blots. Several cloned restriction fragments o f chloroplast DNA were also used as probes in order to attempt to identify the homologies between each o f the bands of this pattern with different regions o f the wild type chloroplast physical map. The hybridization - - - obtained with the various probes were however rather similar with each other: the same bands o f the Southern blots were labeled with all the probes tested so far (fragments EcoH, I, J . . . . . S, T, U). Only the relative density o f labeling
10,
"-5 c}
5'
1
2
3
4
5
6
7
8
Length (um)
• Fig. 3. Frequence distribution of lengths of minicircular molecules from the chloroplast DNA from Y3BUD. A total of 50 molecules was analyzed.
Fig. 4. Agarose gel electrophoresis of chloroplast DNA prepared from Y3BUD. Hybridization of the resulting Southern blots with chloroplast probes. Chloroplast DNA from Y3BUD (lanes Mini C) was fractionated on a 0.7% agarose gel with EcoR 1 digests of wild type chloroplast DNA as reference (= Ref). The material fluorescent under UV + Ethidium bromide was photographed (extreme left and right lanes), then transfered to nitrocellulose filters and hybridized with nick-translatedwild type chloroplast DNA (ctDNA lane) or cloned restriction fragments of chloroplast DNA (lanes EcoJ, H and PL). Letters B, F, H, J, L, P point to the restriction fragments of the reference digest hybridized by the cloned probes (extremeleft); numbers 1 to 6 point to Y3BUD DNA bands moving faster than the unresolved high molecular weight DNA (HMW) (extreme right). Autoradiographs were exposed for 2 days; in the ease of Y3BUD DNA hybridized with EcoJ, H and P+L 5 days (right lanes) brought additional details
122 differed from probe to probe: for instance band 1 was more labeled than the main band of unresolved high molecular weight chloroplast DNA with probes of chloroplast DNA and EcoH fragment, with probe EcoJ these two bands were equally labeled and with probes EcoP+L band 1 was less labeled.
Discussion The isolation of the light (p = 1.686) component of total DNA from the mutant Y3BUD yielded essentially bona fide chloroplast DNA since the material thus obtained hybridized strongly with radioactive chloroplast probes and showed the presence of typical large molecules of 42 #m. Low buoyant density however is not a criterion sufficient to characterize the minicircles detected among our DNA preparations as chloroplastic. Hybridizations with wild type chloroplast DNA and cloned chloroplast probes should have provided stronger arguments to identify the chloroplastic nature of some minicircles at least. The specificity of the hybridizations is apparent from the labeling of reference chloroplast DNA fragments with the corresponding cloned probes (and only with the corresponding probes, without important cross-hybridizations) and also from the fact that not all but only some bands visible in the gel hybridize with the probes. Now almost similar hybridization patterns were surprisingly obtained for identical Southern blots with all the different clones of restriction fragments scattered around the physical map of chloroplast DNA (Hallick 1982). One cannot therefore assume total homology between each of the probes and the hybridizing minicircles. The observed homologies could be limited to short sequences repeated along the wild type chloroplast genome. Such sequences have been demonstrated in the case of Chlamydomonas (Rochaix and Malnoe 1978; Gelvin and Howell 1980); they are believed to cause zerotime binding of denatured chloroplast DNA fragments to hydroxylapatite in renaturation experiments and extensive cross-hybridization between chloroplast DNA restriction fragments. On one hand some snap-back reassociation of denatured chloroplast DNA is apparent in the case of Euglena (Rawson and Boerma 1976; Chelm at al. 1977; preceding paper), but on the other hand Koller and Delius (1982) did not observe more that two stem-loop figures of inverted repeats along the entire molecule of denatured chloroplast DNA. Moreover cross-hybridization between restriction fragments are minimal with chloroplast DNA of Euglena, making the occurence of repeats questionable. The exact nature of the sequences involved in the observed hybridizations remains thus to be identified.
P. Heizmann et al.: Minicircles Homologouswith Chloroplast DNA These hybridizations are probably of the same nature as those suggesting non-stoechiometry in reassociation kinetics of cp- mutant DNA. The detailed anatomy of the minicircular molecules will have to be elucidated before a realistic model can be presented to explain their relationship with chloroplast DNA structure, replication and mutagenesis, their biogenesis and biological function. This problem is of theoretical and practical importance since its solution should allow to better understand the control of replication and reiteration of chloroplast genomes; it might also provide us with potential chloroplast vectors for genetic engeneering.
Acknowledgements. We thank Mr. P. M. Malet and Mrs. C. FaureSchwob for technical assistance, and Drs. G. Bernardi and J. Doly for helpful discussions and advices. This work was partly supported by a C.N.R.S. grant (A.T.P. n° 8068).
References Chelm BK, Hoben PJ, Hallick RB (1977) Biochem 16:782-785 Chu NM, Oishi KK, Tewari KK (1981) Plasmid 6:279-292 Davis RW, Simon M, Davidson N (1971) Meth Enzymol 21:413 -428 Freyssinet G, Heizmann P, Verdier G, Trabuehet G, Nigon V (1970) PhysiolVeg 10:421-442 Gelvin SB, Howell SH (1979) Mol Gen Genet 173:315-322 Gibson WH, Hershberger CL (1975) Arch Biochem Biophys 168: 8-14 Hallick RB (1982) In: The Biologyof Euglena, vol. 4 BuetowDE (ed) (in press) Heizmann P, Doly J, Hussein Y, Nicolas P, Nigon V, Bernadi G (1981) Bioehim Biophys Acta 653:412-415 Koller B, Delius H (1982) FEBS Letters 139:86-92 Kolodner R, Tewari KK (1975) J Biol Chem 250:8840-8847 Kopecka H, Crouse EJ, Stutz E (1977) Eur J Biochem 72:525535 Manning JE, Richards OC (1972) Biochem 11:2036-2043 Mielenz JR, Hershberger CL (1974) Biochem Biophys Res Comm 58:769-777 Nass MMK, Ben-Shaul Y (1972)Biochim Biophys Acta 272:130 -136 Rawson JRY, Boerma C (1976) Proc Natl Acad Sci USA 73: 2401-2404 Richards OC, ManningJE (1975) In: Lefort-Tran M, Valencia 1/, (eds) Les Cycles Cellulaires. Editions du CNRS, pp 212-221 Rochalx JD, Malnoe P (1978) Cell 15:661-670 Sehiff JA, Epstein HT (1965) In: Locke M (ed) Reproduction: Molecular, Subcellular, Cellular. Academic Press, New York, pp 131-189 Stutz E, Vandrey JP (1971) Proc. Ilnd Intern. Congr; on Photosynth. Forti G, Avron M, Melandri A (eds) pp 2601-2609 Stutz E, Crouse EJ, Graf L (1975) In: Les CyclesCellulalres, pp 255-265 Communicated by R. J. Schweyen Received July 5, 1982
Current Genetics (1982) 6:119-122
© Springer-Verlag 1982
MinicircularDNA Having Sequence Homologies with Chloroplast DNA in a Bleached Mutant of Euglena gracilis Philippe Heizmann, Patrick Ravel-Chapuis, and Victor Nigon Universit6 Claude Bernard Lyon I, D6partement de Biologie G6n6rale, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France
Summary. Chloroplast DNA was isolated from total celMar DNA of a bleached mutant of Euglena gracilis (Y3BUD) by enrichment of the light component (p = 1.686) by repeated CsC1equilibrium centrifugations. Electron microscope visualization of this DNA showed minicircular DNA molecules in addition to large circular molecules (42 #m) identical to wild type chloroplast DNA. They were heterogenous in size and their contour lengths ranged from 0.8 to 8.5 lma. Fractionnation by agarose gel electrophoresis gave several discrete bands. Some of them hybridized with pure chloroplast DNA and with several cloned chloroplast DNA fragments, particularly to ribosomal fragments, while others did not show homology with chloroplast DNA being probably of extrachloroplatic origin. Key words: Minicircular DNA - Chloroplast DNA Bleached mutant
Introduction It is generally accepted that chloroplast DNA constitute homogeneous populations of identical molecules as judged from the combined results of electron microscope visualization, restriction endonuclease mapping and renaturation kinetics analyzes (Manning and Richards 1972; Kopecka et al. 1977; Stutz and Vandrey 1971; Kolodner and Tewari 1975; Chu et al. 1981). Relative variations of chloroplast DNA sequences, presumed to be chloroplast ribosomal DNA on the basis of their buoyant density (p = 1.701-1.691) in CsC1 gradients, were suggested in the course of growth cycle in Euglena; minicircular DNA of various densities (p = Offprint requests to: P. Heizmann
1.700-1.686) have also been described (Gibson and Hershberger 1975; Nass and Ben-Shaul 1972; Manning and Richards 1972; Mielenz and Hershberger 1974). However Stutz et al. (1975) and Kopecka et al. (1977) did not confirm these results after rigourous identification and estimation of ribosomal DNA by hybridization with chloroplast ribosomal RNA and restriction digestion. The case of bleached mutants of Euglena offers a different situation where non-stoechiometry among various chloroplast DNA sequences has been shown to occur: - in the ~ - bleached mutants having low amounts of chloroplast DNA, ribosomal cistrons are amplified relative to non ribosomal sequences (preceding paper); - in the c p - bleached mutants having about as much chloroplast as wild type cells some sequences are more frequent than others. Moreover the restriction pattern of the ribosomal fragments of one of these mutants (Y3BUD) displays the characteristic modifications appearing during mutagenesis (Heizmann et al. 1982). The high content in mutated chloroplast DNA in the strain Y3BUD allowed its isolation for direct analysis. The present paper describes some features of this DNA.
Materials and Methods Strains and Cultures. The wild type reference strain was a Z type strain (1224-5/25 from the G6tfingen Institute Algensammlung); the Y3BUD mutant derived from a bacillaris strain was kindly provided by Prof. J. A. Schiff (Brandeis University). Cells were grown on an organotrophic medium and harvested in late logarithmic phase of growth (Freyssinet et al. 1970). DNA Extraeffon and Analysis. Reference chloroplast DNA from chloroplasts purified by flottation and total DNA preparation from Y3BUD were extracted by mild sarkosyl lysis and phenol/ chloroform treatment according to Richards and Manning (1975). Total DNA from Y3BUD were run on CsC1preparative gradients including 0.5 /~g/mlof Ethidium bromide for UV inspection of the gradients. The material banding above the main nuclear band
13172-8083/82/0006/0119/$ 01.00
120
P. Heizmann et al.: Minicircles Homologous with Chloroplast DNA
Figs 1 and 2. Spreading of chloroplast DNA prepared from Y3BUD by CsC1 centrifugations. Fig. 1: minicircular molecule of 2.0/~m. Fig. 2: minicircular molecules in OC and CCC configurations heterodisperse in size among linear molecules of sheared chloroplast DNA
at the position of wild type chloroplast DNA was pipetted out of the gradient and rebanded two additional times. Agarose gel electrophoresis, Southern transfers, in vitro DNA labeling by nicktranslation have been described or referenced in the preceding paper. The cloned restriction fragments of chloroplast DNA used as probes were excised from their plasmid vector (RSF 2124) in order to increase the label effectively incorporated in the probe and to avoid eventual cross-hybridization of the vector with sequences of DNA from Euglena. DNA samples were spread by the formamide method of Davis et al. (1971), stained with uranyl acetate, shadowed with Pt/Pd and observed with an Hitachi HU12 electron microscope. Lengths of molecules were determined with a map-measurer and replica gratings as standards.
Results Plastid remnants from bleached mutantslike Y3BUD have not been isolated and purified as plain chloroplast so far. We attempted thus to enrich their chloroplast DNA from total DNA preparations by repeated CsC1 preparative centrifugations on the basis of their buoyant density (p = 1.686) lower than that of nuclear DNA (p = 1.707). (Schiff and Epstein 1967). After three cycles o f banding the upper band o f chloroplast DNA represented the major part of the DNA and could be isolated with minimal contamination by nuclear DNA (probably less than 10%).
Electron microscope examination o f the DNA thus prepared showed a large majority (about 80% of the mass of total DNA) of linear molecules with lengths of 10 to 30/am. They could represent either broken chloroplast DNA or nuclear contaminants for a minor part. Some large molecules apparently similar with intact wild type chloroplast DNA (42 #m of contour length) were also visualized. At last numerous minicircular molecules of DNA were observed (Figs. 1 and 2). Both covalently closed and open circular molecules were found. Length measurements of 50 molecules indicated contour length ranging from 0.8 to 8.5/am with plurimodal distribution: there are at least four distinct classes with sizes averaging at 1/am, 2 - 4 / a m , 5 lain and 8/am. The second class itself is very broad and certainly constituted of several overlapping subclasses (Fig. 3). Upon fractionation by agarose gel electrophoresis, these DNA preparations showed discrete bands clearly separated from each other confirming the occurence of several definite classes o f molecule sizes (Fig. 4). At least 6 sharp bands (noted from 1 to 6 on Fig. 4) move faster than the major fluorescent band of unresolved high molecular weight DNA (MW > 15.106 daltons o f the EcoA reference fragment). A diffuse zone also appears between the bands 1 and 2 at the level o f reference fragments EcoF and EcoG.
P. Heizmann et al.: Minicircles Homologous with Chloroplast DNA
121 All the six bands were resistant to b o t h RNase A and to S 1 nuclease treatments b u t disappeared after DNasel digestion: they are thus made o f plain double stranded DNA. Hybridization with total nick-translated chloroplast DNA clearly labeled band 1 and 3, b u t also five other bands not detected under UV-Ethidium bromide fluorescence. On the opposite, bands numbered 2, 4, 5, 6 and the diffuse zone between bands 1 and 2 were not significantly labeled b y the chloroplast probe even after long exposures o f the X-ray films with the Southern blots. Several cloned restriction fragments o f chloroplast DNA were also used as probes in order to attempt to identify the homologies between each o f the bands of this pattern with different regions o f the wild type chloroplast physical map. The hybridization - - - obtained with the various probes were however rather similar with each other: the same bands o f the Southern blots were labeled with all the probes tested so far (fragments EcoH, I, J . . . . . S, T, U). Only the relative density o f labeling
10,
"-5 c}
5'
1
2
3
4
5
6
7
8
Length (um)
• Fig. 3. Frequence distribution of lengths of minicircular molecules from the chloroplast DNA from Y3BUD. A total of 50 molecules was analyzed.
Fig. 4. Agarose gel electrophoresis of chloroplast DNA prepared from Y3BUD. Hybridization of the resulting Southern blots with chloroplast probes. Chloroplast DNA from Y3BUD (lanes Mini C) was fractionated on a 0.7% agarose gel with EcoR 1 digests of wild type chloroplast DNA as reference (= Ref). The material fluorescent under UV + Ethidium bromide was photographed (extreme left and right lanes), then transfered to nitrocellulose filters and hybridized with nick-translatedwild type chloroplast DNA (ctDNA lane) or cloned restriction fragments of chloroplast DNA (lanes EcoJ, H and PL). Letters B, F, H, J, L, P point to the restriction fragments of the reference digest hybridized by the cloned probes (extremeleft); numbers 1 to 6 point to Y3BUD DNA bands moving faster than the unresolved high molecular weight DNA (HMW) (extreme right). Autoradiographs were exposed for 2 days; in the ease of Y3BUD DNA hybridized with EcoJ, H and P+L 5 days (right lanes) brought additional details
122 differed from probe to probe: for instance band 1 was more labeled than the main band of unresolved high molecular weight chloroplast DNA with probes of chloroplast DNA and EcoH fragment, with probe EcoJ these two bands were equally labeled and with probes EcoP+L band 1 was less labeled.
Discussion The isolation of the light (p = 1.686) component of total DNA from the mutant Y3BUD yielded essentially bona fide chloroplast DNA since the material thus obtained hybridized strongly with radioactive chloroplast probes and showed the presence of typical large molecules of 42 #m. Low buoyant density however is not a criterion sufficient to characterize the minicircles detected among our DNA preparations as chloroplastic. Hybridizations with wild type chloroplast DNA and cloned chloroplast probes should have provided stronger arguments to identify the chloroplastic nature of some minicircles at least. The specificity of the hybridizations is apparent from the labeling of reference chloroplast DNA fragments with the corresponding cloned probes (and only with the corresponding probes, without important cross-hybridizations) and also from the fact that not all but only some bands visible in the gel hybridize with the probes. Now almost similar hybridization patterns were surprisingly obtained for identical Southern blots with all the different clones of restriction fragments scattered around the physical map of chloroplast DNA (Hallick 1982). One cannot therefore assume total homology between each of the probes and the hybridizing minicircles. The observed homologies could be limited to short sequences repeated along the wild type chloroplast genome. Such sequences have been demonstrated in the case of Chlamydomonas (Rochaix and Malnoe 1978; Gelvin and Howell 1980); they are believed to cause zerotime binding of denatured chloroplast DNA fragments to hydroxylapatite in renaturation experiments and extensive cross-hybridization between chloroplast DNA restriction fragments. On one hand some snap-back reassociation of denatured chloroplast DNA is apparent in the case of Euglena (Rawson and Boerma 1976; Chelm at al. 1977; preceding paper), but on the other hand Koller and Delius (1982) did not observe more that two stem-loop figures of inverted repeats along the entire molecule of denatured chloroplast DNA. Moreover cross-hybridization between restriction fragments are minimal with chloroplast DNA of Euglena, making the occurence of repeats questionable. The exact nature of the sequences involved in the observed hybridizations remains thus to be identified.
P. Heizmann et al.: Minicircles Homologouswith Chloroplast DNA These hybridizations are probably of the same nature as those suggesting non-stoechiometry in reassociation kinetics of cp- mutant DNA. The detailed anatomy of the minicircular molecules will have to be elucidated before a realistic model can be presented to explain their relationship with chloroplast DNA structure, replication and mutagenesis, their biogenesis and biological function. This problem is of theoretical and practical importance since its solution should allow to better understand the control of replication and reiteration of chloroplast genomes; it might also provide us with potential chloroplast vectors for genetic engeneering.
Acknowledgements. We thank Mr. P. M. Malet and Mrs. C. FaureSchwob for technical assistance, and Drs. G. Bernardi and J. Doly for helpful discussions and advices. This work was partly supported by a C.N.R.S. grant (A.T.P. n° 8068).
References Chelm BK, Hoben PJ, Hallick RB (1977) Biochem 16:782-785 Chu NM, Oishi KK, Tewari KK (1981) Plasmid 6:279-292 Davis RW, Simon M, Davidson N (1971) Meth Enzymol 21:413 -428 Freyssinet G, Heizmann P, Verdier G, Trabuehet G, Nigon V (1970) PhysiolVeg 10:421-442 Gelvin SB, Howell SH (1979) Mol Gen Genet 173:315-322 Gibson WH, Hershberger CL (1975) Arch Biochem Biophys 168: 8-14 Hallick RB (1982) In: The Biologyof Euglena, vol. 4 BuetowDE (ed) (in press) Heizmann P, Doly J, Hussein Y, Nicolas P, Nigon V, Bernadi G (1981) Bioehim Biophys Acta 653:412-415 Koller B, Delius H (1982) FEBS Letters 139:86-92 Kolodner R, Tewari KK (1975) J Biol Chem 250:8840-8847 Kopecka H, Crouse EJ, Stutz E (1977) Eur J Biochem 72:525535 Manning JE, Richards OC (1972) Biochem 11:2036-2043 Mielenz JR, Hershberger CL (1974) Biochem Biophys Res Comm 58:769-777 Nass MMK, Ben-Shaul Y (1972)Biochim Biophys Acta 272:130 -136 Rawson JRY, Boerma C (1976) Proc Natl Acad Sci USA 73: 2401-2404 Richards OC, ManningJE (1975) In: Lefort-Tran M, Valencia 1/, (eds) Les Cycles Cellulaires. Editions du CNRS, pp 212-221 Rochalx JD, Malnoe P (1978) Cell 15:661-670 Sehiff JA, Epstein HT (1965) In: Locke M (ed) Reproduction: Molecular, Subcellular, Cellular. Academic Press, New York, pp 131-189 Stutz E, Vandrey JP (1971) Proc. Ilnd Intern. Congr; on Photosynth. Forti G, Avron M, Melandri A (eds) pp 2601-2609 Stutz E, Crouse EJ, Graf L (1975) In: Les CyclesCellulalres, pp 255-265 Communicated by R. J. Schweyen Received July 5, 1982
