Sequence homologies between Escherichia coli and chloroplast ribosomal DNA as seen by heteroduplex analysis

J. Mol. Biol. (1980) 142, 247-261

Sequence Homologies between Escherichia cob and Chloroplast Ribosomal DNA as Seen by Heteroduplex Analysis Halo

DELIUS ANDBARBARAKOLLER

European Molecular Biology Laboratory 69 Heidelberg, Germany (Received 21 March 1980) Circular VViciafaba (broad bean) chloroplast DNA was hybridized to the restriction fragment BurnHI B from the DNA of the transducing phage lambda rifd18, which carries the Eacherichia co.% ribosomal RNA operon rmB. Cytochrome spreading8 of the heteroduplexes show homologies in the 16 S and 23 S rRNA regions, but none in the spacer. The same lambda rifd18 fragment was hybridized to the Vi& cpDNAt Sal1 fragment 3, which contains the Vi&a rRNA operon, resulting in an analogous heteroduplex configuration. Cytochrome spreadinga of this heteroduplex in increasing concentrations of formamide reveal regions of incomplete homologies. Heteroduplexes between the E. coZi rrnD operon, obtained from the recombinant plasmid pBK8, and circular Vi&a cpDNA revealed homologies in the spacer region as well as in the 16 S and 23 S rRNA region. Hybrids between all three types of rDNA and their homologous rRNAs were prepared using the mica adsorption technique. They show that the 23 S, 16 S, and 5 S rRNAs are transcribed from the same strand of Vi& opDNA. The positions of the rRNAs were measured and compared to the heteroduplex structure. It was observed that the E. coli MD operon in the plasmid pBK8 contains two 5 S rRNA sequences near the distal end.

1. Introduction Chloroplasts have their own nucleic acid and protein-synthesizing apparatus, which resemble those of prokaryotic cells in many aspects while showing distinct differences to eukaryotic cytoplasmic and nuclear components. Chloroplast 70 S ribosomes contain 23 S, 16 S and 6 S ribosomal RNA like Escherichia c&i. (For details, see reviews by Whitfeld (1977) and Bedbrook 6 Kolodner (1979).) Comparison of sequences of the 16 S rRNA from maize and from E. coli (Schwan & Koessel 1979; Brosius et al., 1979; Carbon et al., 1979) show extensive homologous stretches alternating with non-homologous positions due to base exchange as well as to deletions and insertions. To compare the whole rRNA operon of chloroplasts with an rRNA operon of bacteria, restriction enzyme fragments of the transducing phage lambda rifd18 and of the recombinant plasmid pBK8 were used as a source for E. coli rRNA genes. Lambda rifd18 was isolated by Kirschbaum & Konrad (1973). The DNA originates t Abbreviations

used: cpDNA,

chloroplast

DNA;

cp-rDNA,

chloroplast

ribosomal

DNA.

241 0022-2836/80/260247-15

$02.00/O

0 1980 Academic

Press Inc. (London)

Ltd.

248

H.

DELIUS

AND

B. KOLLER

to 55% from the E. coli chromosome. It carries the rrnB operon, which includes the sequences of the 23 S, 16 S, 5 S rRNA and of the tRNAilU (Lindahl et al., 1977; Lund et al., 1976). The restriction fragment BamHI B includes the E. coli rrnB operon (Kiss et al., 1978). The plasmid pBK8 was described by Boros et al. (1979). It contains the E. c&i rrnD operon, which codes, for the same rRNA sequences as rrnB but has a different

spacer

et al., 1979).

cpDNAj-, more

region,

Heteroduplexes

which

closely

which

includes of the

rrnB

sequences DNA

are shown here, demonstrate

related

to the rrnD

operon

with

that

for tRNAy

and tRNAt$

the rrnD

chloroplast

of E. coli than

(Young

and with Vicia rDNA (cp-rDNA) is

DNA

to the rrnB

operon.

2. Materials and Methods Preparation and chloroplast

of cpDNA from Viciu fuba (broad bean), restriction fragments of cpDNA rRNA were sa described before (Koller & Delius 1980; Koller et al., 1978). (a) Preparation

fragment of lambda rifa18 BamHI BamHI fragment A

B and pBK8

Lambda rifd18 DNA, pBK8 DNA and the E. coli rRNA were gifts from Ibolya Kiss and Pal Venetianer. Lambda rifd18 DNA (10 pg) was digested with BamHI enzyme (New England Biolabs, Beverley, Ma. 0 19 15) in 100 ~1 of enzyme buffer (as described in the New England Biolabs catalogue) and the fragments were separated in cylindrical 0.8% (w/v) agarose gels and eluted using Malachite green/polyacrylamide columns as described by Koller et al. (1978). pBK8 DNA was digested with BamHI enzyme in the same way. After digestion the mixture was made 1% (w / v ) in sodium dodecyl sulphate, passed over a Sepharose column (45 mm x 3 mm) equilibrated with 10 mM-TriseHCl (pH 7*4), 1 mMEDTA, and used directly for hybridization.

(b) Preparation or the mixture

of heteroduplexes between lambda rif d18 Ban-&I fragment of pBKS BamHI fragmenk and circdar Vicia cpDNA

B

Vicia cpDNA supercoils were used aa obtained in ethidium bromide/CsCl gradient fractions at a concentration of 0.1 to 1 pg/ml. A mixture of 17 ~1 of supercoil fraction plus 21 ~1 of formamide was heated by immersing the tubes in boiling water for 1 min; 4 ~1 of fragment DNA at a concentration of about 10 pg/ml were added and heating was continued for 1 min. After incubating the mixture at 40°C for 1 h it was passed through a Sephadex G-100 column (45 mm x 3 mm) equilibrated with the spresding mixture: 30% formamide, 0.1 M-T& * HCI (pH 7.5), 1 mm-EDTA. The eluted DNA was spread after addition of 0.1 mg cytochrome c/ml (Sigma, Type V), and 0.06 c(g PM2 DNA/ml as a length standard (M, = 6.64 x 106; Stueber & Bujard, 1977). The hypophase contained 10 y. formamide, 0.01 M-Tris*HCl (pH 7.8), 1 mM-EDTA. Samples were picked up on Parlodioncovered grids, stained with uranyl acetate as described by Davis & Hyman (1971), and rotary shadowed with platinum and carbon. (c) Preparation fragment

of heteroduplsxea between, lambda rifd18 BamHI B and Vicia cpDNA SalI fragment 36

Sal1 fragment 3 of Vicia cpDNA (18 ~1 at a concn of about 2 to 4 pg/ml in 10 mMTris *HCl (pH 7.5)) 1 mM-EDTA) and lambda rifd18 BamHI fragment B (2 ~1 at a concn of 20 to 40 pg/ml) were mixed with 30 ~1 of formamide, and heated in a boiling waterbath for 1 min. Then 10 ~1 of saturated CsCl were added, and the DNA was reannealed for 1 h at 4O’C. The mixture was passed through a Sephadex column and spread as described above. For the analysis of partial homologies, the reannealed mixture was passed through t See footnote

to p. 247.

E. coZi/CHLOROPLAST

rDNA

HETERODUPLEXES

249

Sephadex G-100 columns, which were equilibrated with spreading mixtures containing 45% or 60% formamide in 0.1 M-Tris*HCl (pH 7*5), 1 mM-EDTA. The hypophasecontained 15% or 30% formamide in O-1 M-Tris*HCI (pH 7*8), 1 mM-EDTA, respectively (Davis & Hyman, 1971). (d) Preparation.

of

hybrids between rDNA

fragments

and rRNA

For the preparation of DNA/RNA hybrids, denatured DNA was mixed with rRNA from Vi& or from E. c&i. The final concentration of DNA and RNA was about 2 to 4 pg/ml of each. A portion (40 ~1) was dialysed in a hollow fibre device (THF 12, Reichelt Chemie-Technik, Heidelberg, West Germany) against a buffer containing 80 y0 formamide, 0.1 M-HEPES (pH 7.3), 0.4 M-N&I, O*Ol M-EDTA (Chow et al., 1977). The dialysed sample was incubated for 2 h at 50°C in a piece of Teflon tubing sealed with pins. The reannealed nucleic acids were passed through a Sephadex G-100 column (45 mm x 3 mm) equilibrated with 10 m&%-sodium phosphate, 1 mM-EDTA (pH 7-O). A peak fraction of 40 ~1 was collected, mixed with 1 ~1 of bacteriophage T4 gene 32 protein (2 mg/ml) and incubated for 5 min. The complexes were fixed by addition of 4 ~1 of 1% glutaraldehyde and incubation for another 15 min at 37°C. The samples were passed through a Sepharose CL-2B column of the same dimensions, equilibrated with 0.01 y0 glutaraldehyde, 4 mMmagnesium acetate. The peak fraction or portions of it were diluted with 4 mm-magnesium acetate. After addition of phage PM2 DNA to a final concentration of 0.06 pg/ml, t,he sample was placed on a piece of Parafilm and covered with a sheet of freshly cleaved mica (about 1.5 cm x 1.5 cm) as described by Portmann & Koller (1976). After 5 min of adsorption, the piece of mica was washed in water for 1 h, dried after 2 washes in absohlte ethanol and rotary shadowed with platinum from an angle of about 3”. Carbon was evaporated on top of the platinum. The film was floated off on water and picked LIP from below on 400-mesh copper grids.

(e) Electron microscopy

and measurements

Photographs were taken with a Philips 301 electron microscope at a magnification of 3400 x for cytochrome spreadings and 11,000 x for mica adsorptions. Measurements were done on the IO-fold or l&fold enlarged negatives using an X-Y stage (Bruehl, Nuremburg, West Germany) connected to a Wang 720 calculator.

3. Results Heteroduplex between

related

homologies

analysis

is an efficient

DNAs

in the electron

between

two E. coli rRNA

(a) Heteroduplexes

tool

to visualize

extended

sequence

homologies

microscope. This method was used to study operons and chloroplast rDNA.

between the Vicia cp-rDNA

and E. coli rrnB

operon

Denatured lambda rifd18 BamHI fragment B DNA, which includes the rrnB operon, was reannealed with denatured circular Vicia cpDNA. The heteroduplex is shown in Figure 1. The BamHI fragment B is hybridized to the single-stranded circle, showing a non-homologous single-stranded end of the fragment, followed by an homology in the 16 S rRNA region, a non-homologous asymmetrical spacer region and an homology in the 23 S rRNA region, followed by a shorter single strand of the fragment. To ensure that this heteroduplex structure is indeed the consequence of homologies in the rRNA genes, the lambda rifd18 BamHI fragment B was reannealed with the Vi& cpDNA XaZI fragment 3, which contains the rRNA sequence (Koller & Delius, 1980). The resulting heteroduplex spread from 30% formamide is shown in Figure 2(a). The heteroduplex shows the same double-stranded regions corresponding

H.

DELIUS

AND

B.

KOLLER

FIG. 1. Cytochrome spreading of a heteroduplex between circular rif*l8 BarnHI B. Arrows indicate homologies in the 23 S and 18 S rRNA genes.

Vi&

cpDNA

and

lambda

E. coZi/CHLOROPLAST

rDNA

HETERODUPLEXES

(a)

(b)

(cl

(dl

251

FIG. 2. Cytochrome spreadings of heteroduplexes. (a) Vi& cpDNA Sal1 3b and lambda rifd18 BarnHI B fragments in normal spreadings. (b) The same heteroduplex spread from 45% formamide. (c) The same heteroduplex spread from 60% formamide. (d) Spinach cpDNA BgZI 3 and lambda rifd18 BanzHI B in a 30 yc formamide spreading.

to the homologies in the 16 S and 23 S genes. These heteroduplexes were measured. Several heteroduplex maps (standardized to the length of the Vicia cpDNA XalI 3b fragment) are shown in the upper part of Figure 3. The single-strand regions which belong to the chloroplast fragment could be identified by comparison with the length measurements done on R-loops as described earlier (Koller & Delius, 1980) and on rDNA/DNA hybrids (see below). The upper histogram in Figure 3 is derived by summing the single-stranded regions in the epDNA Salf 3b fragment. The measured sizes of the single and double-stranded regions are given in the schematic drawing of the heteroduplex shown in Figure 4(a). About 25% mismatches have been detected by comparing the sequences of E. coli and maize 16 S rRNA (Schwarz & Koessel, 1979,198O). In order to visualize such regions in the heteroduplexes between the Vicia cpDNA and the E. coli rRNA operon, these heteroduplexes, prepared in the same way, were spread with cytochrome c at increasing concentrations of formamide, a technique which had been used by Davis & Hyman (1971) to study regions of partial homology in phage T3/T7 hetero-

262

H.

I

I

DELIUS

1 16s

AND

1

B. KOLLER

i 235

FIQ. 3. Maps and histograms of heteroduplexes between Vi& cpDNA x Sal1 3 and lambda rifd18 BumHI B. The thin lines represent double-stranded, the thick lines single-stranded DNA. The shorter of the opposite single strands is placed centrally on top of the longer one in the upper half of each map. (The shorter stretches in this case represent lambda rifd18 BamHI B DNA, see Figs 4 and 8). The maps are standardized to the total length of the Vicia cpDNA Sal1 3b fragment of 7.92 x 1Oe daltons or 11,970 base-pairs (Koller & Delius, 1980). The histograms are constructed from the single-strand stretches of this fragment. The upper histogram represents measurements on DNA spread from 30% formamide. The lower histogram represents measurements done on DNA spread from 45% and 60% formamide. The maps of 46% and 60% formamide spreadings were pooled and are arranged in the order of increasing single-strand content.

E. coZi/CHLOROPLAST Lambda 1285~11L2 ~1521r132

(a)

HETERODUPLEXES

x &mHI

8

565?74 !T 2613t256 L. . . *I e-2453t209

I j

,

rifd18

rDNA

1633t168

Vi&

cpDNA

p8K8x

x Sol1

BarnHI

263

932351 7 I 375LtL20 3b

A

(b)

pBK8

xBumH1

A

133Sl l3Wl 279Lt235

2399.8 -i

Cc)

I’

1602tl25

t t

d39t96

Viciu

cpDNA

.-.

709i57

529!67

FIG. 4. Schematic drawings of the length measurements of heteroduplexes. (a) V’icia cpDNA and lambda rif”l8 BamHI B. (b) Vicia cpDNA and pBK8 BornHI A, spread from 30% formamide, not accounting for the small loop structure in the spacer region. (c) The same as (b) but including heteroduplex structures in the spacer region found in spreadings from 20% formamide (see Fig. 6 (e) to (f). The lengths and standard deviations are given in nucleotides. Thin lines represent single strands, thick lines double strands. Total fragment lengths were determined separately by comparison to PM2 DNA using mica adsorptions. Lambda rifd18 BarnHI B has a molecular weight of 4.97x 10B&0.04x 1Oe or 7240558 base-pairs, pBK8 of 5-31 x lOs~O.08 x lo6 or 8032h 121 base-pairs.

duplexes. Figure 2(b) and (c) shows preparations of the heteroduplexes spread from 45% and 60% formamide. Regions within the 16 S and preferentially 23 S rRNA sequence which appear completely double-stranded in 30% formamide form small denaturation loops in 45% formamide and quite extensive denaturation is seen in preparations spread from 60% formamide. Heteroduplex maps that show the increasing separation of strands in molecules spread from the higher concentrations of formamide are shown in the lower part of Figure 3. They are summarized in the lower histogram. A comparison of the two histograms shows that the duplex is especially labile in the region of the 23 S rDNA. Low melting occurs preferentially in the regions marked by the three arrows in the schematic drawing between the two histograms (the positions of the 16 S and 23 S rRNAs are given as determined in the hybrids; see Figs 7 and 8). The 16 S rRNA region appears more stable than the 23 S rRNA region, indicating a lower degree of mismatching. Figure 2(d) shows a heteroduplex between lambda rifd18 BamHI B and spinach cpDNA BglI fragment 3 with a similar structure. This confirms the results obtained by Southern blotting of

264

H.

DELIUS

AND

B. KOLLER

spinach rRNA on lambda rifd18 DNA, which was done in a parallel study by Bohnert et al. (personal communication). No homology was found between the spacer region of the rrnB operon of E. coli and Vicia cp-rDNA.

(b) Heteroduplexes

between Vicia cpDNA

and the E. coli rrnD operon

To check for possible homologies within the 23 S-16 S spacer region of cp-rDNA and a second type of E. coli rRNA operon, heteroduplexes were prepared between the rrnD operon and cp-rDNA. The rrnD operon was cloned in pBR322 by Boros et al. (1979). The recombinant plasmid pBK8 was digested with BamHI enzyme and the mixture of the two resulting fragments was hybridized with Vicia cpDNA. The heteroduplexes found are almost identical to those formed with the rrnB operon with respect to the 16 S and 23 S rRNA but, in addition, a short reannealed sequence is apparent in the spacer region (Fig. 5). The homology is very short and looks almost like a cross-over of two single strands, but it was found regularly in the same position in almost all heteroduplex molecules. Three heteroduplexes between Vicia cpDNA and pBK8 x BamHI A spread from 30% formamide are shown in Figure 6(a), (b) and (c). The homologous stretch is 18Of60 nucleotides away from the end of the 16 S rRNA position and 447191 nucleotides from the 23 S homology. Measurements on this heteroduplex giving the total lengths of the spacer regions are presented in Figure 4(b). When the same heteroduplex sample was spread under less denaturing conditions, using 20% formamide, a second more extended double-stranded region adjacent to the first one on the E. c&i spacer, was apparent (Fig. 6(d), (e) and (f)). A schematic drawing of the Vicia cpDNA/pBK8 x BamHI A heteroduplex including the positions of the additional loops is shown in Figure 4(c). Knowing the sizes of the two spacers, the three loops formed can be assigned to the Vicia spacer DNA, since in any other arrangement the sum of the single-stranded DNA stretches would clearly exceed the length of the pBK8 spacer region. Spreading the heteroduplex rrnB x cprDNA in 20% formamide did not reveal any duplex formation in the spacer region (data not shown). The 16 S rRNA region appears completely double-stranded in all three heteroduplex structures (Fig. 4). In the 23 S rRNA region the duplex is clearly shorter than the 23 S rRNA sequence. The value for the E. coli 23 S-16 S spacer of 723 nucleotides in the pBK8 heteroduplex is much higher than the value of 438 obtained by sequence analysis (Young et al., 1979). This could be due partly to a non-homology in the proximal end of the 23 S rRNA sequence and partly to a stretching of the single strand during the spreading procedure. (c) Measurements on rRNA/DNA

hybrids

For a more accurate determination of the positions of the structural rRNA sequences, all three types of rDNAs were reannealed with their homologous rRNAs, using conditions in which only DNA/RNA hybrids are stable and hardly any doublestranded DNA is formed (Chow et al., 1977). The remaining single strands were complexed with T4 gene 32 protein (Priess et al., 1980). Figure 7 shows mica adsorptions of hybrid molecules exhibiting the thin outline of the hybrid stretches and the thick single strand/gene 32 protein complexes. Vicia cpDNA SalI fragment 3b was

E. coZi/CHLOROPLAST

rDNA

HETERODUPLEXES

255

Pm. 5. Cytochrome spreading of a heteroduplex between circular cpDNA rend pBK8 BalrsHI A. Arrows indicate the homologies in the 23 S and 16 S rRNA genes and in the spacer region (long arrow).

-__-.- .._. .--.._-.--.._-----

- .-.. Id)

FIG. 6. Heteroduplex (a), (b) and (c) Spreading8

structures as described in Fig. 5, at a higher from 30% forma&de. (d), (e) and (f) Spreadinga

magnification. from 20% formamide.

E. coZi/CHLOROPLAST

rDNA

HETERODUPLEXES

251

FIG. 7. Mica adsorptions of r-RNA/DNA hybrids. (a) Vicia cpDNA Sal1 3b hybridized with V&z ep-rRNA. (b) Lambda rifd18 BumHI B hybridized with E. coli rRNA. (c) pBK8 BamHI A hybridized with E. coli rRNA. The arrows point to the thin RNA/DNA hybrid stretches. The single strands were complexed with T4 gene 32 protein.

hybridized with a mixture of high and low-molecular weight chloroplast RNA (Fig. 7(a)). The 23 S, 16 S, and 5 S rRNA hybrid regions are clearly distinguishable. Hybrids of E. coli rRNA with lambda rifd18 BumHI B and with pBK8 BumHI A are shown in Figure 7(b) and (c), respectively. It can be seen that the rrnD operon included in the pBK8 fragment carries two copies of 5 S rRNA sequences. Only one copy is present in the rrnB operon contained in the lambda rifd18 BamHI B fragment. Vicia and E. coli 23 S and 16 S rRNAs were separated from the 5 S rRNA using agarose gel electrophoresis and electrophoretic elution. Hybrids with 23 S and 16 S rRNA alone did not show the small double-stranded regions close to the distal end

H.

268

DELIUS

AND

B. KOLLER

Vicia cpDNA x Sal I 3b/ Vicio cp-rRNA

1593232

2318t37 3116?56

1513t47 Lambda rifd18 x BornHI

1532+55 pBK8 x BornHI

B/E.coh

8

rRNA

2959t95

106+26

A /E.colirRNA

28lr24 435ti3 c 158047 2934250 165

225527 3067+_61 n 137%21

23s

“,2? 1.

23772143

4

ll%l4 5s

Pm. 8. Schematic drawings of the length measurements of mica adsorptions. Thin lines refer to single-stranded DNA, thick lines to standard deviations are given in nuoleotides. Measurements PM2 DNA. The lengths of RNA/DNA hybrids were corrected double-stranded DNA (Prieea et al. 1980).

rRNA/DNA

hybrids

obtained

from

rRNA/DNA hybrids. Lengths and are baaed on the comparison with by a factor of 1.15 in comparison to

of the 23 S rRNA. After addition of isolated 5 S rRNA, the original hybrid pattern could be reconstituted in both Vicia and E. coli operons (data not shown). Length measurements on mica preparations can be done with much higher accuracy than could be obtained with cytochrome spreadings (Priess et al. 1980). Measurements obtained with this method on rRNA/DNA hybrids are shown in Figure 8. The values for the E. wli 16 S and 23 S rRNA and the 16 S-23 S spacer in the pBK8 fragment correspond very well with the values obtained by sequence analysis which are : 1541, 2904, and 438 nucleotides for the 16 S, 23 S, and the spacer region, respectively (Brosius et al., 1979,198O; Carbon et al., 1979; Young et al., 1979). The chloroplast 16 S rRNA has about the same size (1513 nucleotides) as the E. coli 16 S rRNA but the 23 S rRNA appears slightly larger (3116 nucleotides) than the E. c&i 23 S rRNA. Comparing the two E. coli fragments, the most interesting aspect is the duplication of the 5 S rRNA gene at the distal end of the rrnD operon, where two 5 S rRNA hybrids were measured. The lengths of the 23 S-5 S and the 5 S-5 S spacers could not be distinguished, but they are about twice as large as the 23 S-5 S spacer in the rrnB operon. The length of the 5 S rRNA is about the same for all three hybrid stretches in the E. c&i DNA (106 to 110 nucleotides).

4. Discussion The rRNA operon in Viciu chloroplast DNA is not located in an inverted repeat configuration, as in most other chloroplast DNAs (Koller & Delius, 1980). For this reason the E. c&i rDNA fragments can be directly hybridized onto the chloroplast circular DNA (Figs 1 and 3). This would not be possible with molecules having the

E. coZi/CHLOROPLAST

rDNA

HETERODUPLEXES

259

rDNA in an inverted repeat, because the fragment cannot compete against the rapid intramolecular self-annealing of the inverted sequence. The Vi&a rDNA/E. wli rDNA heteroduplexes confirm a high degree of homology in the 16 S rRNA region, which was described for 16 S rRNA of maize and of E. coli as a result of the sequence analysis (Brosius et al., 1979; Carbon et al., 1979 ; Schwarz & Koessel, 1979,198O). The heteroduplex shows that the homology does not extend visibly into the sequences flanking the 16 S rRNA structural gene. The homology in the 23 S rDNA region as measured in the heteroduplexes does not extend across the whole sequence. At both ends the duplex formation is very unstable, indicating a very low degree of homology. This is reflected by the measurements shown in Figure 4(a) and (b), where the values for the 23 S rRNA region show quite large variability and are somewhat shorter than the E. wli 23 S rRNA sequence (2904 nucleotides, Brosius et al., 1980). Other
260

H.

DELIUS

AND

B. KOLLER

and the spinach cp-rDNA could not distinguish between the coding and the complementary strand. The duplex observed in Figures 5 and 6 is possible only if the homologous sequence in the spacer is present in the same orientation as the rRNAs in analogy to the E. wli operon. This allows the assumption that the supposed “tRNAs” are coded on the same strand as the rRNAs, and may be part of one transcription unit. The sizes and sequences of all spacers vary considerably within the different rDNA operons. Comparing the 16 S-23 S spacer of the Vicia Sal1 3b fragment with the corresponding spinach spacer (Fig. 2(a) and (d)), it is apparent that the latter is much the smaller. The mica measurements (Fig. 8) gave smaller sizes for the spacer between 16 S and 23 S hybrid as well as the spacer between 23 S and 5 S in the rrnB operon than for the corresponding spacers in the rrnD operon. In mica preparations very small RyAs (down to 100 nucleotides) hybridized on DNA can be visualized and measured with an accuracy of 20 to 30 nucleotides. The characterization of the 5 S rRNA on the Vicia rDNA operon (Fig. 7(a)) shows that this chloroplast rRNA is constructed in the same way as the E. coli operon: the 16 S, 23 S and 5 S rRNAs are positioned in the same sequence and are coded on the same strand. The additional homology in the spacer region indicates that the chloroplast rRNA operon is more closely related to the group of E. coli rRNA operons represented by the rrnD operon than to the other group (Kenerley et al., 1977) represented by the rrnB operon. The presence of two copies of 5 S rRNA genes in the rrnD operon has not been observed before. It remains to be studied whether this is an artifact resulting from the construction of the recombinant clone, or whether it is to be found in the E. coli genome, and if so whether it is present in other rRNA operons as well. We thank Miss Jill Clarke for her technical

assistance.

REFERENCES Bedbrook, J. R. & Kolodner, R. (1979). RnnzL. Rev. Plant Physiol. 30, 593-620. Bohnert, H. J., Driesel, A. J., Grouse, E. J., Gordon, K., Herrmann, R. G., Steinmetz, A., Mubumbila, M., Keller, M., Burkard, G. & Weil, J. H. (1979). FEBS Letters, 10, 52-56. Boros, I., Kiss, A. & Venetianer, P. (1979). Nucl. Acids Rec. 6, 1817-1830. Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1979). Proc. Nat. Acad. Sci., U.S.A. 75, 4801-4805. Brosius, J., Dull, T. J. & Noller, H. F. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 201-204. Carbon, P., Ehresmann, C., Ehresmann, B., Ebel, J. P. (1979). Eur. J. Biochem. 100, 399-410. Chow, L. T., Roberts, J. M., Lewis, J. B. & Broker, T. R. (1977). Cell, 11, 819-836. Davis, R. W. & Hyman, R. W. (1971). J. Mol. BioZ. 62, 287-301. Deonier, R. C., Ohtsubo, E., Lee, H. J. & Davidson, N. (1974). J. Mol. BioZ. 89, 619-629. Dyer, T. A. & Bowman, C. M. (1979). Biochem. J. 183,595604. Erdmann, V. A. (1978). NucZ. Acids Res. 5, rl-r13. Kenerley, M. E., Morgan, E. A., Post, L., Lindahl, L. & Nomura, M. (1977). J. Bacterial. 132, 931-949. Kirschbaum, J. B. t Konrad, E. B. (1973). J. Bacterial. 116,517-526. Kiss, A., Sain, B., Kiss, I., Boros, I., Udvardy, A. & Venetianer, P. (1978). Gene, 4, 137-152.

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HETERODUPLEXES

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Koller, B. & Delius, H. (1980). Mol. Gen. Genet. 178, 261-269. Koller, B., Delius, H., Buenemann, H., Mueller, W. (1978). Gene, 4,227-239. Lindahl, L., Yanamoto, M. & Nomum, M. (1977). J. Mol. Biol. 109, 23-47. Lund, E., Dahlberg, J. E., Lindahl, L., Jaskunas, S. R., Dennis, P. P. t Nomura, M. (1976). CeE.!, 7, 165177. Portmann, R. & Koller, Th. (1976). Sizth Eur. Congr. Electron Microac., Jerusalem, 12, 546-548. Priess, H., Koller, B., Hess, B. & Delius, H. (1980). Mol. Gem. Gen-et. 178, 27-34. Schwarz, Zs. & Koessel, H. (1979). Nature (London), 279,520-522. Schwarz, Zs. & Koessel, H. (1980). Nature (London), 283, 739-742. Stueber, D. & Bujard, H. (1977). Mol. Gen. Genet. 154,299-303. Whitfeld, P. R. (1977). The Ribonucleic Acid (Steward & Letham, eds), pp. 297-332 Springer-Verlag, New York, Berlin and Heidelberg. Young, R. A., Macklis, R. & Steitz, J. A. (1979). J. B&Z. Chem. 254, 32643271.