Vol. 130, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, May 1977, p. 860-868 Copyright © 1977 American Society for Microbiology
Analysis of Euglena gracilis Chloroplast Deoxyribonucleic Acid with a Restriction Endonuclease, EcoRI JONATHAN R. MIELENZ,1* JOEL J. MILNER, AND CHARLES L. HERSHBERGER2 Department of Microbiology, University ofIllinois, Urbana, Illinois 61801 Received for publication 7 January 1977
Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoPJ) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight ofthe circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosoal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.
Genes of organelles have been identified by their patterns of inheritance (21). Hybridization studies have proven that deoxyribonucleic acid (DNA) of organelles contains cistrons for ribosomal (r) and transfer (t) ribonucleic acid (RNA) (6, 7). Heteroduplex mapping located individual mitochondrial (mt) cistrons for rRNA and several species of tRNA on the separated strands of DNA (33). A new method of physical mapping ofgenes has been provided by enzymes that cleave DNA at specific sites. Restriction endonucleases have been used successfully to locate regions of viral DNA that code for messenger RNA (15), to determine the origin and direction of replication of mt DNA (5, 20), and to compare DNA of organelles from both mammalian (18) and plant (14, 32) populations. Chloroplast (ct) DNA ofEuglena gracilis Z is ideally suited for physical mapping of g ees with restriction endonucleases. The ct DNA has a molecular weight of 92 x 106 (17). The molecule contains one to six cistrons for rRNA as measured by different investigators (7, 1i, 23, 27, 28), and 26 cistrons for tRNA (16, 22), Pure and intact nucleic acids are easily obtained from isolated chloroplasts ofE. gracilis. The number, sizes, and approximate buoyant density ofthe fragments of ct DNA produced by ' Present address: Plant Growth Laboratory, University of California, Davis, CA 95616. 2 Present address: Biochemical Development Division, Eli Lilly and Company, Indianapolis, IN 46206.
restriction endonuclease RI from Escherichia coli (EcoRI) have been determined in this investigation. The number of cistrons for 16S and 238 rRNA on the ct DNA was determined by hybridization of ct DNA with ct rRNA labeled with 125I. The fragments of ct DNA that contained these cistrons were identified by hybridization with ct rRNA. MATERIALS AND METHODS Isolation of DNA and RNA. Euglena was grown mixotrophically on a Hutner medium (11). Chloroplasts were isolated as described by Kissel and Buetow (13), with slight modifications. The ct DNA was extracted from the chloroplasts and purified as described (16). Colicinogenic factor El (ColE1) DNA was isolated fromE. coli JC411 as covalently closed circular DNA (3). Bacteriophage lambda (XvirS5O and XcI857), bacteriophage T4B, and their DNAs were isolated as described previously (4, 10). The et RNA from E. gracilis was extracted from the isolated chloroplaats (13). 16S and 23S rRNA's were purified as described previously (2). Electrophoresis of the RNA through polyacrylamide gels indicated that the 16S and 23S rRNA's were free of detectable levels of contaminating RNA. Digestion with EcoRI and electrophoresis. The EcolI restriction endonuclease was prepared from E, coli RY13 (29) or obtained from Miles Laboratories. The DNA was digested in a buffer containing 25 mM tris(hydroxymethyl)aminomethane, 10 mM MgCl2, and 50 mM NaCl, pH 7.5, with EcoRI at 37°C. Electrophoresis of the DNA and visualization
860
ANALYSIS OF CHLOROPLAST DNA WITH ECORI
VOL. 130, 1977
and photography of the gels of agarose (Sigma) with Polaroid 46L film have been described (29). Quantitation of bands E, I, M, and T. Fluorescence was a linear function of the DNA mass in the range of concentrations used for quantitating bands when the gels were analyzed as described by Depew and Wang (8). Several exposures of gels containing different amounts of DNA were needed to obtain proper concentrations for all the bands. Photographs within the linear range of film response were traced with a Joyce-Loebl MK III B recording microdensitometer. The areas of the peaks of interest and neighboring peaks were determined and normalized for molecular weights of the fragments of DNA. Normalized ratios of areas of bands E, I, M, and T to the areas of neighboring bands yielded the number of repeats in those bands. Buoyant density analysis of restriction fragments. The ct DNA was digested with EcoRI and centrifuged to equilibrium in CsCl in a Beckman type 65 rotor (26). The DNA was fractionated, and the ultraviolet absorbance was monitored (26). Appropriate fractions were pooled as shown, diluted with 0.1 x SSC (standard saline citrate: 0.15 M NaCl, 0.015 M citrate, pH 7.0), and centrifuged in a Beckman SW50.1 rotor at 50,000 rpm for 8 h at 4°C. The DNA pellet was dissolved in 0.1 x SSC and electrophoresed on 0.7% agarose. RNA:DNA hybridization. The ct RNA- was labeled with 125I and hybridized to ct DNA immobilized on nitrocellulose filters as described (9, 16). The ct rRNA was hybridized to restriction fragments in gels of agarose by the procedure of Shinnick et al. (24).
RESULTS Purity and integrity of the ct DNA. Samples of DNA intended for digestion with restriction nucleases must be free of contaminating species of DNA and have a high molecular weight. The ct DNA obtained from chloroplasts is pure ct DNA, uncontaminated with nuclear or mt DNA (16). The size of the ct DNA was examined by analytical boundary sedimentation (10). A single sedimenting boundary was observed with a sedimentation coefficient of 64S. This result compares favorably with the value of 66S expected for relaxed circular DNA with a molecular weight of 92 x 106.
861
The size and structure of the ct DNA were analyzed by electron microscopy. A large proportion of the molecules were relaxed circles, but large linear molecules were observed also. These linear molecules could have been generated by shear forces when the DNA was spread for electron microscopy. The molecular weight of the ct DNA was (92 + 3) x 106 when calculated from measurements of nine relaxed circular molecules relative to relaxed circular ColEl DNA molecules, included as an internal standard. Similar results were obtained by Manning and Richards (17). The data for measuring the molecular weight of ct DNA are available (J. R. Mielenz, Ph.D. thesis, University of Illinois, Urbana, 1976). Analysis of the pattern of restriction fragments of ct DNA. The products of digestion of the ct DNA by EcoRI were resolved by electrophoresis in 0.7% agarose (Fig. 1). Twenty-three bands were observed, as in these duplicate gels. This pattern ofbands was the result of complete digestion because neither prolonged digestion nor three repeated digestions of one sample of ct DNA altered the pattern of bands in Fig. 1. Furthermore, digestion of ct DNA under conditions described by Thomas and Davis (31) or the use of EcoRI from Miles Laboratories also produced the pattern in Fig. 1. Digestion of DNA from bacteriophage XcI857 and plasmid ColEl under the conditions described in Materials and Methods yielded only the fragments produced by cleavage at the hexanucleotide site for
EcoRI (29, 31). No bands smaller than ct W were found when samples with greater amounts of DNA were separated on 1% agarose. Also, the bands in Fig. 1 represent all of the radioactive fragments when 32P-labeled ct DNA is analyzed in the same manner (Walfield and Hershberger, unpublished data), so ct A through ct W represent all the major bands when ct DNA is digested with EcoRI. Bands ct E, I, M, and T were present in higher than stoichiometric amounts. Appropriate photographs of gels were quantitated relative to neighboring bands by determining the areas of peaks obtained from microdensitome-
RI ct DNA t tt t t I I tVM\ ye marker I JKLM NOP QR S T UVW FIG. 1. Electrophoretic pattern of fragments of chloroplast DNA that has been digested with EcoRI. Twenty-three distinct bands were observed, lettered ct A to ct W, as in these duplicate gels. All of the fragments were not recorded on one photograph because the quantities of DNA in the bands varied by over 15-fold. Prints of two different exposures of the same gels were split diagonally and appropriate halves combined to produce
AB
this figure.
CDE F
t I G H
862
re
ter tracings. All four bands were found to be present in about twofold excess when normal-
ized for the molecular weight of the fragments (Table 1). Determinations of molecular weights will be described below. The DNA in these bands could represent repeated DNA sequences or different segments of DNA with equally spaced sites sensitive to EcoRI. With a small sample volume (20 ul) and a long migration (22 cm), sufficient resolution was obtained to detect the separation of ct M into two bands (Fig. 2). Electrophoresis was TABLE 1. Quantitation of bands E, I, M, and T
Test band
E I M T
Mol wt (x 106)
4.70 2.35 1.90 0.95
Neighboring band(s)
used for quantitation
F J, K, and L J, K, L, N, O, and P S
Ratio of normalized areas
obfated/
neighboring band 1.8 2.0 2.0 2.3
-i.H
_o
--
-
*
continued until ct Q and smaller bands had migrated off the gel. With ct I, a double-molar band, as a reference, ct J, K, L, and two additional bands were visible immediately below ct I. The two bands, ct Ml and ct M2, normally migrated as a broad single band that contained twice the amount of DNA found in neighboring bands. Examination of the other double-molar bands, ct E, I, and T, in a similar manner did not give significant separation. Therefore, the 23 bands contain at least 24 different fragments of ct DNA. Determination of the size of the fragments. Chloroplasts of Euglena contain up to 65 copies of ct DNA (17) that could be a single species of DNA or subpopulations of molecules with different sequences. The sum of the molecular weights of the restriction fragments should equal the analytical molecular weight if there is one type of molecule. The sizes of the 24 fragments of ct DNA were determined by coelectrophoresis with restriction fragments of ColEl (29) and bacteriophage XvirS50 DNA. XvirS50, which carries a b2 deletion, yields EcoRI fragments that have the same electrophoretic mobilities as another b2 deletion mutant (31). The sum of the molecular weights of all the fragments of ct DNA is 87.6 x 106 (Table 2) and is within the precision of kinetic complexity and analytical measurements of 92 x 106 (17, 25). Therefore, with respect to the locaTABLE 2. Sizes of the fragments of ct and standard DNA
I
Restriction fragmenta
X,+vb
j
--M M2 N
Mol wt (x 106)
EcoRI re-
15.8 14.5 13.7 13.0 6.30 5.90 4.70C 4.70 4.50 4.20 3.70 3.50 3.05 2.80
AV
striction
Mol wt (x 106)
2.10 J 2.10 K 2.05 XI B L 2.00 C M 1.90C D N 1.70 E 0 1.65 P XH, 1.55 F Q 1.25 Col El R 1.20 S A,,, 1.00 G T 0.95' H U 0.87 0.85 V XIv I 2.35X 0.82 W a Fragments of ct DNA are designated by capital letters. b XA,+ is a composite of fragments held together by the ends that anneal with each other when X DNA is circularized (1). r Bands E, I, M, and T were present in twofold amounts relative to adjacent bands and were included twice in the sum of the molecular weights. A
FIG. 2. Separation of fragment M. EcoRI-digested was electrophoresed through 0.7% agarose until fragments ct Q to W migrated off the gel. Fragments ct G to P are shown in the photograph of this gel. ct M appeared as a double band instead of a single band containing twice as much DNA. The double bands are designated ct M, and ct M2. ct DNA
J. BACTERIOL.
MIELENZ, MILNER, AND HERSHBERGER
VOL. 130, 1977
ANALYSIS OF CHLOROPLAST DNA WITH ECORI
tion of the sites sensitive to EcoRI, the ct DNA of Euglena exists primarily as a population of identical molecules. Determination of the buoyant densities of the fragments. The ct DNA of Euglena contains five segments of different base composition ranging from 22 to 41% guanine plus cytosine (G+C) (26). Because of this heterogeneity of the base composition, the fragments of ct DNA produced by EcoRI digestion should have a range of different densities in CsCl. The ct DNA was digested by EcoRI and analyzed in gradients of CsCl. A preparative CsCl gradient of this sample of DNA produced the profile shown in Fig. 3. The fractions ofthis DNA were pooled into four samples as shown and electrophoresed through 0.7% agarose to produce gels a through d (Fig. 3). Several bands of DNA were located predominantly in a single gel, indicating that they had distinct buoyant densities in CsCl. Major components of one fraction appear as minor components in neighboring fractions because DNAs with different densities form overlapping Gaussian bands in gradients of CsCl. ct A, J, L, Q, R, S, and T were mostly in gel a (Fig. 3) and must have originated from the segment of ct DNA with the lightest density. Gels a and b contained ct C, D, F, G, H, I, and N in nearly equal proportions, suggesting that these fragments have densities close to 1.686 g/cm3. Gel c contained two predominant bands, ct B and ct E, that were also found in other fractions. Gel d contained four predominant fragments, ct K, M, 0, and P, with faint neighboring bands showing that ct K, 0, and P had a high buoyant density in CsCl, above 1.697 g/cm3. Significant amounts of band M were found in gels a, b, and d (Fig. 3), so ct M appeared to have a wide range of buoyant densities. Band ct M contained two fragments as explained above. These results indicate that the two fragments of ct M with almost identical size have different buoyant densities. Gels a and b (Fig. 3) contained one species, ct ML (light), whereas gel d contained ct MH (heavy). We do not know whether ct ML is ct M1 or ct M2 in Fig. 2. Analysis of the gels in Fig. 3 leads to a classification of most of the fragments according to the region of the gradient where the major portion of each fragment is found. The classes contain fragments from fractions of the gradient with densities of 1.686 g/cm3 and less, 1.686 to 1.691 g/cm3, 1.691 to 1.696 g/cm3, and 1.696 to 1.716 g/cm3. Loss of fragments during isolation of DNA. Identification of restriction fragments with different buoyant densities suggests the possibility of losing specific segments when ct DNA is
863
purified by procedures that separate DNA with different base compositions, such as density gradient centrifugation and chromatography on hydroxylapatite. Three different preparations of ct DNA were analyzed to determine the extent of this problem. One of the preparations of DNA was obtained from isolated chloroplasts as pure, intact ct DNA. Two other samples of ct DNA were obtained from whole cells (Mielenz, Ph.D. thesis) and purified to homogeneity using equilibrium density gradient centrifugation to remove contaminating nuclear DNA. The molecular weight of each of the samples of ct DNA was determined, since breakage is a prerequisite for significant loss of DNA with a high buoyant density. The DNA obtained from the isolated chloroplasts had a molecular weight of 92 x 106 as described above. Boundary sedimentation of the second preparation of DNA (preparation B) showed that the DNA had a molecular weight of 37 x 106. The third sample of DNA, preparation C, had sustained a significant amount of breakage and had a molecular weight of 13 x 106. Each of these samples was digested with EcoRI, and the fragment pattern was analyzed. Digestion and electrophoresis on agarose of preparation A yielded the characteristic pattern shown above (Fig. 4a). A microdensitometer scan of photographs of the gels across bands ct I through ct P yielded the adjoining tracing. ct I and ct M yielded large predominant peaks that contained twice the area of adjacent bands, ct J and ct L, when normalized for molecular weight, ct J, K, L, N, 0, and P all yielded similar-size peaks (+8%). Preparation B of ct DNA was digested with EcoRI, and the separated fragments produced gel b (Fig. 4b). No single bands of DNA were missing, but the microdensitometer tracings of bands ct I to ct P revealed a significant change. ct I was the largest peak, whereas ct J, L, and N were found in similar amounts (+10%). ct K and ct 0 were found to be reduced to about 55%, whereas ct P was drastically reduced to 14% of ct N, after normalization for molecular weight. ct M was not present in twofold amounts, but was present at 1.7-fold relative to ct L and ct N. ct ML should not be lost by the methods used to purify this DNA, so the 30% loss apparently was due to the selective loss of ct MH along with ct K, 0, and P. Thus, approximately 50% of the DNA found in ct K, M, 0, and P was lost during preparation of the DNA (Table 3). The pattern of restriction fragments from preparation C of ct DNA showed marked alteration, with four bands missing (Fig. 4c). Comparison of gel c with gel a or b (Fig. 4) showed that ct K, 0, and P were missing. The micro-
As
G H
~ ~-i_t~J _-_
-z
H ,-H
R~~~
.
~\
M
N-~~~~~~~~~-
T-
864
T
d~uvw
VOL. 130, 1977
ANALYSIS OF CHLOROPLAST DNA WITH ECORI
densitometer tracing of bands ct I to ct P confirmed the loss of ct K, 0, and P. ct M was present in amounts similar to ct J and ct L (Table 3). The missing part of ct M is thought to be ct MH, since ct ML should be retained because
.*
865
it has a low buoyant density. In addition to bands ct K, 0, and P, one of the bands, ct A or ct B, was missing. Significant recovery of one of these two large fragments suggests that the missing band was not lost solely by fragmenta-
I
FIG. 4. Loss of fragments by purification on density gradients. The gels show ct DNA that was digested with EcoRI and electrophoresed on 0.7% agarose. Photographs of bands ct I through ct P were traced with a microdensitometer to obtain the profiles. The samples of ct DNA with different molecular weights were purified to homogeneity by equilibrium density gradient ultracentrifugation. (a) ct DNA with a molecular weight of 92 X 106; (b) fragmented ct DNA with a molecular weight of37 x 106; and (c) fragmented ct DNA with a molecular weight of 13 x 106.
FIG. 3. Determination of the approximate buoyant densities of the restriction fragments of ct DNA. The ct DNA was digested with EcoRI and centrifuged to equilibrium in CsCl. Fractionation of the sample yielded the profile of absorbancy at 254 nm (top), with Micrococcus luteus DNA at 1.731 g/cm3. Gels a to d contain DNA from buoyant densities of 1.670 to 1.686 g/cm3, 1.686 to 1.691 g/cm3, 1.691 to 1.696 g/cm3, and 1.696 to 1.716 g/cm3, respectively. Gel a-d contains fragments from complete ct DNA as a reference.
typical of specific hybrids. The temperature for one-half denaturation (T,0) was 78°C. Formation of specific hybrids should be independent of temperature within the interval of 30 to 150C below the T,m. This prediction was verified by measuring hybridization between 50 and 640C. The level of hybridization was the same within this temperature interval. Identification of fragments containing the cistrons for rRNA. Restriction fragments can be used to identify specific cistrons contained on ct DNA. Identification of the restriction fragments with cistrons for 16S and 23S rRNA was selected as a model for testing this approach with ct DNA. The ct rRNA was labeled with 125I to specific activities of 105 to 107 cpm/,ug. This rRNA was hybridized to the restriction fragments of ct DNA contained within a 0.7% agarose gel (24). The bands of DNA were detectable before and after hybridization. An autoradiograph of the gels detected specific hybridization to bands ct B, E, K, M, 0, and P (Fig. 5). No significant hybridization of the rRNA to the other 17 bands of DNA was detected. Similar results were obtained when individual restriction fragments were extracted from the gels (15), bound to nitrocellulose filters, and hybridized to '25I-labeled ct rRNA (Mielenz, Ph.D. thesis).
TABLE 3. Analysis of fragments lost versus molecular weight of the DNA Sam-
pie of Method of puri- Mol
w
No. of restriction fragments
Fragments depleted or miss-
ct DNA
fication
(X 1w)
A
Extraction from isolated chloroplasts Extracted from whole cells
92
24
None
37
24
K
B
C
J. BACTERIOL.
MIELENZ, MILNER, AND HERSHBERGER
866
Same as B
13
ing
MH 0 P 19 A or B K |MH 0
Amt of band miss(% ing of A)
45 30 45 86 100 100 100 100
tion. The missing band was probably ct B since it had a heavier buoyant density than ct A. Hybridization of ct rRNA to ct DNA. The number of cistrons for rRNA located on ct DNA was determined by hybridization of rRNA to ct DNA that was isolated as intact molecules. Since the time course of hybridization (data not shown) indicated that hybrids attained equilibrium in 12 h and were stable for at least 24 h, 18 h was used for subsequent hybridization reactions. The level of ct rRNA hybridized to ct DNA was measured at saturating concentrations of ct rRNA by incubating the ct DNA with several concentrations of rRNA. The concentration of the RNA was varied by 10-fold from 0.5 to 5 RNA/DNA ratio. The plateau was flat and well defined at 3.2% of the DNA hybridized with RNA, as expected if all specific sites on the ct DNA were saturated. Specificity of the reaction was determined by hybridizing the RNA to DNA from E. coli as a heterologous control. The level ofbinding to the control was virtually the same as the binding to blank filters. Specific hybridization was 62.5 times the nonspecific binding. Thermal stability of the hybrid was determined to further ascertain the specificity of hybridization. The hybrids denatured over a narrow temperature range, with a sigmoidal profile of denaturation from 55 to 990C that is
.M -..-"
ir -iff
.1001-
.
..dmkmkl.
I I
a
a
1 It IM It
4M dk dihM
.
DISCUSSION Digestion of Euglena ct DNA with the enzyme EcoRI produces 24 fragments of different sizes. Two fragments (ct ML and ct MH) are nearly identical in size. Three other bands of DNA are also distinctive because they are present in twofold excess relative to neighboring bands. They could result from repeated DNA sequences or from segments with different sequences of nucleotides that contain nearly identical spacing between sites sensitive to EcoRI. The sum of the molecular weights of all the fragments agrees within 5% of the kinetic complexity and the analytical molecular weight of ct DNA of 92 x 106, and suggests that the ct DNA of Euglena exists as a single molecular clone within the cell. Agreement between three
0 9
-
-
v
.
V.
4 it
FIG. 5. Hybridization ofct rRNA to fragments ofct DNA produced by digestion with EcoRI. The top figure shows the autoradiograph ofthe gel after hybridization of 125I1-labeled ct rRNA to the bands ofDNA within the gel . The bottom figure is a photograph of the bands ofDNA contained in the same 0.7% gel of agarose. The contrast in the autoradiograph was reversed during printing.
VOL. 130, 1977
ANALYSIS OF CHLOROPLAST DNA WITH ECORI
methods of measuring the size also indicates that the ct DNA contains a single unique sequence of 92 x 106 daltons rather than tandem repetitions of a shorter sequence. Several restriction fragments of ct DNA have different buoyant densities in CsCl. This observation is consistent with the fact that ct DNA of Euglena contains five segments with different base compositions (26). Seven fragments have densities less than total ct DNA, at 1.686 g/cm3, eight have densities of about 1.686 g/cm3, and two have densities slightly greater than 1.686 g/cm3 (Table 3). Four of the restriction fragments, ct K, MH, 0, and P, contain DNA with a buoyant density greater than 1.696 g/cm3 and can be compared to fraction V of Slavik and Hershberger (26). The DNA in fraction V contains 41% G+C, has a buoyant density of 1.702 g/cm3, and contains 6% of the ct DNA. The sum of the molecular weights of fragments ct K, MH, 0, and P is 7.15 x 106 or 8% of the genome. Therefore, the four restriction fragments and fraction V apparently contain much of the same segments of ct DNA. Fragments ct U, V, and W have not been found in the density gradients. These fragments represent individually about 1% of the ct DNA and are the most difficult to detect. They will form broad bands (or band) in equilibrium gradients because of their low molecular weight. They may be distributed through several fractions of the gradient, further reducing the ability to detect these small fragments. Significant amounts of ct DNA can be lost through purification of ct DNA by techniques that separate DNA by differences in base composition. A similar conclusion has been suggested previously (26, 28), but the missing restriction fragments were not identified. There is a strong inverse correlation between the preferential loss of fragments ct K, M, 0, and P and the molecular weight of the DNA. Greater breakage separates more of the segments with a high buoyant density from the adjoining segments with a low buoyant density. Segments with the highest buoyant density would be lost preferentially compared with DNA of lower buoyant density if the DNA is purified by equilibrium density gradient ultracentrifugation. Fragments ct K, MH, 0, and P are depleted differentially at 45, 30, 45, and 86%, respectively. Since the loss results from differences in the buoyant densities, these fragments can be listed by decreasing buoyant density as ct P, (O and K), MH, with the lower limit for ct MH
867
similar to that in Fig. 1, except that bands ct K, 0, and P appear to be missing. Insufficient experimental detail was presented to deternine the reason for the discrepancy. Furthermore, the gels of Hallick et al. (12) did not appear to contain a sufficient quantity of DNA to allow detection ofthe smallest bands, ct U, V, and W. The ct rRNA hybridizes to 3.2% of the ct DNA; therefore, ct DNA from Euglena contains approximately two copies of the cistrons for 16S and 23S ct rRNA. The measurements in this study were made with ct DNA that was isolated as intact molecules (16) and with highly purified and intact ct rRNA that was extracted from
isolated chloroplast ribosomes (2). The measurements, therefore, accurately define the number of cistrons for ct DNA from E. gracilis. Two copies of the cistrons for ct rRNA may be a common feature of all species of ct DNA because similar results have been obtained with ct DNA from five species of higher plants (30). Hybridization of ct rRNA to the restriction fragments indicates that ct B, E, K, M, 0, and P contain sequences complementary to rRNA. These six fragments have buoyant densities greater than total ct DNA, due in part to the 50% G+C in the rRNA (2). These heavy fragments can be lost or perhaps preferentially enriched if broken ct DNA is purified by procedures that exploit differences in base composition or chemical properties. This result may explain, in part, the variety of published estimates for the number of rRNA cistrons on ct DNA from Euglena (7, 19, 23, 27, 28). Fragments ct B, E, K, M, 0, and P contain more DNA than is required for the two copies of the cistrons for rRNA. The remainder of the DNA in these restriction fragments could include spacer sequences, sequences for precursors of rRNA, and unrelated sequences that are adjacent to cistrons for rRNA. Fragments ct B and ct E have intermediate buoyant densities between total ct DNA and the fragments ct K, M, 0, P, yet ct B and ct E contain part of the cistrons for rRNA. This suggests that the cistrons for rRNA are bounded on at least one side by DNA with a low buoyant density. The data do not allow determination of whether the cistrons for rRNA are located in a cluster or dispersed at several locations on the DNA. All of the cistrons could be accommodated in a single cluster with relatively short sequences of spacer if fragments ct B and ct E contain short segments from either end of the cluster. At least 6.6 x 106 daltons of double-stranded DNA is required to code for at 1.696 g/cm3. Hallick et al. have recently reported the re- two copies of the cistrons for rRNA. Fragments sults of digestion of ct DNA of Euglena with ct K, M, 0, and P have a total molecular weight EcoRI (12). The pattern of fragments shown is of 7.15 x 106.
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MIELENZ, MILNER, AND HERSHBERGER
ACKNOWLEDGMENTS We thank T. Spilhman for providing a sample of EcoRI, M. Connaughton for XvirS50 DNA, N. Slavik for ColEl DNA, and D. Kaiser for the XcI857 phage. This research was supported by the University of Illinois Research Board, research grants GB 35598 and GB 41928 from the National Science Foundation, Public Health Service training grant 00510, and Public Health Service research grant GM 2243 from the National Institute of General Medical Sciences. Since completing this work, we have leamed that Kopecka, Crouse, and Stutz have analyzed ct DNA of Euglena with restriction enzymes. We thank E. Crouse for communication of his data. LITERATURE CITED 1. Allet, B., P. G. N. Jeppesen, K. J. Katagiri, and H. Delius. 1973. Mapping DNA fragments produced by cleavage of ADNA with endonuclease RI. Nature (London) 241:120-123. 2. Avadhani, N. G., and D. E. Buetow. 1972. Isolation of active polyribosomes from the cytoplasm, mitochondria and chloroplasts ofEuglena gracilis. Biochem. J. 128:353-365. 3. Bazaral, M., and D. R. Helinski. 1968. Circular DNA forms of colinogenic factors El, E2 and E3 fromEscherichia coli. J. Mol. Biol. 36:185-194. 4. Bovre, K. and W. Szybalski. 1971. Multistep DNA-RNA hybridization techniques. Methods Enzymol. 21D:350-383. 5. Brown, W. M., and J. Vinograd. 1974. Restriction endonuclease cleavage maps of animal mitochondrial DNAs. Proc. Natl. Acad. Sci. U.S.A. 71:4617-4621. 6. Casey, J., M. Cohen, M. Rabinowitz, H. Fukuhara, and G. S. Getz. 1972. Hybridization of mitochondrial transfer RNA's with mitochondrial and nuclear DNA of grande (wild type) yeast. J. Mol. Biol. 63:431 440. 7. Crouse, E. J., J. P. Vandrey, and E. Stutz. 1974. Hybridization studies with RNA and DNA isolated from Euglena gracilis chloroplasts and mitochondria. FEBS Lett. 42:262-266. 8. Depew, R. E., and J. C. Wang. 1975. Conformational fluctuations of DNA helix. Proc. Natl. Acad. Sci. U.S.A. 72:42754279. 9. Gillespie, D., and S. Spiegelman. 1965. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12:829-842. 10. Gray, H. B., Jr., and J. E. Hearst. 1968. Flexibility of native DNA from the sedimentation behavior as a function of molecular weight and temperature. J. Mol. Biol. 35:111-129. 11. Greenblatt, C. L., and J. A. Schiff. 1959. A pheophytinlike pigment in dark-adapted Euglena gracilis. J. Protozool. 6:23-28. 12. Hallick, R. B., C. Lipper, 0. C. Richards, and W. J. Rutter. 1976. Isolation of a transcriptionally active chromosome from chloroplasts of Euglena gracilis. Biochemistry 15:3039-3045. 13. Kissil, M. S., and D. E. Buetow. 1974. Mass isolation of chloroplasts for preparation of polyribosomes. J. Cell Biol. 63:169a. 14. Levings, C. S., III, and D. R. Pring. 1976. Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize.
J. BACTERIOL.
Science 193:158-160. 15. Lewis, J. B., J. F. Atkins, C. W. Anderson, P. R. Baum, and R. F. Gesteland. 1975. Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments. Proc. Natl. Acad. Sci. U.S.A. 72:1344-1348. 16. McCrea, J. M., and C. L. Hershberger. 1976. Chloroplast DNA codes for transfer RNA. Nucleic Acids Res. 3:2005-2018. 17. Manning, J. E., and 0. C. Richards. 1972. Isolation and molecular weight of circular chloroplast DNA in Euglenagracilis. Biochim. Biophys. Acta 259:285-296. 18. Potter, S. S., J. E. Newbold, C. A. Hutcheson III, and M. H. Edgell. 1975. Specific cleavage analysis of mammalian mitochondrial DNA. Proc. Natl. Acad. Sci. U.S.A. 72:44954500. 19. Rawson, J. R. Y., and R. Haselkorn. 1973. Chloroplast ribosomal RNA genes in the chloroplast DNA ofEuglena gracilis. J. Mol. Biol. 77:125-132. 20. Robberson, D. L., D. A. Clayton, and J. F. Morrow. 1974. Cleavage of replicating forms of mitochondrial DNA by EcoRI endonuclease. Proc. Natl. Acad. Sci. U.S.A. 71:4447-4451. 21. Sager, R. 1972. Cytoplasmic genes and organelles. Academic Press Inc., New York. 22. Schwartzbach, S. D., L. I. Hecker, and W. E. Barnett. 1976. Transcriptional origin of Euglena chloroplast tRNAs. Proc. Natl. Acad. Sci. U.S.A. 73:1984-1988. 23. Scott, N. S. 1973. Ribosomal RNA cistrons in Euglena gracilis. J. Mol. Biol. 81:327-336. 24. Shinnick, T. M., E. Lund, 0. Smithies, and F. R. Blattner. 1975. Hybridization oflabeled RNA to DNA in agarose gels. Nucleic Acids Res. 2:1911-1929. 25. Slavik, N. S., and C. L. Hershberger. 1975. The kinetic complexity of Euglena gracilis chloroplast DNA. FEBS Lett. 52:171-174. 26. Slavik, N. S., and C. L. Hershberger. 1976. The intemal structural organization of chloroplast DNA from Euglena gracilis Z. J. Mol. Biol. 103:563-581. 27. Stutz, E., E. J. Crouse, and L. Graf. 1974. Does gene amplification occur within the chloroplast DNA of developing chloroplasts of Euglena gracilis? International Colloquium on Cell Cycles. C.N.R.S., Gif-surYvette, France. 28. Stutz, E., and J. P. Vandrey. 1971. Ribosomal DNA satellite of Euglena gracilis chloroplast DNA. FEBS Lett. 17:277-280. 29. Tanaka, T., and B. Weisblum. 1975. Construction of a colicin El-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bacteriol. 121:354-362. 30. Thomas, J. R., and K. K. Tewari. 1974. Conservation of 70S ribosomal RNA genes in the chloroplast DNAs of higher plants. Proc. Natl. Acad. Sci. U.S.A. 71:31473151. 31. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 32. Vedel, F., F. Quetier, and M. Bayen. 1976. Specific cleavage of chloroplast DNA from higher plants by EcoRl restriction nuclease. Nature (London) 263:440442. 33. Wu, M., and N. Davidson. 1972. Expression ofthe mitochondrial genome in HeLa cells. J. Mol. Biol. 71:8193.
JOURNAL OF BACTERIOLOGY, May 1977, p. 860-868 Copyright © 1977 American Society for Microbiology
Analysis of Euglena gracilis Chloroplast Deoxyribonucleic Acid with a Restriction Endonuclease, EcoRI JONATHAN R. MIELENZ,1* JOEL J. MILNER, AND CHARLES L. HERSHBERGER2 Department of Microbiology, University ofIllinois, Urbana, Illinois 61801 Received for publication 7 January 1977
Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoPJ) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight ofthe circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosoal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.
Genes of organelles have been identified by their patterns of inheritance (21). Hybridization studies have proven that deoxyribonucleic acid (DNA) of organelles contains cistrons for ribosomal (r) and transfer (t) ribonucleic acid (RNA) (6, 7). Heteroduplex mapping located individual mitochondrial (mt) cistrons for rRNA and several species of tRNA on the separated strands of DNA (33). A new method of physical mapping ofgenes has been provided by enzymes that cleave DNA at specific sites. Restriction endonucleases have been used successfully to locate regions of viral DNA that code for messenger RNA (15), to determine the origin and direction of replication of mt DNA (5, 20), and to compare DNA of organelles from both mammalian (18) and plant (14, 32) populations. Chloroplast (ct) DNA ofEuglena gracilis Z is ideally suited for physical mapping of g ees with restriction endonucleases. The ct DNA has a molecular weight of 92 x 106 (17). The molecule contains one to six cistrons for rRNA as measured by different investigators (7, 1i, 23, 27, 28), and 26 cistrons for tRNA (16, 22), Pure and intact nucleic acids are easily obtained from isolated chloroplasts ofE. gracilis. The number, sizes, and approximate buoyant density ofthe fragments of ct DNA produced by ' Present address: Plant Growth Laboratory, University of California, Davis, CA 95616. 2 Present address: Biochemical Development Division, Eli Lilly and Company, Indianapolis, IN 46206.
restriction endonuclease RI from Escherichia coli (EcoRI) have been determined in this investigation. The number of cistrons for 16S and 238 rRNA on the ct DNA was determined by hybridization of ct DNA with ct rRNA labeled with 125I. The fragments of ct DNA that contained these cistrons were identified by hybridization with ct rRNA. MATERIALS AND METHODS Isolation of DNA and RNA. Euglena was grown mixotrophically on a Hutner medium (11). Chloroplasts were isolated as described by Kissel and Buetow (13), with slight modifications. The ct DNA was extracted from the chloroplasts and purified as described (16). Colicinogenic factor El (ColE1) DNA was isolated fromE. coli JC411 as covalently closed circular DNA (3). Bacteriophage lambda (XvirS5O and XcI857), bacteriophage T4B, and their DNAs were isolated as described previously (4, 10). The et RNA from E. gracilis was extracted from the isolated chloroplaats (13). 16S and 23S rRNA's were purified as described previously (2). Electrophoresis of the RNA through polyacrylamide gels indicated that the 16S and 23S rRNA's were free of detectable levels of contaminating RNA. Digestion with EcoRI and electrophoresis. The EcolI restriction endonuclease was prepared from E, coli RY13 (29) or obtained from Miles Laboratories. The DNA was digested in a buffer containing 25 mM tris(hydroxymethyl)aminomethane, 10 mM MgCl2, and 50 mM NaCl, pH 7.5, with EcoRI at 37°C. Electrophoresis of the DNA and visualization
860
ANALYSIS OF CHLOROPLAST DNA WITH ECORI
VOL. 130, 1977
and photography of the gels of agarose (Sigma) with Polaroid 46L film have been described (29). Quantitation of bands E, I, M, and T. Fluorescence was a linear function of the DNA mass in the range of concentrations used for quantitating bands when the gels were analyzed as described by Depew and Wang (8). Several exposures of gels containing different amounts of DNA were needed to obtain proper concentrations for all the bands. Photographs within the linear range of film response were traced with a Joyce-Loebl MK III B recording microdensitometer. The areas of the peaks of interest and neighboring peaks were determined and normalized for molecular weights of the fragments of DNA. Normalized ratios of areas of bands E, I, M, and T to the areas of neighboring bands yielded the number of repeats in those bands. Buoyant density analysis of restriction fragments. The ct DNA was digested with EcoRI and centrifuged to equilibrium in CsCl in a Beckman type 65 rotor (26). The DNA was fractionated, and the ultraviolet absorbance was monitored (26). Appropriate fractions were pooled as shown, diluted with 0.1 x SSC (standard saline citrate: 0.15 M NaCl, 0.015 M citrate, pH 7.0), and centrifuged in a Beckman SW50.1 rotor at 50,000 rpm for 8 h at 4°C. The DNA pellet was dissolved in 0.1 x SSC and electrophoresed on 0.7% agarose. RNA:DNA hybridization. The ct RNA- was labeled with 125I and hybridized to ct DNA immobilized on nitrocellulose filters as described (9, 16). The ct rRNA was hybridized to restriction fragments in gels of agarose by the procedure of Shinnick et al. (24).
RESULTS Purity and integrity of the ct DNA. Samples of DNA intended for digestion with restriction nucleases must be free of contaminating species of DNA and have a high molecular weight. The ct DNA obtained from chloroplasts is pure ct DNA, uncontaminated with nuclear or mt DNA (16). The size of the ct DNA was examined by analytical boundary sedimentation (10). A single sedimenting boundary was observed with a sedimentation coefficient of 64S. This result compares favorably with the value of 66S expected for relaxed circular DNA with a molecular weight of 92 x 106.
861
The size and structure of the ct DNA were analyzed by electron microscopy. A large proportion of the molecules were relaxed circles, but large linear molecules were observed also. These linear molecules could have been generated by shear forces when the DNA was spread for electron microscopy. The molecular weight of the ct DNA was (92 + 3) x 106 when calculated from measurements of nine relaxed circular molecules relative to relaxed circular ColEl DNA molecules, included as an internal standard. Similar results were obtained by Manning and Richards (17). The data for measuring the molecular weight of ct DNA are available (J. R. Mielenz, Ph.D. thesis, University of Illinois, Urbana, 1976). Analysis of the pattern of restriction fragments of ct DNA. The products of digestion of the ct DNA by EcoRI were resolved by electrophoresis in 0.7% agarose (Fig. 1). Twenty-three bands were observed, as in these duplicate gels. This pattern ofbands was the result of complete digestion because neither prolonged digestion nor three repeated digestions of one sample of ct DNA altered the pattern of bands in Fig. 1. Furthermore, digestion of ct DNA under conditions described by Thomas and Davis (31) or the use of EcoRI from Miles Laboratories also produced the pattern in Fig. 1. Digestion of DNA from bacteriophage XcI857 and plasmid ColEl under the conditions described in Materials and Methods yielded only the fragments produced by cleavage at the hexanucleotide site for
EcoRI (29, 31). No bands smaller than ct W were found when samples with greater amounts of DNA were separated on 1% agarose. Also, the bands in Fig. 1 represent all of the radioactive fragments when 32P-labeled ct DNA is analyzed in the same manner (Walfield and Hershberger, unpublished data), so ct A through ct W represent all the major bands when ct DNA is digested with EcoRI. Bands ct E, I, M, and T were present in higher than stoichiometric amounts. Appropriate photographs of gels were quantitated relative to neighboring bands by determining the areas of peaks obtained from microdensitome-
RI ct DNA t tt t t I I tVM\ ye marker I JKLM NOP QR S T UVW FIG. 1. Electrophoretic pattern of fragments of chloroplast DNA that has been digested with EcoRI. Twenty-three distinct bands were observed, lettered ct A to ct W, as in these duplicate gels. All of the fragments were not recorded on one photograph because the quantities of DNA in the bands varied by over 15-fold. Prints of two different exposures of the same gels were split diagonally and appropriate halves combined to produce
AB
this figure.
CDE F
t I G H
862
re
ter tracings. All four bands were found to be present in about twofold excess when normal-
ized for the molecular weight of the fragments (Table 1). Determinations of molecular weights will be described below. The DNA in these bands could represent repeated DNA sequences or different segments of DNA with equally spaced sites sensitive to EcoRI. With a small sample volume (20 ul) and a long migration (22 cm), sufficient resolution was obtained to detect the separation of ct M into two bands (Fig. 2). Electrophoresis was TABLE 1. Quantitation of bands E, I, M, and T
Test band
E I M T
Mol wt (x 106)
4.70 2.35 1.90 0.95
Neighboring band(s)
used for quantitation
F J, K, and L J, K, L, N, O, and P S
Ratio of normalized areas
obfated/
neighboring band 1.8 2.0 2.0 2.3
-i.H
_o
--
-
*
continued until ct Q and smaller bands had migrated off the gel. With ct I, a double-molar band, as a reference, ct J, K, L, and two additional bands were visible immediately below ct I. The two bands, ct Ml and ct M2, normally migrated as a broad single band that contained twice the amount of DNA found in neighboring bands. Examination of the other double-molar bands, ct E, I, and T, in a similar manner did not give significant separation. Therefore, the 23 bands contain at least 24 different fragments of ct DNA. Determination of the size of the fragments. Chloroplasts of Euglena contain up to 65 copies of ct DNA (17) that could be a single species of DNA or subpopulations of molecules with different sequences. The sum of the molecular weights of the restriction fragments should equal the analytical molecular weight if there is one type of molecule. The sizes of the 24 fragments of ct DNA were determined by coelectrophoresis with restriction fragments of ColEl (29) and bacteriophage XvirS50 DNA. XvirS50, which carries a b2 deletion, yields EcoRI fragments that have the same electrophoretic mobilities as another b2 deletion mutant (31). The sum of the molecular weights of all the fragments of ct DNA is 87.6 x 106 (Table 2) and is within the precision of kinetic complexity and analytical measurements of 92 x 106 (17, 25). Therefore, with respect to the locaTABLE 2. Sizes of the fragments of ct and standard DNA
I
Restriction fragmenta
X,+vb
j
--M M2 N
Mol wt (x 106)
EcoRI re-
15.8 14.5 13.7 13.0 6.30 5.90 4.70C 4.70 4.50 4.20 3.70 3.50 3.05 2.80
AV
striction
Mol wt (x 106)
2.10 J 2.10 K 2.05 XI B L 2.00 C M 1.90C D N 1.70 E 0 1.65 P XH, 1.55 F Q 1.25 Col El R 1.20 S A,,, 1.00 G T 0.95' H U 0.87 0.85 V XIv I 2.35X 0.82 W a Fragments of ct DNA are designated by capital letters. b XA,+ is a composite of fragments held together by the ends that anneal with each other when X DNA is circularized (1). r Bands E, I, M, and T were present in twofold amounts relative to adjacent bands and were included twice in the sum of the molecular weights. A
FIG. 2. Separation of fragment M. EcoRI-digested was electrophoresed through 0.7% agarose until fragments ct Q to W migrated off the gel. Fragments ct G to P are shown in the photograph of this gel. ct M appeared as a double band instead of a single band containing twice as much DNA. The double bands are designated ct M, and ct M2. ct DNA
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ANALYSIS OF CHLOROPLAST DNA WITH ECORI
tion of the sites sensitive to EcoRI, the ct DNA of Euglena exists primarily as a population of identical molecules. Determination of the buoyant densities of the fragments. The ct DNA of Euglena contains five segments of different base composition ranging from 22 to 41% guanine plus cytosine (G+C) (26). Because of this heterogeneity of the base composition, the fragments of ct DNA produced by EcoRI digestion should have a range of different densities in CsCl. The ct DNA was digested by EcoRI and analyzed in gradients of CsCl. A preparative CsCl gradient of this sample of DNA produced the profile shown in Fig. 3. The fractions ofthis DNA were pooled into four samples as shown and electrophoresed through 0.7% agarose to produce gels a through d (Fig. 3). Several bands of DNA were located predominantly in a single gel, indicating that they had distinct buoyant densities in CsCl. Major components of one fraction appear as minor components in neighboring fractions because DNAs with different densities form overlapping Gaussian bands in gradients of CsCl. ct A, J, L, Q, R, S, and T were mostly in gel a (Fig. 3) and must have originated from the segment of ct DNA with the lightest density. Gels a and b contained ct C, D, F, G, H, I, and N in nearly equal proportions, suggesting that these fragments have densities close to 1.686 g/cm3. Gel c contained two predominant bands, ct B and ct E, that were also found in other fractions. Gel d contained four predominant fragments, ct K, M, 0, and P, with faint neighboring bands showing that ct K, 0, and P had a high buoyant density in CsCl, above 1.697 g/cm3. Significant amounts of band M were found in gels a, b, and d (Fig. 3), so ct M appeared to have a wide range of buoyant densities. Band ct M contained two fragments as explained above. These results indicate that the two fragments of ct M with almost identical size have different buoyant densities. Gels a and b (Fig. 3) contained one species, ct ML (light), whereas gel d contained ct MH (heavy). We do not know whether ct ML is ct M1 or ct M2 in Fig. 2. Analysis of the gels in Fig. 3 leads to a classification of most of the fragments according to the region of the gradient where the major portion of each fragment is found. The classes contain fragments from fractions of the gradient with densities of 1.686 g/cm3 and less, 1.686 to 1.691 g/cm3, 1.691 to 1.696 g/cm3, and 1.696 to 1.716 g/cm3. Loss of fragments during isolation of DNA. Identification of restriction fragments with different buoyant densities suggests the possibility of losing specific segments when ct DNA is
863
purified by procedures that separate DNA with different base compositions, such as density gradient centrifugation and chromatography on hydroxylapatite. Three different preparations of ct DNA were analyzed to determine the extent of this problem. One of the preparations of DNA was obtained from isolated chloroplasts as pure, intact ct DNA. Two other samples of ct DNA were obtained from whole cells (Mielenz, Ph.D. thesis) and purified to homogeneity using equilibrium density gradient centrifugation to remove contaminating nuclear DNA. The molecular weight of each of the samples of ct DNA was determined, since breakage is a prerequisite for significant loss of DNA with a high buoyant density. The DNA obtained from the isolated chloroplasts had a molecular weight of 92 x 106 as described above. Boundary sedimentation of the second preparation of DNA (preparation B) showed that the DNA had a molecular weight of 37 x 106. The third sample of DNA, preparation C, had sustained a significant amount of breakage and had a molecular weight of 13 x 106. Each of these samples was digested with EcoRI, and the fragment pattern was analyzed. Digestion and electrophoresis on agarose of preparation A yielded the characteristic pattern shown above (Fig. 4a). A microdensitometer scan of photographs of the gels across bands ct I through ct P yielded the adjoining tracing. ct I and ct M yielded large predominant peaks that contained twice the area of adjacent bands, ct J and ct L, when normalized for molecular weight, ct J, K, L, N, 0, and P all yielded similar-size peaks (+8%). Preparation B of ct DNA was digested with EcoRI, and the separated fragments produced gel b (Fig. 4b). No single bands of DNA were missing, but the microdensitometer tracings of bands ct I to ct P revealed a significant change. ct I was the largest peak, whereas ct J, L, and N were found in similar amounts (+10%). ct K and ct 0 were found to be reduced to about 55%, whereas ct P was drastically reduced to 14% of ct N, after normalization for molecular weight. ct M was not present in twofold amounts, but was present at 1.7-fold relative to ct L and ct N. ct ML should not be lost by the methods used to purify this DNA, so the 30% loss apparently was due to the selective loss of ct MH along with ct K, 0, and P. Thus, approximately 50% of the DNA found in ct K, M, 0, and P was lost during preparation of the DNA (Table 3). The pattern of restriction fragments from preparation C of ct DNA showed marked alteration, with four bands missing (Fig. 4c). Comparison of gel c with gel a or b (Fig. 4) showed that ct K, 0, and P were missing. The micro-
As
G H
~ ~-i_t~J _-_
-z
H ,-H
R~~~
.
~\
M
N-~~~~~~~~~-
T-
864
T
d~uvw
VOL. 130, 1977
ANALYSIS OF CHLOROPLAST DNA WITH ECORI
densitometer tracing of bands ct I to ct P confirmed the loss of ct K, 0, and P. ct M was present in amounts similar to ct J and ct L (Table 3). The missing part of ct M is thought to be ct MH, since ct ML should be retained because
.*
865
it has a low buoyant density. In addition to bands ct K, 0, and P, one of the bands, ct A or ct B, was missing. Significant recovery of one of these two large fragments suggests that the missing band was not lost solely by fragmenta-
I
FIG. 4. Loss of fragments by purification on density gradients. The gels show ct DNA that was digested with EcoRI and electrophoresed on 0.7% agarose. Photographs of bands ct I through ct P were traced with a microdensitometer to obtain the profiles. The samples of ct DNA with different molecular weights were purified to homogeneity by equilibrium density gradient ultracentrifugation. (a) ct DNA with a molecular weight of 92 X 106; (b) fragmented ct DNA with a molecular weight of37 x 106; and (c) fragmented ct DNA with a molecular weight of 13 x 106.
FIG. 3. Determination of the approximate buoyant densities of the restriction fragments of ct DNA. The ct DNA was digested with EcoRI and centrifuged to equilibrium in CsCl. Fractionation of the sample yielded the profile of absorbancy at 254 nm (top), with Micrococcus luteus DNA at 1.731 g/cm3. Gels a to d contain DNA from buoyant densities of 1.670 to 1.686 g/cm3, 1.686 to 1.691 g/cm3, 1.691 to 1.696 g/cm3, and 1.696 to 1.716 g/cm3, respectively. Gel a-d contains fragments from complete ct DNA as a reference.
typical of specific hybrids. The temperature for one-half denaturation (T,0) was 78°C. Formation of specific hybrids should be independent of temperature within the interval of 30 to 150C below the T,m. This prediction was verified by measuring hybridization between 50 and 640C. The level of hybridization was the same within this temperature interval. Identification of fragments containing the cistrons for rRNA. Restriction fragments can be used to identify specific cistrons contained on ct DNA. Identification of the restriction fragments with cistrons for 16S and 23S rRNA was selected as a model for testing this approach with ct DNA. The ct rRNA was labeled with 125I to specific activities of 105 to 107 cpm/,ug. This rRNA was hybridized to the restriction fragments of ct DNA contained within a 0.7% agarose gel (24). The bands of DNA were detectable before and after hybridization. An autoradiograph of the gels detected specific hybridization to bands ct B, E, K, M, 0, and P (Fig. 5). No significant hybridization of the rRNA to the other 17 bands of DNA was detected. Similar results were obtained when individual restriction fragments were extracted from the gels (15), bound to nitrocellulose filters, and hybridized to '25I-labeled ct rRNA (Mielenz, Ph.D. thesis).
TABLE 3. Analysis of fragments lost versus molecular weight of the DNA Sam-
pie of Method of puri- Mol
w
No. of restriction fragments
Fragments depleted or miss-
ct DNA
fication
(X 1w)
A
Extraction from isolated chloroplasts Extracted from whole cells
92
24
None
37
24
K
B
C
J. BACTERIOL.
MIELENZ, MILNER, AND HERSHBERGER
866
Same as B
13
ing
MH 0 P 19 A or B K |MH 0
Amt of band miss(% ing of A)
45 30 45 86 100 100 100 100
tion. The missing band was probably ct B since it had a heavier buoyant density than ct A. Hybridization of ct rRNA to ct DNA. The number of cistrons for rRNA located on ct DNA was determined by hybridization of rRNA to ct DNA that was isolated as intact molecules. Since the time course of hybridization (data not shown) indicated that hybrids attained equilibrium in 12 h and were stable for at least 24 h, 18 h was used for subsequent hybridization reactions. The level of ct rRNA hybridized to ct DNA was measured at saturating concentrations of ct rRNA by incubating the ct DNA with several concentrations of rRNA. The concentration of the RNA was varied by 10-fold from 0.5 to 5 RNA/DNA ratio. The plateau was flat and well defined at 3.2% of the DNA hybridized with RNA, as expected if all specific sites on the ct DNA were saturated. Specificity of the reaction was determined by hybridizing the RNA to DNA from E. coli as a heterologous control. The level ofbinding to the control was virtually the same as the binding to blank filters. Specific hybridization was 62.5 times the nonspecific binding. Thermal stability of the hybrid was determined to further ascertain the specificity of hybridization. The hybrids denatured over a narrow temperature range, with a sigmoidal profile of denaturation from 55 to 990C that is
.M -..-"
ir -iff
.1001-
.
..dmkmkl.
I I
a
a
1 It IM It
4M dk dihM
.
DISCUSSION Digestion of Euglena ct DNA with the enzyme EcoRI produces 24 fragments of different sizes. Two fragments (ct ML and ct MH) are nearly identical in size. Three other bands of DNA are also distinctive because they are present in twofold excess relative to neighboring bands. They could result from repeated DNA sequences or from segments with different sequences of nucleotides that contain nearly identical spacing between sites sensitive to EcoRI. The sum of the molecular weights of all the fragments agrees within 5% of the kinetic complexity and the analytical molecular weight of ct DNA of 92 x 106, and suggests that the ct DNA of Euglena exists as a single molecular clone within the cell. Agreement between three
0 9
-
-
v
.
V.
4 it
FIG. 5. Hybridization ofct rRNA to fragments ofct DNA produced by digestion with EcoRI. The top figure shows the autoradiograph ofthe gel after hybridization of 125I1-labeled ct rRNA to the bands ofDNA within the gel . The bottom figure is a photograph of the bands ofDNA contained in the same 0.7% gel of agarose. The contrast in the autoradiograph was reversed during printing.
VOL. 130, 1977
ANALYSIS OF CHLOROPLAST DNA WITH ECORI
methods of measuring the size also indicates that the ct DNA contains a single unique sequence of 92 x 106 daltons rather than tandem repetitions of a shorter sequence. Several restriction fragments of ct DNA have different buoyant densities in CsCl. This observation is consistent with the fact that ct DNA of Euglena contains five segments with different base compositions (26). Seven fragments have densities less than total ct DNA, at 1.686 g/cm3, eight have densities of about 1.686 g/cm3, and two have densities slightly greater than 1.686 g/cm3 (Table 3). Four of the restriction fragments, ct K, MH, 0, and P, contain DNA with a buoyant density greater than 1.696 g/cm3 and can be compared to fraction V of Slavik and Hershberger (26). The DNA in fraction V contains 41% G+C, has a buoyant density of 1.702 g/cm3, and contains 6% of the ct DNA. The sum of the molecular weights of fragments ct K, MH, 0, and P is 7.15 x 106 or 8% of the genome. Therefore, the four restriction fragments and fraction V apparently contain much of the same segments of ct DNA. Fragments ct U, V, and W have not been found in the density gradients. These fragments represent individually about 1% of the ct DNA and are the most difficult to detect. They will form broad bands (or band) in equilibrium gradients because of their low molecular weight. They may be distributed through several fractions of the gradient, further reducing the ability to detect these small fragments. Significant amounts of ct DNA can be lost through purification of ct DNA by techniques that separate DNA by differences in base composition. A similar conclusion has been suggested previously (26, 28), but the missing restriction fragments were not identified. There is a strong inverse correlation between the preferential loss of fragments ct K, M, 0, and P and the molecular weight of the DNA. Greater breakage separates more of the segments with a high buoyant density from the adjoining segments with a low buoyant density. Segments with the highest buoyant density would be lost preferentially compared with DNA of lower buoyant density if the DNA is purified by equilibrium density gradient ultracentrifugation. Fragments ct K, MH, 0, and P are depleted differentially at 45, 30, 45, and 86%, respectively. Since the loss results from differences in the buoyant densities, these fragments can be listed by decreasing buoyant density as ct P, (O and K), MH, with the lower limit for ct MH
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similar to that in Fig. 1, except that bands ct K, 0, and P appear to be missing. Insufficient experimental detail was presented to deternine the reason for the discrepancy. Furthermore, the gels of Hallick et al. (12) did not appear to contain a sufficient quantity of DNA to allow detection ofthe smallest bands, ct U, V, and W. The ct rRNA hybridizes to 3.2% of the ct DNA; therefore, ct DNA from Euglena contains approximately two copies of the cistrons for 16S and 23S ct rRNA. The measurements in this study were made with ct DNA that was isolated as intact molecules (16) and with highly purified and intact ct rRNA that was extracted from
isolated chloroplast ribosomes (2). The measurements, therefore, accurately define the number of cistrons for ct DNA from E. gracilis. Two copies of the cistrons for ct rRNA may be a common feature of all species of ct DNA because similar results have been obtained with ct DNA from five species of higher plants (30). Hybridization of ct rRNA to the restriction fragments indicates that ct B, E, K, M, 0, and P contain sequences complementary to rRNA. These six fragments have buoyant densities greater than total ct DNA, due in part to the 50% G+C in the rRNA (2). These heavy fragments can be lost or perhaps preferentially enriched if broken ct DNA is purified by procedures that exploit differences in base composition or chemical properties. This result may explain, in part, the variety of published estimates for the number of rRNA cistrons on ct DNA from Euglena (7, 19, 23, 27, 28). Fragments ct B, E, K, M, 0, and P contain more DNA than is required for the two copies of the cistrons for rRNA. The remainder of the DNA in these restriction fragments could include spacer sequences, sequences for precursors of rRNA, and unrelated sequences that are adjacent to cistrons for rRNA. Fragments ct B and ct E have intermediate buoyant densities between total ct DNA and the fragments ct K, M, 0, P, yet ct B and ct E contain part of the cistrons for rRNA. This suggests that the cistrons for rRNA are bounded on at least one side by DNA with a low buoyant density. The data do not allow determination of whether the cistrons for rRNA are located in a cluster or dispersed at several locations on the DNA. All of the cistrons could be accommodated in a single cluster with relatively short sequences of spacer if fragments ct B and ct E contain short segments from either end of the cluster. At least 6.6 x 106 daltons of double-stranded DNA is required to code for at 1.696 g/cm3. Hallick et al. have recently reported the re- two copies of the cistrons for rRNA. Fragments sults of digestion of ct DNA of Euglena with ct K, M, 0, and P have a total molecular weight EcoRI (12). The pattern of fragments shown is of 7.15 x 106.
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MIELENZ, MILNER, AND HERSHBERGER
ACKNOWLEDGMENTS We thank T. Spilhman for providing a sample of EcoRI, M. Connaughton for XvirS50 DNA, N. Slavik for ColEl DNA, and D. Kaiser for the XcI857 phage. This research was supported by the University of Illinois Research Board, research grants GB 35598 and GB 41928 from the National Science Foundation, Public Health Service training grant 00510, and Public Health Service research grant GM 2243 from the National Institute of General Medical Sciences. Since completing this work, we have leamed that Kopecka, Crouse, and Stutz have analyzed ct DNA of Euglena with restriction enzymes. We thank E. Crouse for communication of his data. LITERATURE CITED 1. Allet, B., P. G. N. Jeppesen, K. J. Katagiri, and H. Delius. 1973. Mapping DNA fragments produced by cleavage of ADNA with endonuclease RI. Nature (London) 241:120-123. 2. Avadhani, N. G., and D. E. Buetow. 1972. Isolation of active polyribosomes from the cytoplasm, mitochondria and chloroplasts ofEuglena gracilis. Biochem. J. 128:353-365. 3. Bazaral, M., and D. R. Helinski. 1968. Circular DNA forms of colinogenic factors El, E2 and E3 fromEscherichia coli. J. Mol. Biol. 36:185-194. 4. Bovre, K. and W. Szybalski. 1971. Multistep DNA-RNA hybridization techniques. Methods Enzymol. 21D:350-383. 5. Brown, W. M., and J. Vinograd. 1974. Restriction endonuclease cleavage maps of animal mitochondrial DNAs. Proc. Natl. Acad. Sci. U.S.A. 71:4617-4621. 6. Casey, J., M. Cohen, M. Rabinowitz, H. Fukuhara, and G. S. Getz. 1972. Hybridization of mitochondrial transfer RNA's with mitochondrial and nuclear DNA of grande (wild type) yeast. J. Mol. Biol. 63:431 440. 7. Crouse, E. J., J. P. Vandrey, and E. Stutz. 1974. Hybridization studies with RNA and DNA isolated from Euglena gracilis chloroplasts and mitochondria. FEBS Lett. 42:262-266. 8. Depew, R. E., and J. C. Wang. 1975. Conformational fluctuations of DNA helix. Proc. Natl. Acad. Sci. U.S.A. 72:42754279. 9. Gillespie, D., and S. Spiegelman. 1965. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12:829-842. 10. Gray, H. B., Jr., and J. E. Hearst. 1968. Flexibility of native DNA from the sedimentation behavior as a function of molecular weight and temperature. J. Mol. Biol. 35:111-129. 11. Greenblatt, C. L., and J. A. Schiff. 1959. A pheophytinlike pigment in dark-adapted Euglena gracilis. J. Protozool. 6:23-28. 12. Hallick, R. B., C. Lipper, 0. C. Richards, and W. J. Rutter. 1976. Isolation of a transcriptionally active chromosome from chloroplasts of Euglena gracilis. Biochemistry 15:3039-3045. 13. Kissil, M. S., and D. E. Buetow. 1974. Mass isolation of chloroplasts for preparation of polyribosomes. J. Cell Biol. 63:169a. 14. Levings, C. S., III, and D. R. Pring. 1976. Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize.
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