Characterization and mapping of mitochondrial ribosomal RNA and mitochondrial DNA in Drosophila melanogaster

Cell, Vol . 9, 6 1 5-625, December 1976 (Part 1), Copyright ©1976 by MIT

Characterization and Mapping of Mitochondrial Ribosomal RNA and Mitochondrial DNA in Drosophila melanogaster Carol K . Klukas" and Igor B . Dawidt Carnegie Institution of Washington Department of Embryology 115 West University Parkway Baltimore, Maryland 21210

Summary We report the isolation and some properties of the mitochondrial ribosomal RNA (mt-rRNA) of Drosophila melanogaster, and a restriction map of the mtDNA of this organism which shows the position of the rRNA genes and of the A + T-rich region in the DNA . The mt-rRNAs are about 860 and 1500 nucleotides long and have the unusual composition of about 20% G+C . Some 25% of the mtDNA is very rich in A+T and is visualized as a contiguous early melting region in denaturation mapping . We mapped the three cleavage sites of the restriction endonuclease Hae III and the four sites of Hind III on mtDNA relative to each other and relative to the early melting region . The rRNA genes have been positioned on this map . The two rRNA genes are next to each other, separated by a gap of about 160 bases . We determined the polarity of the rRNA molecules : transcription proceeds in the direction small-to-large rRNA . Despite great divergence in nucleotide composition and sequence, the properties of mt-rRNA and the arrangement of mt-rRNA genes are very similar in Drosophila and vertebrate animals . Introduction The mitochondrial genetic system has been studied in a number of organisms . In all metazoan animals, mtDNA is a circular molecule of 15,000-18,000 base pairs (bp), which codes for two mitochondrial rRNA molecules, a set of tRNAs, and poly(A)-containing RNAs (Borst, 1972 ; Dawid, 1972a ; Hirsch and Penman, 1974 ; Ojala and Attardi, 1974) . Considerable characterization of the DNA and RNA, and some mapping studies of DNA regions coding for certain RNAs have been carried out with the mitochondrial nucleic acids of vertebrate animals, but much less information is available on the corresponding materials from invertebrates . Only sea urchin mtDNA (Matsumoto et al ., 1974), the mitochondrial ribosome of the locust (Kleinow, Neupert, and Miller, 1974), and the poly(A) RNA of Drosophila (Hirsch, Spradling Penman, 1974) have been studied in some detail . "'Present address : King's College, 26-29 Drury Lane, Department of Biophysics, London WC2B 5RL, England . 'To whom all correspondence should be addressed .

We have chosen to study the mitochondria) DNA and RNA of Drosophila melanogaster, a representative invertebrate animal whose genome is currently under intense investigation . D . melanogaster mtDNA is a circle of 18,400 bp, has an average G + C content of 22%, and contains a long region which is largely or entirely composed of A and T (Bultmann and Laird, 1973 ; Peacock et al ., 1973 ; Polan et al ., 1973 ; Wolstenholme, 1973) . There has been no characterization of Drosophila mt-rRNA . In this paper, we report the size, composition, and some physical properties of mt-rRNA of D . melanogaster, and a restriction map of the mtDNA which shows the locations of the rRNA genes and of the A + T-rich region in the DNA . We compare these results with the available information on mtDNA and mtRNA in vertebrate animals . Results Characterization of mt-rRNA SDS-acrylamide gels of RNA isolated from mitochondria of adult D . melanogaster run at room temperature revealed four ultraviolet-absorbing bands and a slight band of DNA in preparations not treated with DNAase (Figure 1) . Cytoplasmic 18S and 28S rRNA, identified by comparison with parallel gels containing D . melanogaster rRNA, were always present . The two remaining bands, present in about equimolar quantities, were designated large and Lg 28S

I

I

Sm

RT

I8S

10 0

Figure 1 . Analysis of mt-rRNA by SDS-Polyacrylamide Gel Electrophoresis About 10 pg RNA extracted from the mitochondria of adult D . melanogaster were electrophoresed in 2 .4% acrylamide gels for 3 hr at 5 ma per gel . Experiments were performed at either room temperature or at 10°C as indicated . Gels were scanned at 265 nm, and the absorbance traces are shown . The direction of migration was from left to right .

Cell 616

Table 1 . Nucleotide Composition of D . melanogaster RNA RNA

N

Cytidylic Acid

Adenylic Acid

Guanylic Acid

Uridylic Acid

% G+C 17 .6 ± 0 .2 20 .9 ± 0 .4

mt-Lg

3

6 .8 ± 0 .4

41 .0 ± 1 .3

10 .9 ± 0 .2

41 .4 ± 1 .1

mt-Sm

4

8 .2 ± 0 .8

39 .6 ± 0 .8

12 .7 ± 0 .5

39 .6 + 1 .1

small mitochondrial rRNA (mt-rRNA) for reasons summarized in the Discussion . When identical gels were run at 10°C (Figure 1), the mobility of the mtrRNAs increased such that both ran faster than 1 8S rRNA . The mt-rRNAs were purified by sedimentation through sucrose-SDS gradients . The large and small mt-rRNAs sediment at 13S and 10 .5S, respectively, relative to E . coli 23S and 16S RNAs as standards . RNA was recovered from peak fractions, and the purity of each rRNA was verified by gel electrophoresis . To estimate their molecular weights, these RNAs were separately spread for electron microscopy together with E . coli 23S RNA, and their lengths were measured . This experiment led to molecular weights of 522,000 and 300,000 daltons for the two mt-rRNAs . Purified mt-rRNAs were also electrophoresed in 5% acrylamide gels in 98% formamide, with E . coli 23S and 16S rRNA as molecular weight standards . This method led to values of 530,000 and 200,000 daltons . For the large mt-rRNA, the two estimates agree well ; since no suitable standard of the size of the small mt-rRNA was available in the gel electrophoresis, we consider the value of 300,000 daltons, based on length measurement, to be more precise for the small mt-rRNA . Nucleotide Composition of mt-rRNA 32 P-mt-rRNA from cultured cells was isolated by sucrose-SDS gradients . Two RNA species sedimenting at 13S and 10 .5S were found, which were identical in sedimentation behavior and electrophoretic mobility to the RNA species of adult fly mitochondria. The 32 P-RNA recovered from sucrose gradient peaks was further purified by electrophoresis in formamide-acrylamide gels, and the RNA was eluted from peak slices . Nucleotide composition analysis was performed on an aliquot of each 32P-RNA, and the remaining 32 P-RNA was further purified by electrophoresis in aqueous acrylamide gels . The RNAs migrated as single bands in these gels . The nucleotide composition of peak fractions was analyzed . The composition of each mt-rRNA was the same when determined with eluates from the formamide gels or aqueous gels, supporting the conclusion that the RNAs were pure . The values were therefore pooled, and the averages are presented in Table 1 . Since the results depicted uncommonly low G + C contents, we also analyzed 18S RNA isolated from the mitochondrial preparation (where it occurs

as a contaminant) and of separately prepared cytoplasmic rRNA . The values for 18S RNA from both sources agreed closely with each other and with earlier analyses of cytoplasmic rRNA (Tartof and Perry, 1970) . Thus no artifactual modification of RNA occurred during the preparation of mitochondria and of mtRNA . Restriction Endonuclease Digestion of mtDNA mtDNA of D . melanogaster has previously been isolated as circular molecules, and its molecular weight has been determined by electron microscopy to be close to 12 x 10 6 daltons (Wolstenholme, 1973 ; Peacock et al ., 1973) . We have also measured the molecular weight of this DNA by electron microscopy and have obtained in two experiments values of 12 .16 + 0 .22 x 10 6 (17) and 11 .89 ± 0 .32 x 10 6 (51) . From these and the above-cited measurements, we derive a mean value of 12 .15 x 10 6 or 18,400 by for the genome size of D . melanogaster mtDNA . Samples of mtDNA were digested with Hind III, Hae III, and Eco RI, and analyzed by agarose gel electrophoresis (Table 2) . In addition, we found that D . melanogaster mtDNA has no Sma I cleavage site . The sizes of Hind III and Hae III fragments were also determined by electron microscopy . There appears to be a systematic underestimation of size in our electrophoretic determinations, possibly due to the low G + C content of mtDNA . The data of Table 2 agree with the earlier determination of the sizes of Hae III fragments of mtDNA by Manteuil, Hamer, and Thomas (1975) . The Hind III and Hae III sites were used as reference points in mapping experiments . Denaturation Mapping of Restriction Fragments Peacock et al . (1973) have published electron micrographs of heat-denatured mtDNA which reveal an early melting region rich in A+T . We repeated this denaturation mapping using alkali at four pH values ranging from pH 10 .6 to pH 11 .05 . The partially denatured molecules were analyzed by electron microscopy . At pH 10 .6, the early melting region included close to 25% of the total length of the circle, while very little denaturation was observed in other parts of the DNA . At pH 10 .75 and pH 10 .9, additional small denaturation bubbles were observed in the DNA, so that 57% of the molecule was denatured at pH 10 .9 . However, the distinctive early melting region remained unchanged and in-



mtDNA and mt-rRNA in Drosophila 61 7

Table 2 . Size of Restriction Fragments of D . melanogaster mtDNA Hind III

Hae III

Gel

EMb

Fraction of Genome

Gel

EM

Fraction of Genome

Eco RI

Fragment A

7,6500

7,940 ± 290

0 .44

8490

8910 f 409

0 .49

10,150

B

5,150

5,394 f 210

0 .30

5460

5740 ± 330

0 .32

4,770

C

4,170

4,545 f 197

0 .25

3330

3390 ± 180

0 .19

1,620

D

424

(424)

17,394

18,300

Sum

Gel

0 .02

760

17,280

18,040

17,300

Sizes of fragments are given in base pairs . bin the electron microscope analysis, 335 Hind III and 317 Hae III fragments were measured . The mean ± standard deviation is given .

cluded only 25% of the circle length . At pH 11 .05, the DNA was totally denatured . Since the largest denaturation bubble had a constant size in a relatively broad pH range, it could be used as a reference point for further mapping studies . A complete digest of mtDNA with Hae III was partly denatured at pH 10 .6 and examined in the electron microscope . The large denaturation bubble was located in the largest Hae III fragment (A fragment) . Of the 57 molecules in the other size classes (B and C fragments), all but two were fully native . The fraction of the total mtDNA length located in the double-stranded region on each side of the denaturation bubble in fragment A was determined to be 7% and 17% of genome length . The orientation of the early melting bubble relative to Hae III fragments B and C was investigated by denaturation mapping of a partial Hae Ill digest (Figure 2) . The results demonstrate that the short (7%) double-stranded tab is next to the C fragment, and the long (17%) tab is next to the B fragment . Denaturation mapping with a Hind III digest showed that the bubble is contained in the B fragment, which is barely longer than the bubble itself . Figure 3 shows traces of partially denatured molecules from a Hind III partial digest . The bubble is located in the Hind III B fragment and is only slightly asymmetric in its placement with the longer tab attached to the A fragment . The B fragment represents 30% of the mtDNA length (Table 2), 25% of which is occupied by the early melting bubble . The double-stranded tabs are about 3% and 2% long (Figure 3) . The small Hind III D fragment will be incorporated into the map at a later point . The map of the denaturation bubble and the Hind III and Hae III sites could be superimposed on each other in two different ways, as shown in Figure 4 . A distinction between the two possible alignments could be made on the basis of the fragments produced in a double digest . Such a digest was analyzed by gel electrophoresis and showed clearly that model a is

C

0 0a

I 0

A

0

0 .2

B

0

0

0.4

0.6

0.8

1 .0

GENOME LENGTH

Figure 2 . Denaturation Map of a Partial Hae III Digest of mtDNA The DNA was partially denatured and examined by electron microscopy . The molecules were measured and aligned at the denaturation bubble . The top molecule is a circle . A single A fragment is shown from a complete digest which had been subjected to partial denaturation . Open bars represent denatured regions ; single lines represent double-stranded regions . The average model for the Hae III map is shown at the bottom .

the correct one . The actual sizes of fragments in the double digest are incorporated into Figure 7 below, and may be compared with the predictions in Figure 4 . Mapping of mt-rRNA Genes by Observation of RNA-DNA Hybrids in the Electron Microscope The location of the mtDNA regions that code for the mt-rRNAs were mapped with respect to the Hind III and Hae III cleavage sites through two types of RNA-DNA hybridization experiments . In each experiment, nicked circular mtDNA and an equal amount of mtDNA completely digested with Hind III or Hae III were mixed, denatured, and then renatured in the presence of excess mt-rRNA . The mixture was spread and examined in the electron microscope . The molecules of interest were



Cell 618

single-stranded circles with one long doublestranded region resulting from hybridization of a restriction fragment to the circle, as well as one or more short duplex regions, possibly representing RNA-DNA hybrids . Such molecules would show the relationship of the mt-rRNA genes to each other and to two restriction endonuclease cleavage sites . Electron micrographs of DNA circles of this sort, one with a Hind III fragment and one with an Hae III fragment hybridized, are shown in Figure 5 . In these spreadings, other possible types of hybrids were also observed, including double-stranded circles, double-stranded restriction fragments, small duplex regions on single-stranded circles or on long single-stranded pieces, and so on . However, only molecules of one clearly defined class were photographed and analyzed-that is, those which contained one long and one or more shorter double-stranded regions . All traces were linearized as shown in Figure 6 by breaking the circle in such a way that the long double-stranded region was plotted on the left, followed by the shorter of its two adjacent singlestranded regions . Before plotting, the length of each single-stranded region was multiplied by 0 .95 to correct for the difference in linear density between single- and double-stranded DNA as determined by a comparison of the lengths of SV40 DNA and 4X174 DNA in these spreadings . In the experiment involving the Hae III fragments, 21 of the 49 molecules fell into one distinct class : the long double-stranded region was within the size expected for one of the Hae III fragments (the B fragment), and there were two shorter doublestranded regions which were very close to each A

B

C

other and separated by a small gap . We believe that the two closely apposed duplex regions represented the mt-rRNAs, since this arrangement is known in the mtDNA of HeLa cells (Wu et al ., 1972) and Xenopus (Wellauer and Dawid, 1973) . These molecules were selected for the analysis (Figure 6) . Among the other molecules that are not shown, many are consistent with the same regular patternfor example, some had a single duplex region at the position of the RNA-DNA duplex, possibly because the gap between the rRNAs was not visible ; others had a short duplex region in this area, possibly due to the hybridization of only one of the two rRNAs . The remaining molecules showed no regular pattern . In the molecules shown in Figure 6, in all but three cases, the larger rRNA duplex is proximal to the B fragment duplex . The general pattern in the three exceptions (dots in Figure 6) is consistent with the pattern in the other 18, but we excluded the three molecules from the analysis . The average map involving the Hae III B fragment in Figure 6 is based on the 18 molecules selected by these strict criteria . While this mode of analysis introduces several assumptions, the results are tested below by independent means . In the corresponding experiment performed with Hind III fragments, again one consistent class of molecules was apparent which contained a long duplex region corresponding to the Hind III C fragment . Molecules were selected by the criteria listed above, and the resulting average map is shown in Figure 6 . The molecular weight of the single-stranded gap between the two mt-rRNAs was calculated using 4X174 DNA as the length standard . Data from the Hind III and Hae III experiments were combined in this calculation (n = 26) to yield a value of 152 f 27 nucleotides . .7



a t

.7

b t

5.4

2 .7

2 .0

l I 1 { l

t

5 .4

2 .7

3 .4

3 .8

5 .2

t

.5

3 .4

+ 1 1 { 1

t

f

{

t

Figure 4 . Models for the Position of Hind III and Hae III Cleavage Sites in mtDNA

0.2

0.4

GENOME

0.6

0.8

1 .0

LENGTH

Figure 3 . Denaturation Map of a Partial Hind III Digest of mtDNA See legend to Figure 2 for explanation .

The early melting region is represented by an open bar . Arrows above the line indicate Hind III sites, and below the line, Hae III sites . Numbers indicate the expected sizes in kilo base pairs of fragments produced in Hind III-Hae II double digestion of mtDNA . In constructing these models, the slight asymmetry of the early melting region within the Hind III B fragment was ignored because consideration of this asymmetry did not change the order of the predicted cleavage sites, and only changed the predicted sizes of the double digest fragments by 1% of genome length .

mtDNA 619

and mt-rRNA

in Drosophila

In plotting Figure 6, the linear density of the RNADNA hybrids was assumed to be the same as that of duplex DNA, since no appropriate standard was available. The average lengths of the duplex regions correspond to 1300 and 800 bases for the two rRNAs. Molecular weights of hybrid regions measured in this way may be too low (for example, see Forsheit, Davidson, and Brown, 1974). We therefore expanded the rRNA gene regions of the final model of Drosophila mtDNA to correspond to the measured sizes of the rRNAs themselves, 1500 and 860 bases (see Figure 7 below). It should be possible by superposition to combine the mt-rRNA maps (Figure 6) with the denaturationrestriction site map (Figure 4a) to obtain a consistent combined map. Four possible superpositions exist, and only one of these is consistent. This map (Figure 7, below) is confirmed and corrected in detail on the basis of an experiment which we describe next.

Figure

5. Electron

Micrographs

of mt-rRNA-mtDNA

Hybrid

Hybridization of ‘*+mt-rRNAs to Restriction Fragments of mtDNA To test the validity of the map derived by electron microscopy, mt-rRNAs were labeled with 1251, and each RNA was hybridized separately to Hind III and Hae III fragments that had been separated on agarose gels and transferred to membrane filters. Each digest was electrophoresed and transferred in duplicate. One filter with each enzyme digest was hybridized with large and one with small rz51-mtrRNA. This experiment confirmed the maps derived by electron microscopy, first by the fact that the Hae III B fragment and the Hind III C fragment showed very low levels of hybridization, and second, in the details of hybridization of the different RNAs and DNA fragments. To quantitate the results of this experiment, the bands were cut out and the radioactivity was determined. The results are presented in Table 3. The fraction of each mt-rRNA that hybridizes to each restriction fragment allows

Molecules

Denatured circular mtDNA was annealed to Hae Ill- or Hind Ill-digested DNA and to mt-rRNA, and analyzed in the electron microscope, Single-stranded mtDNA circles which contained a long double-stranded region resulting from hybridization of a restriction fragment, as well as one or more short double-stranded regions possibly representing DNA-RNA hybrid, were photographed. Two such molecules are shown, one with the Hae Ill B fragment, and one with the Hind Ill C fragment duplex, Interpretive drawings below the electron micrographs indicate the position of the large and small mt-rRNAs (Lg and Sm) and of the restriction fragment (Hae B and Hin C).

Cell 620

the placement of one restriction site within that rRNA gene region. The results with the Hind III and Hae III digest are highly consistent with each other and confirm the map based on electron microscopy, but suggest that the rRNA genes are 200300 bp farther away from the early melting bubble. The data of Table 3 allow a test for the size of the gap between the rRNA genes. The DNA fragment in a Hind Ill-Hae III double digest which contains parts of both rRNA genes is 790 bp long (see Figure 7). From the sizes of the two rRNAs and the fractions which hybridize to the different restriction fragments (Table 3), we calculate that 420 bp of the 790 bp fragment are gene for the large rRNA, and 200 bp are gene for the small rRNA. This leaves 170 bp, in close agreement with the estimation of the size of the gap by electron microscopy (152 bp). Figure 7 quotes the mean of these values (160 bp). The above ‘*51-RNA-DNA hybridization experiment was repeated on a Hind III digest run on a 1.2% agarose gel to map the very small Hind III D fragment. We know from an agarose gel of a partial digest (not shown) that the D fragment is located adjacent to the A fragment. Hybridization of r251-mtrRNA to the gel transfers which include the Hind III D fragment established that this fragment does not contain ribosomal sequences and thus must map between the A and C fragments rather than between A and B. Figure 7 shows a map of D. melanogaster mtDNA which incorporates all the available information. Hoe B

The distances measured by electron microscopy were adjusted slightly (l-2%) on the basis of the gel transfer hybridization, and the Hind III D fragment was inserted. The polarity of the rRNAs is indicated on the basis of the experiments reported below. The Direction of rRNA Transcription Polarity was determined by hybridization of labeled mt-rRNA to the single-stranded tails generated in mtDNA fragments by exonucleases. The approach follows the work of Dawid and Wellauer (1976). The experiments made use of exonucleases of known specificities: exonuclease Ill from E. coli is specific for the 3’ end of the DNA (Richardson, Lehman, and Kornberg, 1964), and the exonuclease coded by bacteriophage h is specific for the 5’ end (Little, 1967). Figure 8 shows models of a portion of the mtDNA containing the rRNA genes. The model assumes one of the two possible polarities of the RNA molecules (the correct one); an analogous model may be constructed for the opposite polarity. A Hind III digest of mtDNA and, separately, an Hae III digest were treated with exonuclease III from E. coli or with h exonuclease. Digestion proceeded under the conditions described previously (Dawid and Wellauer, 1976) until about 1000 nucleotides were released from each end. The partially single-stranded DNA molecules were then loaded onto membrane filters, and parallel filters were hybridized with 125llabeled large or small mt-rRNA. The results are given in Table 4, which we compare with the predictions derived from the model of Figure 8. When the DNA was not treated with exonuclease but denatured before loading onto filters, both rRNAs hybridized and yielded a ratio of 0.55-0.6 for the binding of small-to-large RNA. Treatment of a Hind III digest with exonuclease III should digest the anticoding strand leftward from the Hind III site, thereby exposing the coding strand for the small rRNA. No coding Table 3. mtDNA Restriction Fragment

Hin C

I 5

0

5 MOLECULAR

Figure 6. Maps of Single-Stranded Regions Containing a Restriction

IO WEIGHT,

‘25l-mt-rRNA

Hybridized

to

Large mt-rRNA cm (96)

Restriction

Fragments

Small mt-rRNA cpm (%)

Hae Ill A

2,720 (28)

60,900

C

7,050 (72)

2,300

15

of

(96) (4)

KB

mtDNA Circles with Fragment and mt-rRNA

Duplex

Hybrid molecules with a Hind III digest were prepared as described in the legend to Figure 5. The molecules are circles, but are presented as linear maps. Thin lines represent single-stranded DNA; thick lines represent duplex. The dots are explained in the text. The average map of the molecules containing the Hind III B fragment is shown below the individual molecules. At the bottom, the average map of the analogous experiment with an Hae Ill digest is shown. This average is based on nine molecules.

Hind

Ill A B

11,120

(100) 0’

10,650 34,250

(24) (76j

Separate digests of mtDNA with Hae Ill and Hind Ill were subjected to gel electrophoresis; the bands were transferred to filters; and replicate filters were hybridized with either large or small W-mtrRNA. The bands were detected by autoradiography and cut out, and the radioactivity was determined. The low level of radioactivity bound to Hae Ill B was subtracted from the values of Hae ill A and C; similarly, Hind Ill C was used as a blank for Hind Ill A and B.

mtDNA and mt-rRNA in Drosophila 62 1

strand for the large rRNA should be exposed, since rightward digestion hydrolyzes coding strand and exposes anticoding sequences . In agreement with this model, the exonuclease Ill-treated Hind III di1 60 860 5

gest binds small rRNA only (Table 4) . Treatment of an Hae III digest with exonuclease Ill should also hydrolyze the anticoding strand leftward, exposing a short region of DNA sequences coding for the large rRNA and a longer region coding for the small rRNA . The hybridization ratio should therefore be higher than in DNA not treated with exonuclease . This is true (Table 4), although the ratio rises even more than would be expected . Treatment of an Hae III digest with A exonuclease should digest the anticoding strand rightward and should expose only coding sequences for the large rRNA . Table 4 shows that much more large than small rRNA is bound by this sample, although the ratio is not zero as predicted . The fourth sample (Hind III digest treated with A exonuclease) was not available for analysis . The results are highly consistent with the polarity shown in the models of Figures 7 and 8 . Discussion Properties of mt-rRNA Mitochondria of D . melanogaster contain two RNA species in equimolar ratio with a size similar to that of mt-rRNA in vertebrate animals (see Dawid, 5'

3'

D Hin Figure 7 . Map of Drosophila melanogaster mtDNA Hae III cleavage sites are designated by arrows from the inside of the circle, and Hind III cleavage sites by arrows from the outside . Restriction fragments are designated in bold letters . The open bar represents the early melting region, and the cross-hatched bars the position of the mt-rRNA genes . The 3' and 5' ends of the rRNA molecules are indicated . The map is based on electron microscopy and hybridization of rRNA with restriction fragments as described in the text . The numbers inside the circle are the lengths in base pairs of the products of a Hind III-Hae III double digest . On the outside of the circle, the lengths of certain other regions of interest are indicated . The total genome size is 18,400 by or 12 .15 x 106 daltons .

`„

5'

3'

5'

3'

5'

I=;!3'

3'

5'

3'

5'

5'

3'

5'

3'

Hin

Hae

Figure 8 . Model for the Test of Polarity of mt-rRNA A region of the mtDNA molecule is shown which codes for rRNA (see Figure 7) . The strands continue left and right beyond the Figure. The positions of the relevant Hind III and Hae III sites are shown . The rRNAs are shown as dashed lines ; the coding strand is a heavy line, and the anticoding strand a light one . The polarity shown in this model is the one supported by our experiments .

Table 4 . Hybridization of mt-rRNA to Exonuclease-Digested Restriction Fragments of mtDNA RNA Hybridized (cpm)

Restriction Endonuclease

Exonuclease

Small rRNA

Large rRNA

Hind III

None

6,853

12,336

Exonuclease III

5,377

0

None

5,103

8,536

0 .6

Exonuclease III

4,882

666

7 .3

X Exonuclease

436

3,280

Hae III

Sm/Lg 0 .55 00

0 .13

About 0 .2 pg of DNA were loaded onto membrane filters for each hybridization . Samples not treated with exonuclease were denatured before loading, but exonuclease-treated DNA was not denatured, so that only single-stranded tails of DNA molecules hybridized . Hybridization with 1251-labeled RNA was carried out as described in Experimental Procedures . The RNAs were used at concentrations of 0 .05 and 0 .25 µg/ml for small and large rRNA, respectively, so that the level of radioactivity in the two hybridization reactions was equal (see Experimental Procedures) . The values listed are the average of duplicate determinations and are corrected for binding of RNA to blank filters .

Cell 622

1972a) . These RNAs are quantitatively the major RNAs of high molecular weight in mitochondria, except for some cytoplasmic rRNA, and they show a pronounced dependence of electrophoretic mobility on temperature (Figure 1) and on gel concentration (data not shown) . In these respects, the two RNAs resemble mt-rRNAs from vertebrate animals so closely that we conclude that they are the Drosophila mt-rRNAs, even though we have not isolated them from mitochondrial ribosomes . Drosophila mt-rRNAs behave in sedimentation and electrophoresis in an unexpected way, even more so than vertebrate animal mt-rRNAs (Dawid, 1972a) . For example, the large rRNA sediments at 13S relative to E . coli rRNA markers, but it co-migrates with 23S rRNA in aqueous gels at room temperature . The slow sedimentation and slow electrophoretic mobility suggest that Drosophila mt-rRNA has little secondary structure at room temperature, so that it offers more hydrodynamic resistance than other RNAs of similar size . These physical properties emphasize the desirability of a general designation for mtRNA not dependent upon sedimentation rate ; calling the large mt-rRNA "13S RNA" would obscure its similar size and general analogy to vertebrate animal large mt-rRNAs which sediment at 16S-17S . The composition of Drosophila mt-rRNA is unusual, and the very low G + C content may explain in part the hydrodynamic behavior of this RNA . The G + C content of the Drosophila RNA may be the lowest reported for any rRNA (see compilation by Lava-Sanchez, Amaldi, and La Posta, 1972 ; and for mt-rRNA, Borst and Grivell, 1971 ; Dawid, 1972a) . The biological significance of the overall composition of rRNA is not clear, but we wish to point out a correlation between the compositions of cytoplasmic and mitochondrial rRNA of animals . All vertebrate animal cytoplasmic rRNAs have a high G + C content with an average of about 60% . The mtrRNAs of these species have a G + C content of about 40% . Drosophila differs from the vertebrate and many invertebrate animals in that its cytoplasmic rRNA has a G + C content close to 40% . In this case, the mt-rRNA is even lower in G + C, maintaining a similar relation to the cytoplasmic rRNA as in the vertebrates . Properties of mtDNA D . melanogaster mtDNA is larger than most other animal mtDNAs, but it contains a section very rich in A + T which may not contribute any information . The place of this A + T-rich segment in the evolution of Drosophila mtDNA has been studied in detail by Fauron and Wolstenholme (1976) .

We have been unable to find D loops (Kasamatsu, Robberson, and Vinograd, 1971) in Drosophila mtDNA in spite of an extensive search . Closed circular DNA was isolated from embryos, young larvae, and adult flies, and maintained in 0 .3 M salt or higher until the DNA was fixed with glyoxal and prepared for electron microscopy . No D loop was ever seen . We observe D loops in high fractions of the mtDNA molecules of Xenopus or other animals . While such loops could still have escaped detection for unknown reasons, we suggest that D loops may not be a "holding point" in the replication of Drosophila mtDNA as they are in many other animal mtDNAs (Kasamatsu and Vinograd, 1974) . This suggestion is supported by our preliminary observation that Drosophila mtDNA has a very low superhelix density, with a value not measured precisely . Kasamatsu et al . (1971) suggested that the extent of D loop synthesis may be determined by the superhelix density of the DNA such that the DNA is largely relaxed when the D loop is completed . This hypothesis is compatible with the finding that Xenopus mtDNA has a higher negative superhelix density and a longer D loop than mouse mtDNA (Hallberg, 1974), and with the low superhelix density and apparent absence of D loops in D . melanogaster mtDNA . Evolutionary Conservation in the Organization of Animal mtDNA Animal mtDNAs are circles of very similar size, but the primary nucleotide sequences evolve at a fairly rapid rate . The mtDNAs of two Xenopus species differ by an average of 25% of their nucleotides (Dawid, 1972b), and similar degrees of divergence were observed in mammalian mtDNAs by Jakovcic, Casey, and Rabinowitz (1975) . Our preliminary experiments show that homology between the mtDNAs of Drosophila and vertebrate animals is detectable but quite low . In contrast, the arrangement of the rRNA genes is identical in the animal mtDNAs where it has been studied . Close proximity of the rRNA genes with a gap of 120-160 by separating the two genes holds true for the mtDNAs of HeLa cells (Wu et al ., 1972), X . laevis (Wellauer and Dawid, 1973), and X . mulled (J . L . Ramirez and I . B . Dawid, unpublished observations) . This constancy of arrangement in the face of extensive sequence changes may suggest that the two rRNAs are transcribed as part of a common precursor . Aloni and Attardi (1971) have provided evidence for the transcription of large RNAs from HeLa mtDNA, but the relation of these transcripts to the rRNAs has not been investigated in detail . We have determined the polarity of the coding strand of Drosophila mtDNA in the rRNA region and have shown that transcription is directed from the

mtDNA and mt-rRNA in Drosophila 6 23

smaller to the larger rRNA . The same polarity'has been found in Xenopus mt-rRNA (J . L . Ramirez and I . B . Dawid, unpublished results) . Evidence from many sources is mounting that transcription of rRNA genes in procaryotes, eucaryotic nuclei, chloroplasts, and now mitochondria is always in the direction small-to-large rRNA (see Lewin, 1976 ; Dawid and Wellauer, 1976) . This arrangement of rRNA genes may be a truly universal feature of genomes . Experimental Procedures Rearing and Harvesting of Organisms D . melanogaster Ore-R were obtained from K . Tartof and reared in population cages according to the method of Travaglini and Tartof (1972) . Large quantities of embryos were obtained by allowing eggs to be laid for 18-24 hr periods in yeast paste spread over agar plates . Embryos were then collected by washing the paste through 80-mesh screen . All embryonic stages and a few hatched larvae were present in these preparations . Culture of Embryonic Cells in Vitro and Labeling with 32 P04 Schneider's D . melanogaster embryonic cell line 2 (Schneider, 1972) was maintained in Schneider's revised Drosophila medium (GIBCO) supplemented with 5 mg/ml Bacto-Peptone (Difco), 15% fetal bovine serum (Flow Laboratories), 5 mg/ml L-glutamine, and 0 .01% phenol red (GIBCO) . Cells were labeled by replacing the medium 1-2 days before harvest with Schneider's revised medium made with tricine buffer rather than phosphate and supplemented with 15-20 pCi/mI 32 P0 4 (New England Nuclear) . Cells were harvested by gently flushing them from the bottom of the flask with a Pasteur pipette. Preparation of Mitochondria Adult flies were homogenized by grinding in a mortar and pestle . Embryos and cultured cells were disrupted by homogenizing in a Dounce or glass-teflon homogenizer . Mitochondria were then prepared essentially according to the method of Dawid (1966) . Preparation and Sucrose Gradient Centrifugation of mtRNA RNA was extracted from adult fly mitochondria by the method of Solymosy et al (1968) . RNA from mitochondria of cultured cells was extracted according to Brown and Littna (1964) . Usually, RNA was treated with DNAase (RNAase-free ; Worthington) before separation or analysis by sedimentation or electrophoresis . Sucrose gradients were run under the conditions described by Dawid (1972c) . Large and small mt-rRNAs separated by two cycles of sucrose gradient centrifugation were labeled with 1251 by the method of Commerford (1971), as modified by Prensky, Steffensen, and Hughes (1973) and by P . Gage (personal communication) . Preparation of mtDNA Purified mitochondria from 20 g of embryos were resuspended in 10 ml 0 .15 M NaCl, 0 .1 M EDTA, 0 .1 M Tris-HCI (pH 8 .5), and lysed with 2% SDS . The lysate was warmed to room temperature, homogenized gently with a Dounce homogenizer, and centrifuged at 30,000 rpm for 30 min in a Beckman SW50 .1 rotor. Ethidium bromide (EB ; Calbiochem) was added to the supernatant to 0 .1 %, and then solid CsCI was added to 1 M . The lysate was chilled in ice and centrifuged at 10,000 rpm for 10 min . Solid CsCI was added to the supernatant to a density of 1 .62 g/cm3' and the samples were centrifuged to equilibrium (2 .5-3 days) at 33,000 rpm in a Beckman 40 or 65 rotor. The lower band formed by the closed circular mtDNA was visible when the gradients were observed in ultraviolet light ; this band was collected and rebanded in EB-CsCI .

The EB was extracted with isopropanol . Electron microscopic analysis of this mtDNA showed it to be homogenous closed circular DNA . Nicking of Closed Circular mtDNA mtDNA (5 pg/ml) was singly nicked with 8 pg/ml DNAase in the presence of 300 µg/ml EB by incubation at 30°C for 15 min (Greenfield, Simpson, and Kaplan, 1975) . The digestion was stopped by adding 50 mM EDTA, and the mixture was chilled and extracted with phenol . The aqueous phase was extracted several times with ether . Electron microscopic analysis of mtDNA nicked in this way and partially denatured with alkali showed that it still contained 10-20% closed circular DNA, and that most molecules contained only a single nick . Gel Electrophoresis RNA was analyzed in 2 .4% polyacrylamide gels containing SDS (Weinberg et al ., 1967) and in 5% acrylamide gels in 98% formamide (Staynov, Pinder, and Gratzer, 1972) . Gels were traced in a Joyce Chromoscan or stained with Stains-all (Dahlberg, Dingman, and Peacock, 1969) . DNA was electrophoresed in 0 .8 or 1 .2% agarose vertical slab gels (Sharp, Sugden, and Sambrook, 1973) . A Hind III digest of bacteriophage A DNA (Wellauer et al ., 1974) was used as molecular weight standard . Gels were stained with EB and photographed on an ultraviolet light box . The procedure of Southern (1975) was used to elute DNA from agarose gels onto millipore filters . DNA containing millipore filters were hybridized with mtRNA in 0 .6 M NaCl, 0 .06 M sodium citrate, 0 .16 M Tris-HCI (pH 8 .0), 20 mM EDTA, 30% formamide, 0 .1%SDS, and 0 .1 µg/ml 1251-mt-rRNA for 24 hr at 37°C . The small mt-rRNA had a specific activity of 17 x 106 cpm/ttg, and the large mt-rRNA 3 .5 x 106 cpm/µg . The filters were washed, treated with RNAase, and exposed to X-ray film . Bands were then cut out, and the radioactivity was determined in a liquid scintillation spectrometer. Determination of Nucleotide Composition This analysis was performed with 32P-labeled RNA according to the method of Markham and Smith (1952) . Restriction Endonuclease Digestion Endonuclease Hind III (Smith, 1974) was a gift from D . Brown ; Hae III (Roberts et al ., 1975) was a gift from R . Brown ; and Eco RI (Greene et al ., 1974) was a gift from R . Reeder. Sma I was prepared according to C . Mulder (personal communication) and donated by J . L. Ramirez, The enzymes were used under the conditions described in the above references . Electron Microscopy Duplex DNA was spread for electron microscopy from formamide (Davis, Simon, and Davidson, 1971) . The DNA was stained with uranyl acetate prior to shadowing with platinum-palladium . SV40 DNA was included as a standard containing 5000 by (Wellauer et al ., 1974) . Photographs were taken in an HU-11 El electron microscope and were projected at a final magnification of 150,000200,000X . Traces were measured with an electronic graphics calculator (Numonics) . Denaturation mapping was performed by exposing DNA to alkali for 10-15 min in the presence of 9% formaldehyde (Inman and Schnds, 1970), using 18 mM EDTA as the only buffer (Carroll and Brown, 1976) . The DNA solution was then neutralized and spread in 50 mM EDTA, 6% formaldehyde, 35% formamide, 50 ug/ml cytochrome c, over a water hypophase . OX174 was included as a single-stranded DNA of 5200 bases (Wu et al ., 1972) . RNA-DNA hybrids were prepared for electron microscopic analysis by a modification of the procedure of Forsheit et al . (1974) . Singly nicked (and an equal amount of restriction endonuclease digested) mtDNA was denatured in 95% formamide, 10 mM EDTA

Cell 624

(pH 8 .5) at 37°C for 10 min . The DNA was annealed with RNA at room temperature for 1 hr with 0 .5 µg/ml mtDNA, 2 pg/ml large and 1 pg/ml small mt-rRNA, 0 .5 M Tris-HCI, 50 mM EDTA (pH 8 .5), and 40% formamide . The mixture was diluted 5 times, molecular weight standards were added, and the sample was spread from 40% formamide onto a water hypophase . Acknowledgments We thank Dr . P . K . Wellauer for his contribution to the measurement of RNA and for help with electron microscopy ; Dr . K . D . Tartof for advice on raising Drosophila ; and Dr . W . B . Upholt for reading the manuscript . The majority of this work has been taken from a thesis submitted by C . K . K. to the Johns Hopkins University as part of the requirements for the degree of doctor of philosophy . C . K . K . was supported by an NIH training grant, and this work was supported in part by an NIH research grant . Received July 2, 1976 ; revised August 16, 1976 References Aloni, Y ., and Attardi, G . (1971) . Expression of the mitochondrial genome in Hela cells : II . Evidence for complete transcription of mitochondrial DNA. J . Mol . Biol . 55, 251-267 . Borst, P . (1972) . Mitochondrial nucleic acids . Ann . Rev . Biochem . 41, 333-376 . Borst, P ., and Grivell, L . A . (1971) . Mitochondrial ribosomes . FEBS Letters 13, 73-88 . Brown, D . D ., and Littna, E . (1964) . RNA synthesis during the development of Xenopus laevis, the South African clawed toad . J . Mol . Biol . 8, 669-687 . Bultmann, H ., and Laird, C . D . (1973) . Mitochondrial DNA from Drosophila melanogaster . Biochim . Biophys. Acta 299, 196-209 . Carroll, D ., and Brown, D. D . (1976), Repeating units of Xenopus laevis oocyte-type 5S DNA are heterogeneous in length . Cell 7, 467-475 . Commerford, S . L . (1971) . Iodination of nucleic acids in vitro . Biochemistry 10, 1993-2000 . Dahlberg, A . E ., Dingman, C . W., and Peacock, A . C ., (1969) . Electrophoretic characterization of bacterial polyribosomes in agarosepolyacrylamide composite gels . J . Mol. Biol . 41, 139-147 . Davis, R . W., Simon, M ., and Davidson, N . (1971) . Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids . Methods Enzymol . 21, 413-428 . Dawid, I . B . (1966) . Evidence for the mitochondrial origin of frog egg cytoplasmic DNA . Proc. Nat . Acad . Sci . USA 56, 269-276 . Dawid, I . B . (1972a) . Mitochondrial protein synthesis . In Mitochondria/Biomembranes (Amsterdam : North-Holland), pp . 35-51 . Dawid, I . B . (1972b) . Evolution of mitochondrial DNA sequences in Xenopus . Dev. Biol . 29, 139-151 . Dawid, I . B . (1972c) . Mitochondrial RNA in Xenopus laevis : I . The expression of the mitochondrial genome . J . Mol . Biol . 63, 201-216 . Dawid, I . B ., and Wellauer, P . K . (1976) . A reinvestigation of 5' -°3' polarity in 40S ribosomal RNA precursor of Xenopus laevis . Cell 8, 443-448 . Fauron, C . M .-R ., and Wolstenholme, D . R . (1976) . Structural heterogeneity of mitochondrial DNA molecules within the genus Drosophila . Proc . Nat . Acad . Sci . USA, in press . Forsheit, A . B ., Davidson, N ., and Brown, D . D . (1974) . An electron microscope heteroduplex study of the ribosomal DNAs of Xenopus laevis and Xenopus mulled . J . Mol . Biol . 90, 301-314 . Greene, P. J ., Betlach, M . C., Goodman, H . M ., and Boyer, H . W. (1974). The EcoRl restriction endonuclease . Methods Mol . Biol . 7, 87-111 .

Greenfield, L ., Simpson, L ., and Kaplan, D . (1975) . Conversion of closed circular DNA molecules to single-nicked molecules by digestion with DNAase I in the presence of ethidium bromide . Biochim . Biophys . Acta 407, 365-375 . Hallberg, R . L. (1974) . Mitochondrial DNA in Xenopus laevis oocytes : I . Displacement loop occurrence . Dev . Biol . 38, 346-355 . Hirsch, M ., and Penman, S . (1974) . The messenger-like properties of the poly(A)+ RNA in mammalian mitochondria . Cell 3, 335-339 . Hirsch, M ., Spradling, A ., and Penman, S . (1974) . The messengerlike poly(A)-containing RNA species from the mitochondria of mammals and insects . Cell 1, 31-35 . Inman, R . B ., and Schnos, M . (1970) . Partial denaturation of thymine- and 5-bromouracil-containing A DNA in alkali . J . Mol . Biol . 49, 93-98 . Jakovcic, S ., Casey, J ., and Rabinowitz, M . (1975) . Sequence homology between mitochondrial DNAs of different eukaryotes . Biochemistry 14, 2043-2050 . Kasamatsu, H ., and Vinograd, J . (1974) . Replication of circular DNA in eukaryotic cells . Ann . Rev . Biochem . 43, 695-719 . Kasamatsu, H ., Robberson, D . L ., and Vinograd, J . (1971) . A novel closed-circular mitochondrial DNA with properties of a replicating intermediate . Proc . Nat . Acad . Sci . USA 68, 2252-2257 . Kleinow, W ., Neupert, W ., and Miller, F. (1974) . Electron microscope study of mitochondrial 60S and cytoplasmic 80S ribosomes from Locusta migratoria . J . Cell Biol . 62, 860-875 . Lava-Sanchez, P . A ., Amaldi, F ., and La Posta, A . (1972) . Base composition of ribosomal RNA and evolution . J . Mol . Evol . 2, 44-55 . Lewin, B . (1976) . Order and spacing of ribosomal RNA genes . Nature 260, 574-576 . Little, J . W . (1967) . An exonuclease induced by bacteriophage A . J . Biol . Chem . 242, 679-686 . Manteuil, S ., Hamer, D . H ., and Thomas, C . A ., Jr . (1975) . Regular arrangement of restriction sites in Drosophila DNA . Cell 5, 413-422 . Markham, R ., and Smith, J . D . (1952). The structure of ribonucleic acid : I . Cyclic nucleotides produced by ribonuclease and by alkaline hydrolysis . Biochem . J . 52, 552-557. Matsumoto, L ., Kasamatsu, H ., Pik6, L ., and Vinograd, J . (1974) . Mitochondrial DNA replication in sea urchin oocytes . J . Cell Biol . 63, 146-159 . Ojala, D ., and Attardi, G . (1974) . Identification and partial characterization of multiple discreet polyadenylic acid-containing RNA components coded for by Hela cell mitochondrial DNA . J . Mol . Biol . 88, 205-219 . Peacock, W. J ., Brutlag, D ., Goldring, E ., Appels, R ., Hinton, C . W ., and Lindsley, D . L. (1973) . The organization of highly repeated DNA sequences in Drosophila melanogaster chromosomes . Cold Spring Harbor Symp. Quant . Biol . 38, 405-416 . Polan, M . L., Friedman, S ., Gall, J . G ., and Gehring, W . (1973) . Isolation and characterization of mitochondrial DNA from Drosophila melanogaster . J . Cell Biol . 56, 580-589 . Prensky, W ., Steffenson, D. M ., and Hughes, W . L . (1973) . The use of iodinated RNA for gene localization . Proc . Nat . Acad . Sci . USA 70, 1860-1864 . Richardson, C . C., Lehman, I . R ., and Kornberg, A . (1964) . A deoxyribonucleic acid phosphatase exonuclease from Escherichia coli . J . Biol . Chem . 239, 251-258 . Roberts, R . J ., Breitmeyer, J . B ., Tabachnik, N . F ., and Myers, P . A . (1975) . A second specific endonuclease from Haemophilus aegyptius. J . Mol . Biol . 91 . 121-123 . Schneider, I . (1972) . Cell lines derived from late embryonic stages of Drosophila melanogaster, J . Embryol . Exp . Morphol . 27, 353-365 .

mtDNA and mt-rRNA in Drosophila 625

Sharp, P . A ., Sugden, B ., and Sambrook, J . (1973) . Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis . Biochemistry 12, 3055-3063 . Smith, H . 0 . (1974) . Restriction endonuclease from H . influenzae RD . Methods Mol . Biol . 7, 71-84 . Solymosy, F ., Fedorcsak, I ., Gulyas, A ., Farkas, G . L ., and Ehrenberg, L . (1968) . A new method based on the use of diethyl pyrocarbonate as a nuclease inhibitor for the extraction of undegraded nucleic acid from plant tissues . Eur . J . Biochem . 5, 520-527 . Southern, E . M . (1975) . Detection of specific sequences among DNA fragments separated by gel electrophoresis . J . Mol . Biol . 98, 503-517 . Staynov, D . Z ., Pinder, J . C ., and Gratzer, W. B . (1972) . Molecular weight determination of nucleic acids by gel electrophoresis in nonaqueous solutions . Nature New Biol . 235, 108-110 . Tartof, K . D ., and Perry, R . P . (1970) . The 5S RNA genes of Drosophila melanogaster . J . Mol . Biol . 51, 171-183 . Travaglini, E . C ., and Tartof, K, D . (1972) . A method for mass culturing large numbers of Drosophila . Drosophila Information Service 48, 157 . Weinberg, R . A ., Loening, U ., Willems, M ., and Penman, S . (1967) . Acrylamide gel electrophoresis of Hela cell nucleolar RNA . Proc . Nat . Aced . Sci . USA 58, 1088-1095 . Wellauer, P . K ., and Dawid, I . B . (1973) . Measurement of mitochondrial RNA and RNA-DNA hybrids by electron microscopy . Carnegie Institution Yearbook 72, 45-46 . Wellauer, P . K ., Reeder, R . H ., Carroll, D ., Brown, D . D ., Deutch, A ., Higashinakagawa, T ., and Dawid, I . B . (1974) . Amplified ribosomal DNA from Xenopus laevis has heterogenous spacer lengths . Proc . Nat . Aced . Sci . USA 71, 2823-2827 . Wolstenholme, D . R . (1973) . Replicating DNA molecules from eggs of Drosophila melanogaster. Chromosome 43, 1-18 . Wu, M ., Davidson, N ., Attardi, G ., and Aloni, Y . (1972) . Expression of the mitochondrial DNA in Hela cells : XIV . The relative positions of the 4S RNA genes and of the ribosomal RNA genes in mitochondria) DNA . J . Mol . Biol . 71, 81-93 .