53
Biochimica et Biophysica Acta, 561 (1979) 53--58 Q Elsevier/North-Holland Biomedical Press
BBA 99347
BASE COMPOSITION H E T E R O G E N E I T Y OF EUGLENA GRACILIS C H L O R O P L A S T DNA
PATRICK W. GRAY and RICHARD B. HALLICK
Department of Chemistry, University of Colorado, Boulder, CO 80309 (U.S.A.) (Received April 24th, 1978}
Key words: Chloroplast DNA; Restriction nuclease; rRNA gene; Base composition; (Euglena)
Summary
Euglena gracilis chloroplast DNA has an average b u o y a n t density of 1.685 gm/cm 3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized b y equilibrium b u o y a n t density centrifugation. The chloroplast DNA can be characterized as containing t w o major b u o y a n t density components. A segment of 17 kbp, representing 13% of the genome and containing the r R N A genes is 43--44 mol% G • C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G • C content. Introduction
Euglena gracilis chloroplast DNA exists as a covalently closed, superhelical duplex molecule [1]. The size of this genome has been estimated as 140 kbp from electron microscope c o n t o u r length measurements [ 1 ], and 130 kbp from data on electrophoretic mobilities of restriction nuclease fragments [2]. Depending on the conditions of cell culture, there are 500--2000 copies of chloroplast DNA per cell [3,4]. The DNA has an average b u o y a n t density in neutral CsC1 of 1.685 g/cm 3 [5,6,7], corresponding to an average base composition of 75 mol% A • T, and 25 mol% G • C nucleotide pairs. Evidence for heterogeneity of base composition in the chloroplast DNA has come from t w o lines of experimentation. First, a satellite component, p = 1.700--1.701 g/cm 3, is observed when partially sheared DNA is sedimented to equilibrium in CsC1 [1,8,9]. This satellite DNA contains the sequences coding for chloroplast rRNA [8,9]. The r R N A region should be G • C-rich compared to the genome since the r R N A base composition is 42 mol% G • C [10]. Abbreviation: kbp, kilobase pairs.
54 Second, differential thermal denaturation profiles of the DNA have been interpreted in terms of multiple base composition components with G . C contents ranging from 22--42 mol% [11,12]. Recently a detailed restriction endonuclease map of Euglena chloroplast DNA has been described [2]. The r R N A gene region was located on this map as three tandemly repeated 5.6 kbp segments, each of which contains a 16and 23-S r R N A gene. In the present study chloroplast DNA was digested with the enzymes Pst I and Bam HI. The resulting fragments were purified, and characterized b y equilibrium sedimentation in neutral CsC1. From the resulting b u o y a n t densitites, base compositional heterogeneities within this genome have been located. Methods Isolation of covalently closed circular chloroplast DNA from Euglena, endonuclease digestions, and electrophoresis of chloroplast DNA have been described [2,4,13,14]. DNA fragments were isolated from agarose gels for b u o y a n t density analysis b y centrifugation of agarose/DNA mixtures to equilibrium in KI [15]. Samples were subsequently dialyzed against 0.01 M Tris-HC1, 0.005 M NaC1, pH 7.5, collected b y ethanol precipitation, and redissolved in 0.01 M Tris-HC1, 0.005 M NaC1. Optical grade CsC1 was added until the n2D° was 1.400. Samples of 0.5--2.0 pg DNA were centrifuged in an AN-F rotor in a Spinco Model E analytical ultracentrifuge equipped with a photoelectric scanner. Centrifugation was for 48 h at 40 000 rev./min and 25°C. Micrococcus lysodeikticus DNA was included with the sample as a density standard. Mol% G . C base compositions were calculated according to the m e t h o d of Schildkraut et al. [16]. Results
Buoyant densities o f intact and restriction nuclease digested DNA The result of an equilibrium b u o y a n t density centrifugation of chloroplast DNA, isolated as covalently closed circular DNA, is shown in Fig. 1. The DNA bands in a sharp peak at p = 1.685 g/cm 3, consistent with previously reported results [1,5,6--8,17]. There is no apparent contamination of the DNA with either nuclear DNA, which has a p = 1.708 g/cm 3 [5--7], or mitochondrial DNA, which has a p = 1.689 g/cm 3 [18--20]. It has previously been shown that chloroplast DNA, randomly sheared to an average size of 8--10 kbp has a component, enriched in G • C base pairs compared to total chloroplast DNA, which contains the r R N A genes [8,9]. This r R N A c o m p o n e n t is more clearly resolved when intact chloroplast DNA is specifically fragmented with restriction nucleases. As shown in Fig. 1, t w o major b u o y a n t density components, p = 1.680 and 1.689 g/cm 3, are present in the Pst I digestion mixture of chloroplast DNA. The calculated [16] base compositions for these DNAs are 21 and 30 mol% G • C, respectively. The relative areas under the t w o peaks are 0.61 and 0.39 of the total for the 21 and 30 mol% G • C components. The previously mapped, G • C-rich r R N A gene region is known to comprise an internal 17 kbp segment in the Pst A fragment [2].
55
f P,t,,, 1.689 1.68(
_
rL_
1.689
I J
L.
1.680~
/
._J k_ 1.685
1.731
Pst D $6B1
Pst E 1.660 ~
~
j
L..,
t /
k. 1.731
Fig. 1. Equilibrium buoyant density centrifugation of intact ( b o t t o m ) and Pst I digested (top) Euglena chloroplast DNA. Calculated buoyant densities, in g/cm 3 are indicated for each peak. M. lysodeikticus DNA, p = 1.731 g/cm 3, was added to each sample as a standard. Fig. 2. Equilibrium buoyant density centrifugation of isolated Pst I fragments of Euglena chloroplast DNA. Calculated buoyant density for each fragment, in g]cm 3, is indicated for each experiment. M. lysodiekticus DNA standard was also added to each sample (1.731 g/cm3).
Pst A is 0.41 of the genome, nearly the same proportion as the DNA peak of 30 mol% G • C. Direct evidence that the 30 mol% G • C density c o m p o n e n t in the Pst I digestion mixture is Pst A is described below.
Buoyant densities of isolated Pst I fragments To determine the base composition of individual Pst I fragments, analytical equilibrium b u o y a n t density measurements were made for each DNA. The resulting profiles are illustrated in Fig. 2. The four smallest Pst fragments, B, C, D, and E o f 35, 25, 10 and 6.9 kbp all have approximately the same b u o y a n t density, p = 1.680--1.681 g/cm 3. This corresponds to 20--21 mol% G • C base composition. These four fragments, which are linked C-B-D-E on the genome, total 77 kbp, accounting for 0.59 of the 130 kbp DNA. This fraction of the DNA is in agreement with the relative a m o u n t of the genome as 20--21 mol% G • C in the b u o y a n t density analysis of unfractionated Pst I products (Fig. 1). The largest Pst I fragment, Pst A, has a b u o y a n t density of 1.689 g/cm 3, or 30 mol% G • C. Also apparent in the Pst A profile (Fig. 2) is a minor peak. The minor peak is believed to be Pst B, which runs close to Pst A on agarose gels [2]. The a m o u n t of Pst B contamination is variable. In three different experiments the minor peak was 24, 30, and 33% of the DNA sample. We have been unable to obtain complete, preparative electrophoretic separation of DNAs larger than 30 kbp. Nevertheless it can be concluded that the peak at p = 1.689 g/cm 3 is Pst A, and that the higher G • C c o m p o n e n t in the unfractionated digestion mixture is due entirely to Pst A. Based on these b u o y a n t density results it is possible to describe two components of base composition in Euglena chloroplast DNA. Approximately 60% of the genome, defined b y the linked Pst C-B-D-E [2] has an average 20--21
56 mol% G • C content. The remaining 40%, defined by Pst A, and containing the rRNA genes, has an average 30 mol% G . C content. These two components account for the known 25 mol% G • C content of the intact genome.
Buoyant density of the rRNA repeat The chloroplast r R N A genes have been mapped as a triple tandem repeat of 5.6 kbp segments, approximately in the middle of the 53 kbp Pst A fragment [2]. This r R N A gene region should have a higher G • C base composition than that of Pst A, since r R N A has a base composition of 42 mol% G • C [10]. This was tested on chloroplast DNA fragmented with restriction endonuclease Bam HI. Bam HI cleaves each rRNA repeat at a single site [2]. The resulting products include two copies of a 5.6 kbp fragment, Bam E, which contains the entire coding and spacer region for the 16-S and 23-S rRNAs [2]. To determine the b u o y a n t density of Bam E, chloroplast DNA was digested with Bam HI, and the Bam E fragments were isolated as described above. The resulting equilibrium b u o y a n t density profile is shown in Fig. 4. Bam E has a b u o y a n t density of 1.702 g/cm 3, corresponding to 44 mol% G • C. Since the 5.6 kilobase Bam E D N A sequence is repeated three times, 17 kbp of the 53 kbp Pst A has this base content. The remaining 36 kbp has a calculated base content of 23 mol% G • C. This conclusion was confirmed following b u o y a n t density analysis of unfractionated, Bam HI-digested Euglena chloroplast DNA. Three of the six products, Bam D, E and E, are adjacent. These fragments total 18 kbp, represent 0.14 of the genome, and contain greater than 95% of the r R N A gene region. An equilibrium b u o y a n t density profile for this experiment is shown in Fig. 3. There are two major Bam HI b u o y a n t density components. The higher density DNA,
21"/,
1.181
1.700
1.702
1.731
F i g . 3. B u o y a n t d e n s i t y d e t e r m i n a t i o n for Euglena c h l o r o p l a s t r R N A region. Equilibrium density profiles for isolated Barn E ( b o t t o m ) , and for B a r n H I digested, u n f r a c t i o n a t e d D N A ( t o p ) are s h o w n . M l y s o d e i k . ticus D N A w a s a d d e d to each sample as a s t a n d a r d . F i g . 4. B a r n H I and Pst I cleavage sites [ 2 ] , ~ n d regions of base c o m p o s i t i o n h e t e r o g e n e i t y i n t o o l % G • C base pairs o f Euglena chlorplast D N A .
57 p = 1.700 g/cm 3, has a calculated base composition of 43 mol% G • C. The integrated area under this peak represents 0.16 of the total DNA. This DNA arises from the Bam D, E and E fragments. The major component, p = 1.681 g/cm 3, has a calculated 21 mol% G • C content. This DNA represents the fragments Bam A, B, and C [2,14]. The relative area under this peak (0.84) is consistent with the fraction of the genome due to Bam A, B, and C (0.86) predicted from restriction nuclease mapping results [2,14]. These t w o base composition components account for the known 25 mol% G • C content of the intact genome. Furthermore, the regions of base composition heterogeneity determined for the Bam HI fragments are in exact agreement with the results from the study of Pst I fragments. A summary of the relationship between regions of differing base composition and restriction nuclease cleavage sites is illustrated in Fig. 4. Discussion In this report we have described the relationship between regions of different base composition in Euglena chloroplast DNA, and loci on this genome defined b y restriction endonuclease cleavage sites. The chloroplast DNA is composed of t w o components of different base composition. A contiguous region of 113 kbp in the 130 kbp genome has an average base composition of 20--21 mol% G . C . The remaining 17 kbp, which contains the three sets of tandemly repeated rRNA genes, has an average base composition of 44 mol% G • C. The DNA base composition of the rRNA region has also been studied b y Rawson et al. [21]. The base compositions of the Barn D and E fragments, amplified on bacterial plasmids, were found to be 40 and 43 mol% G . C, respectively, in agreement with the present results. Two previous reports on base composition heterogeneity in Euglena chloroplast DNA were based on differential thermal denaturation studies. Crouse et al. [11] have described two major DNA melting regions, corresponding to components of 25 and 44 mol% G • C, in reasonable qualitative agreement with our findings. Slavik and Hershberger [12] however, have concluded that Euglena chloroplast DNA contains five components of base composition of 22, 25, 31, 36 and 41 mol% G • C. They also report that these components represent 40, 27, 16, 10 and 7% of the genome, respectively. The segments are described as being present in relatively large tandem segments, rather than interspersed through the genome. The 31 and 36 mol% G- C components would represent 26% of the genome, or 34 kbp. If such components were present we would have found evidence for their location from the b u o y a n t density analysis of purified Pst B, C, D and E, since the largest of these fragments is the size of the predicted 31 and 36 mol% G" C components. Our results are n o t consistent with the conclusions of Slavik and Hersberger [12], because no large DNA region of > 2 2 mol% G • C is found outside of the r R N A region. Furthermore, the a m o u n t of the genome of > 4 0 mol% G • C seems to have been underestimated by these workers [12]. In summary, previously described base composition heterogeneity in the chloroplast genome can n o w be quantitatively explained in terms of a restriction nuclease cleavage site map. The results described in this paper will hope-
58 fully provide the framework for additional, more detailed physical studies on Euglena chloroplast DNA.
Acknowledgements This work was supported by NIH Grant GM 21351, and in part by NIH Biomedical Research Support Grant RR07013 to the University of Colorado. R.B.H. is the recipient of NIH Research Career Development Award K04 GM00372. We would like to thank Dr. John Heumann for his assistance with the analytical ultracentrifugation studies.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Manning, J.E. and Richaxds, O.C. (1972) Biochim. Biophys. Acta 259, 285--296 Gray, P.W. and Hallick, R.B. (1978) Biochemistry 17, 284--289 Rawson, J.R.Y. and Boerma, C. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2401--2404 Chelm, B.K., Hoben, P.J. and Hallick, R.B. (1977) Biochemistry 16, 782--786 Brawerman, G. and Eisenstadt, J.M. (1964) Biochim. Biophys. A c t a 9 1 , 4 7 7 - - 4 8 5 Ray, D.S. and Hanawalt, P. (1964) J. Mol. Biol. 9, 812--824 Edelman, M., Cowan, C.A., Epstein, H.T. and Schiff, J.A. (1964) Proc. Natl. Acad. Sci. U.S. 52, 1214--1219 Stutz, E. and Vandrey, J.P. (1971) FEBS Lett. 17, 277--280 Rawson, J.R.Y. and Haselkorn, R. (1973) J. Mol. Biol. 77, 125--132 Scott, N.S. (1976) P h y t o c h e m i s t r y 15, 1207--1213 Crouse, E., Vandrey, J. and Stutz, E. (1974) Proc. of the Third Int. Cong. on Photosynth. pp. 1775-1786. Elsevier, A m s t e r d a m Slavik, N.S. and Hershberger, C.L. (1976) J. Mol. Biol. 103, 563--581 Wilson, G.A. and Young, F.E. (1975) J. Mol. Biol. 97, 123--125 Gray, P.W. and Hallick, R.B. (1977) Biochemistry 16, 1665--1671 Blin, N., Gabain, A.V. and Bujard, H. (1975) FEBS Lett. 53, 84--86 Schildkraut, C.L., Marmur, J. and Dory, P. (1962) J. Mol. Biol. 4, 430--443 Stolarsky, L., Slavik, N. and Hershberger, C.L. (1973) Biochim. Biophys. Acta 335, 30--34 Manning, J.E., Wolstenholme, D.R. Ryan, R.S., Hunter, J.A. and Richards, O.C. (1971) Proc. Natl. Aead. Sci. U.S. 68, 1169--1173 Fonty, G., Crouse, E.J., Stutz, E. and Bernardi, G. (1975) Eur. J. Biochem. 54, 367--372 Krawiec, S. and Eisenstadt, J.M. (1970) Biochim. Biophys. Acta 217, 120--131 Rawson, J.R.Y., Kushner, S.R., Vapnek, D., Alton, N.K. and Boerma, C.L. (1978) Gene 3, 191--209
Biochimica et Biophysica Acta, 561 (1979) 53--58 Q Elsevier/North-Holland Biomedical Press
BBA 99347
BASE COMPOSITION H E T E R O G E N E I T Y OF EUGLENA GRACILIS C H L O R O P L A S T DNA
PATRICK W. GRAY and RICHARD B. HALLICK
Department of Chemistry, University of Colorado, Boulder, CO 80309 (U.S.A.) (Received April 24th, 1978}
Key words: Chloroplast DNA; Restriction nuclease; rRNA gene; Base composition; (Euglena)
Summary
Euglena gracilis chloroplast DNA has an average b u o y a n t density of 1.685 gm/cm 3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized b y equilibrium b u o y a n t density centrifugation. The chloroplast DNA can be characterized as containing t w o major b u o y a n t density components. A segment of 17 kbp, representing 13% of the genome and containing the r R N A genes is 43--44 mol% G • C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G • C content. Introduction
Euglena gracilis chloroplast DNA exists as a covalently closed, superhelical duplex molecule [1]. The size of this genome has been estimated as 140 kbp from electron microscope c o n t o u r length measurements [ 1 ], and 130 kbp from data on electrophoretic mobilities of restriction nuclease fragments [2]. Depending on the conditions of cell culture, there are 500--2000 copies of chloroplast DNA per cell [3,4]. The DNA has an average b u o y a n t density in neutral CsC1 of 1.685 g/cm 3 [5,6,7], corresponding to an average base composition of 75 mol% A • T, and 25 mol% G • C nucleotide pairs. Evidence for heterogeneity of base composition in the chloroplast DNA has come from t w o lines of experimentation. First, a satellite component, p = 1.700--1.701 g/cm 3, is observed when partially sheared DNA is sedimented to equilibrium in CsC1 [1,8,9]. This satellite DNA contains the sequences coding for chloroplast rRNA [8,9]. The r R N A region should be G • C-rich compared to the genome since the r R N A base composition is 42 mol% G • C [10]. Abbreviation: kbp, kilobase pairs.
54 Second, differential thermal denaturation profiles of the DNA have been interpreted in terms of multiple base composition components with G . C contents ranging from 22--42 mol% [11,12]. Recently a detailed restriction endonuclease map of Euglena chloroplast DNA has been described [2]. The r R N A gene region was located on this map as three tandemly repeated 5.6 kbp segments, each of which contains a 16and 23-S r R N A gene. In the present study chloroplast DNA was digested with the enzymes Pst I and Bam HI. The resulting fragments were purified, and characterized b y equilibrium sedimentation in neutral CsC1. From the resulting b u o y a n t densitites, base compositional heterogeneities within this genome have been located. Methods Isolation of covalently closed circular chloroplast DNA from Euglena, endonuclease digestions, and electrophoresis of chloroplast DNA have been described [2,4,13,14]. DNA fragments were isolated from agarose gels for b u o y a n t density analysis b y centrifugation of agarose/DNA mixtures to equilibrium in KI [15]. Samples were subsequently dialyzed against 0.01 M Tris-HC1, 0.005 M NaC1, pH 7.5, collected b y ethanol precipitation, and redissolved in 0.01 M Tris-HC1, 0.005 M NaC1. Optical grade CsC1 was added until the n2D° was 1.400. Samples of 0.5--2.0 pg DNA were centrifuged in an AN-F rotor in a Spinco Model E analytical ultracentrifuge equipped with a photoelectric scanner. Centrifugation was for 48 h at 40 000 rev./min and 25°C. Micrococcus lysodeikticus DNA was included with the sample as a density standard. Mol% G . C base compositions were calculated according to the m e t h o d of Schildkraut et al. [16]. Results
Buoyant densities o f intact and restriction nuclease digested DNA The result of an equilibrium b u o y a n t density centrifugation of chloroplast DNA, isolated as covalently closed circular DNA, is shown in Fig. 1. The DNA bands in a sharp peak at p = 1.685 g/cm 3, consistent with previously reported results [1,5,6--8,17]. There is no apparent contamination of the DNA with either nuclear DNA, which has a p = 1.708 g/cm 3 [5--7], or mitochondrial DNA, which has a p = 1.689 g/cm 3 [18--20]. It has previously been shown that chloroplast DNA, randomly sheared to an average size of 8--10 kbp has a component, enriched in G • C base pairs compared to total chloroplast DNA, which contains the r R N A genes [8,9]. This r R N A c o m p o n e n t is more clearly resolved when intact chloroplast DNA is specifically fragmented with restriction nucleases. As shown in Fig. 1, t w o major b u o y a n t density components, p = 1.680 and 1.689 g/cm 3, are present in the Pst I digestion mixture of chloroplast DNA. The calculated [16] base compositions for these DNAs are 21 and 30 mol% G • C, respectively. The relative areas under the t w o peaks are 0.61 and 0.39 of the total for the 21 and 30 mol% G • C components. The previously mapped, G • C-rich r R N A gene region is known to comprise an internal 17 kbp segment in the Pst A fragment [2].
55
f P,t,,, 1.689 1.68(
_
rL_
1.689
I J
L.
1.680~
/
._J k_ 1.685
1.731
Pst D $6B1
Pst E 1.660 ~
~
j
L..,
t /
k. 1.731
Fig. 1. Equilibrium buoyant density centrifugation of intact ( b o t t o m ) and Pst I digested (top) Euglena chloroplast DNA. Calculated buoyant densities, in g/cm 3 are indicated for each peak. M. lysodeikticus DNA, p = 1.731 g/cm 3, was added to each sample as a standard. Fig. 2. Equilibrium buoyant density centrifugation of isolated Pst I fragments of Euglena chloroplast DNA. Calculated buoyant density for each fragment, in g]cm 3, is indicated for each experiment. M. lysodiekticus DNA standard was also added to each sample (1.731 g/cm3).
Pst A is 0.41 of the genome, nearly the same proportion as the DNA peak of 30 mol% G • C. Direct evidence that the 30 mol% G • C density c o m p o n e n t in the Pst I digestion mixture is Pst A is described below.
Buoyant densities of isolated Pst I fragments To determine the base composition of individual Pst I fragments, analytical equilibrium b u o y a n t density measurements were made for each DNA. The resulting profiles are illustrated in Fig. 2. The four smallest Pst fragments, B, C, D, and E o f 35, 25, 10 and 6.9 kbp all have approximately the same b u o y a n t density, p = 1.680--1.681 g/cm 3. This corresponds to 20--21 mol% G • C base composition. These four fragments, which are linked C-B-D-E on the genome, total 77 kbp, accounting for 0.59 of the 130 kbp DNA. This fraction of the DNA is in agreement with the relative a m o u n t of the genome as 20--21 mol% G • C in the b u o y a n t density analysis of unfractionated Pst I products (Fig. 1). The largest Pst I fragment, Pst A, has a b u o y a n t density of 1.689 g/cm 3, or 30 mol% G • C. Also apparent in the Pst A profile (Fig. 2) is a minor peak. The minor peak is believed to be Pst B, which runs close to Pst A on agarose gels [2]. The a m o u n t of Pst B contamination is variable. In three different experiments the minor peak was 24, 30, and 33% of the DNA sample. We have been unable to obtain complete, preparative electrophoretic separation of DNAs larger than 30 kbp. Nevertheless it can be concluded that the peak at p = 1.689 g/cm 3 is Pst A, and that the higher G • C c o m p o n e n t in the unfractionated digestion mixture is due entirely to Pst A. Based on these b u o y a n t density results it is possible to describe two components of base composition in Euglena chloroplast DNA. Approximately 60% of the genome, defined b y the linked Pst C-B-D-E [2] has an average 20--21
56 mol% G • C content. The remaining 40%, defined by Pst A, and containing the rRNA genes, has an average 30 mol% G . C content. These two components account for the known 25 mol% G • C content of the intact genome.
Buoyant density of the rRNA repeat The chloroplast r R N A genes have been mapped as a triple tandem repeat of 5.6 kbp segments, approximately in the middle of the 53 kbp Pst A fragment [2]. This r R N A gene region should have a higher G • C base composition than that of Pst A, since r R N A has a base composition of 42 mol% G • C [10]. This was tested on chloroplast DNA fragmented with restriction endonuclease Bam HI. Bam HI cleaves each rRNA repeat at a single site [2]. The resulting products include two copies of a 5.6 kbp fragment, Bam E, which contains the entire coding and spacer region for the 16-S and 23-S rRNAs [2]. To determine the b u o y a n t density of Bam E, chloroplast DNA was digested with Bam HI, and the Bam E fragments were isolated as described above. The resulting equilibrium b u o y a n t density profile is shown in Fig. 4. Bam E has a b u o y a n t density of 1.702 g/cm 3, corresponding to 44 mol% G • C. Since the 5.6 kilobase Bam E D N A sequence is repeated three times, 17 kbp of the 53 kbp Pst A has this base content. The remaining 36 kbp has a calculated base content of 23 mol% G • C. This conclusion was confirmed following b u o y a n t density analysis of unfractionated, Bam HI-digested Euglena chloroplast DNA. Three of the six products, Bam D, E and E, are adjacent. These fragments total 18 kbp, represent 0.14 of the genome, and contain greater than 95% of the r R N A gene region. An equilibrium b u o y a n t density profile for this experiment is shown in Fig. 3. There are two major Bam HI b u o y a n t density components. The higher density DNA,
21"/,
1.181
1.700
1.702
1.731
F i g . 3. B u o y a n t d e n s i t y d e t e r m i n a t i o n for Euglena c h l o r o p l a s t r R N A region. Equilibrium density profiles for isolated Barn E ( b o t t o m ) , and for B a r n H I digested, u n f r a c t i o n a t e d D N A ( t o p ) are s h o w n . M l y s o d e i k . ticus D N A w a s a d d e d to each sample as a s t a n d a r d . F i g . 4. B a r n H I and Pst I cleavage sites [ 2 ] , ~ n d regions of base c o m p o s i t i o n h e t e r o g e n e i t y i n t o o l % G • C base pairs o f Euglena chlorplast D N A .
57 p = 1.700 g/cm 3, has a calculated base composition of 43 mol% G • C. The integrated area under this peak represents 0.16 of the total DNA. This DNA arises from the Bam D, E and E fragments. The major component, p = 1.681 g/cm 3, has a calculated 21 mol% G • C content. This DNA represents the fragments Bam A, B, and C [2,14]. The relative area under this peak (0.84) is consistent with the fraction of the genome due to Bam A, B, and C (0.86) predicted from restriction nuclease mapping results [2,14]. These t w o base composition components account for the known 25 mol% G • C content of the intact genome. Furthermore, the regions of base composition heterogeneity determined for the Bam HI fragments are in exact agreement with the results from the study of Pst I fragments. A summary of the relationship between regions of differing base composition and restriction nuclease cleavage sites is illustrated in Fig. 4. Discussion In this report we have described the relationship between regions of different base composition in Euglena chloroplast DNA, and loci on this genome defined b y restriction endonuclease cleavage sites. The chloroplast DNA is composed of t w o components of different base composition. A contiguous region of 113 kbp in the 130 kbp genome has an average base composition of 20--21 mol% G . C . The remaining 17 kbp, which contains the three sets of tandemly repeated rRNA genes, has an average base composition of 44 mol% G • C. The DNA base composition of the rRNA region has also been studied b y Rawson et al. [21]. The base compositions of the Barn D and E fragments, amplified on bacterial plasmids, were found to be 40 and 43 mol% G . C, respectively, in agreement with the present results. Two previous reports on base composition heterogeneity in Euglena chloroplast DNA were based on differential thermal denaturation studies. Crouse et al. [11] have described two major DNA melting regions, corresponding to components of 25 and 44 mol% G • C, in reasonable qualitative agreement with our findings. Slavik and Hershberger [12] however, have concluded that Euglena chloroplast DNA contains five components of base composition of 22, 25, 31, 36 and 41 mol% G • C. They also report that these components represent 40, 27, 16, 10 and 7% of the genome, respectively. The segments are described as being present in relatively large tandem segments, rather than interspersed through the genome. The 31 and 36 mol% G- C components would represent 26% of the genome, or 34 kbp. If such components were present we would have found evidence for their location from the b u o y a n t density analysis of purified Pst B, C, D and E, since the largest of these fragments is the size of the predicted 31 and 36 mol% G" C components. Our results are n o t consistent with the conclusions of Slavik and Hersberger [12], because no large DNA region of > 2 2 mol% G • C is found outside of the r R N A region. Furthermore, the a m o u n t of the genome of > 4 0 mol% G • C seems to have been underestimated by these workers [12]. In summary, previously described base composition heterogeneity in the chloroplast genome can n o w be quantitatively explained in terms of a restriction nuclease cleavage site map. The results described in this paper will hope-
58 fully provide the framework for additional, more detailed physical studies on Euglena chloroplast DNA.
Acknowledgements This work was supported by NIH Grant GM 21351, and in part by NIH Biomedical Research Support Grant RR07013 to the University of Colorado. R.B.H. is the recipient of NIH Research Career Development Award K04 GM00372. We would like to thank Dr. John Heumann for his assistance with the analytical ultracentrifugation studies.
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