Current Genetics

Current Genetics (1983) 7:7-12

© Springer-Verlag 1983

Molecular Cloning and Characterization of the Chloroplast Ribosomal RNA Genes from Spirodela oligorhiza Ronald J. A. Keus, Dick J. Roovers, Harm van Heerikhuizen, and Gert S. P. Groot Biochemical Laboratory, Free University, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Summary. The organization of the chloroplast ribosomal RNA genes in Spirodela oligorhiza has been determined. We have therefore characterized two cloned BamHI fragments containing the genes for the large and small rRNA genes, and two PstI fragments, each containing one of the two large, invertedly repeated regions of chloroplast DNA. Characterization was performed by restriction endonuclease mapping, DNA/RNA hybridization and electronmicroscopic R-loop analysis. The results show that the rRNA genes are present in the order 16S, 23S, 4.5S and 5S. The 16S (1.5 kb) and 23S (2.9 kb) rRNA genes are separated by a spacer of 2.25 kb. There appears to be a close resemblance between the organization of the chloroplast rRNA genes in Spirodela oligorhiza and other higher plants so far examined. Key words: Chloroplast DNA - Spirodela oligorhiza Ribosomal RNA genes - Physical map

Introduction Spirodela oligorhiza is a small aquatic higher plant. Its chloroplasts contain covalently closed circular DNA with a circumference of 54 pm corresponding to 182 kbp (van Ee et al. 1980). This size is approximately 20% larger than that of most of the other higher plant chloroplast DNAs (Herrmann and Possingham 1980). The chloroplast rRNA genes are located in the invertedly repeated region in the chloroplast DNA (van Ee et al. 1982) as is the case in many other higher green plants (Bedbrook et al. 1977; Dyer and Bedbrook 1980; Rochaix Abbreviations: cp = chloroplast; rRNA = ribosomal RNA; (k)bp = (kilo) base pairs; TPNS = tri-isopropyl naphtalene sulfonic acid; DTT = dithiothreitol Offprint requests to." G. S: P. Groot

and Malnoe 1978; Crouse et al. 1978; Whitfield et al. 1978; Thompson et al. 1981; Fluhr and Edelman 1981). The organization of the chloroplast rRNA genes from higher plants (5'-16S-23S-4.5S-5S-3') including the presence of the genes for two tRNAs in the spacer between 16S and 23S rRNA gene resembles very much the organization of the rRNA genes in prokaryotes. Prokaryotes however lack the 4.5S rRNA gene. Although for a long time no function of 4.5S rRNA was known, more and more evidence appears that there is a relatively high homology between 4.5S rRNA from chloroplasts and the last 100 nncleotides of the 3' terminus of E. coli 23S rRNA. This suggests a functional homology of the two regions. The spacer between the 4.5S rRNA coding region and the 23S coding region could be merely an insertion in an originally continuous 23S rRNA gene (Malnoe and Rochaix 1978; Wildeman et al. 1980; Takaiwa et al. 1980; Edwards et al. 1981). We have investigated the structural organization of the rRNA operon on cpDNA ofSpirodela oligorhiza since preliminary experiments indicated that the spacer region between the 16S and 23S rRNA genes was considerably shorter than that found in other plants despite the fact, that the molecular weight of total Spirodela cpDNA is much larger than commonly found (van Ee et al. 1981). Moreover we initiated this study in order to investigate possible regulatory sequences on cpDNA that could be implicated as transcriptional start- and stopsignals or as processing signals involved in the maturation of the primary rRNA transcript. Materials and Methods Growth of Plants and Isolation of cpDNA and RNA Spirodela oligorhiza was grown and cpDNA was prepared as described by van Ee et al. (1980). cpRNAs were isolated as follows:

8 500 g of Spirodela oligorhiza were homogenized in 1,500 ml homogenizing buffer containing: 0.45 M mannitol, 50 mM Tris HC1 pH 8.0, 10 mM MgC12 and 10 mM EGTA in a blendor during 3 x 5". After homogenization the homogenate was filtered through miracloth and centrifuged during 5' at 1,000 x g. The resulting pellet was washed once with 10 ml homogenization buffer and recentrifuged. The pellet was resuspended in TPNS-buffer containing 20 g TPNS, 5 g NaC1, 2.12 g Tris HC1 and 3.8 g EGTA per liter (pH 7.6). The chloroplasts were lysed by pipetting several times in a small bore pipet. The lysate was centrifuged to precipitate the starch for 10' at 4,000 x g. The supernatant was extracted twice with phenol-cresol (van Ommen et al. 1977) and once with phenol-chloroform-isoamylalcohol 25 : 24 : 1. The nucleic acids were precipitated with 2.5 volumes ethanol 96% and 0.1 volume 3 M NaAe (pH 4.8). Separation of 16S and 23S rRNA was carried out on a 1.25% agarose gel, using buffer containing 90 mM Tris HC1 (pH 8.3), 90 mM boric acid and 2.5 mM EDTA. 4.5S RNA and 5S RNA were separated on a 10% polyacrylamide gel using the same buffer. The separated RNAs were eluted from gel slices in an Isco model 1750 sample concentrator in the same electrophoresis buffer.

Labeling and Hybridization of RNAs and DNAs 5' end-labeling of the rRNAs was performed as described by Donis-Keller et al. (1977). Nick-translation of DNA fragments was carried out as described by Jeffreys and Flavell (1977). Blotting of DNA fragments onto nitrocellulose paper (Sartorius 15 cm x 15 cm 0.1 um pore size) was performed according to Southern (1975). For hybridization with labeled RNAs we used the method of van Ommen et al. (1977).

Molecular Cloning BamHI fragments from chloroplast DNAs were generated under conditions specified by the supplier. Plasmid pBR322 was also cut with BamHI or with BamHI and EcoRI. The insert from pSpocl8 was cut free from pBR322 and digested with EcoRI to generate two Bam-Eco fragments to be cloned in pBR322 and one Eco-Eco fragment to be cloned in EcoRI digested pBR328. Ligation was carried out with 1 ~zgplasmid or 0.15/~g cosmid and fragments in a 4 : 1 molar ratio (2 : 1 for cosmid cloning) in 20 mM Tris pH 7.4, 10 mM MgC12, 0.6 mM ATP, 1 mg/ml BSA (nuclease free), DTT 10 mM and 0.1 ~zl T4 DNA ligase (Boehringer) up to avol. of 20 ~1, during 16 h at 10 °C. In vitro packaging and cloning was carried out according to Grosveld et al. (1981). Selection of Bam and Bam Eco clones was performed on "twin" BHI agar plates, one containing ampicillin (50 ~zg/ml) the other containing tetracyclin (12.5 ug/ml). Colonies that appeared to be ampicillin resistant and tetracyclin sensitive were further cultured. Selection of cosmid clones took place mainly as described above, but now tetracyclin resistant ampicillin sensitive clones were selected. Selection of Eco-Eco clones took place on the basis of chloramphenicol sensitivity and ampicillin resistance.

R-looping and Electronmieroseopy pSpoc 70 DNA, digested with PstI (0.3 ~zg), pSpoc 18 DNA, digested with BamHI (0.3 ~tg) or cos PstA DNA (0.8 ~zg) was hybridized with purified 16S RNA (0.3 ~zg), purified 23S RNA (0.3 ~g) or total cpRNA (4 ~g) respectively. Hybridization was

R . J . A . Keus et al.: Chloroplast Ribosomal RNA Genes carried out for 4 h at 54 °C in sealed capillaries in 0.1 M Pipes (pH 7.8) containing 10 mM EDTA 0.4 M NaC1 and 70% (v/v) formamide in a final volume of 25 ~1. Samples were spread for electronmicroscopy by the formamide/cytochrome e method and rotary shadowed as described before (Veldman et al. 1981). Photographs were taken with a Zeiss EM 109 electronmicro scope. Contour lengths were measured with a Hewlett Packard 9874A digitizer coupled to a 9825A calculator. We have used the restriction fragment nomenclature as devized by van Ee et al. (1980): fragments produced by BamHI are referred to as BA, BB, BC etc. in the order of declining size. Similarly fragment produced by SmaHI or PstI are referred to as SmA, SmB or PA, PB etc.

Results and Discussion 1. Clones Used in this Study In order to analyse the organization o f the r R N A o p e r o n on c p D N A o f Spirodela oligorhiza, we isolated f r o m a clone b a n k o f B a m H I restriction fragments t w o plasmids containing the 23 S r R N A gene ( p S p o c l 8, containing BH, 6.2 kb in size) and the 16S r R N A g e n e ( p S p o c 7 0 containing BM, 3.2 kb in size) respectively. T h e y were identified b y hybridizing 23S r R N A and 16S r R N A , labeled w i t h [a2p], to S o u t h e r n blots o f size-fractionated restriction fragments o f the cloned D N A (results n o t shown). Moreover we have used the S m a H I fragment (SmH) isolated f r o m a cosmid containing the PstA (PA) fragment o f Spirodela cpDNA. The SmH fragment contains the 3' end o f the 16S r R N A gene and the 5' end o f the 23S r R N A gene and therefore the whole intergenic region (spacer region) b e t w e e n these t w o genes.

2. Characterization o f pSpoel 8 In order to characterize the BH fragment in clone p S p o c 18 we have m a d e use o f the fact, that BH contains two recognition sequences for E c o R I . The two Bam-Eco fragm e n t s and the Eco Eco fragment were subcloned t o produce p S p o c l 8 L , p S p o c l 8 M and p S p o c l 8R respectively. Figure 1 shows that clone p S p o c l 8M contains genetic i n f o r m a t i o n n o t only for 23S r R N A , b u t also for the two small r R N A s viz. 5S r R N A and 4.5S r R N A . In this figure, results are shown o f the h y b r i d i z a t i o n o f the restriction fragments p r o d u c e d b y Sau3A, T a q l and M s p l w i t h 23S, 5S and 4.5S r R N A respectively. In order to define the localization o f these genes we have ordered the restriction e n z y m e recognition sites using the SmithBimstiel t e c h n i q u e ( S m i t h and Birnstiel 1976) on an E c o R I - X h o fragment labeled at the E c o R I site. The results are summarized in Fig. 2. The position o f the 4.5S r R N A is defined m o r e precisely on the basis o f a conserved PvuII-site within the gene and the k n o w n length o f the 4.5S R N A . A conserved I-IindIII size just outside

R. J. A. Keus et al.: Chloroplast Ribosomal RNA Genes

9

Fig. I a-f. Hybridization of labelled 4.5S, 5S or 23S rRNA to restriction fragments of pSpoc18M (KL18-SDM) or pSpocl 8R (KL18-SDR). a pSpoc18M digested with TaqI (T) or EcoRI (E) lanes 1, 3, 5 and 7 are photographs of the ethidiumbromide stained gels; lanes 2, 4, 6, 8 are autoradiograms. The hybridizing probe is indicated on top of the figure, b As in A but digested with MspI (M) instead of TaqI. c As in A but digested with Sau3A (S) instead of TaqI. d pSpocl8R digested with MspI with (+) or without (-) BamHI was hybridized with labelled 23S rRNA. Lanes 1 and 5 are autoradiograms, lanes 2 and 4 are photographs of the ethidiumbromide stained gels; lane 3 is a marker lane (pBR322 digested with MspI). e As in D but digested with TaqI instead of MspI. f As in D but digested with Sau3A instead of MspI

the 5S gene is used to locate the 5S rRNA gene more precisely (sequences to be published by our group). The location of the 23S rRNA was determined b y electronmicroscopic analysis of R-loops prepared by hybridizing 23S rRNA to pSpocl8, cut out with PstI, Sac and Sma, or to BH, excised from this plasmid. An example is given in Fig. 3a. According to electronmicroscopic results, the size of the 23S rRNA coding region is 2,900 bp (Table 1)

and the gene is continuous. These findings are consistent with findings in other green plants examined so far.

3. Characterization of pSpoc 70 A fine structure map of pSpoc70 was constructed in an analogous way as for pSpoc 18. Figure 4 shows the restric-

10

R.J.A. Keus et al.: Chloroplast Ribosomal RNA Genes • • •

500 SDM

SDL

SDR

BamBam Barn //Eco

MspI Sau 3A Taq

Eco ~\Srna

~

16S

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/

/i

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~

, ?

"'.

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x\ Eco

'

~

'

I

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200

m

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H~ Taq MSp

Fig. 2. Organization of the rRNA operon on cpDNAof Spirodela oligorhiza. In the Eco-Ecofragmentall restriction sites for HindIII, PvuII, Sau3A, TaqI and MspI are indicated. In the Eco-Bamfragment only the restriction sites in the 5'-terminal region of the 23S rRNA gene are shown

tion fragments hybridizing with 16S rRNA. Figure 5 shows the fine structure map. We have determined the direction of transcription by hybridizing the 50 nucleotide ColE1 fragment of E. coli 16S rRNA to Southern blots of pSpoc70. The ColE1 fragment contains the 3'-end of the E. eoli 16S rRNA (Baan and van Charldorp 1976) and is highly homologous to the 3'-end of chloroplast 16S rRNA. As can be deduced from Fig. 4, and Fig. 5, the 3' end of the 16S rRNA coding region located approximately 140 bp away from the left end of the BM fragment hybridizes with the ColE1 fragment. The direction of transcription is then towards the 23S rRNA gene. A more precise location of the 16S rRNA gene was determined by electronmicroscopic analysis of R-loops formed between rRNA and pSpoc70. An example is given in Fig. 3b. The size thus obtained for the 16S rRNA coding regions is 1,500 bp (see Table 1), which equals that reported for other cp 16S rRNAs.

4. The 16S-23S Spacer Region

a

I

I

0.25/U

b

!

!

O.25/u

Fig. 3a and b. Electron micrographs of R-loops formed between a the BH fragment cut out of pSpoc18 and purified 23S rRNA or b the BM fragment cut out ofpSpoc70 and purified 16SrRNA

The enzyme PstI cuts outside the inverted repeat region of Spirodela cpDNA, producing two large fragments (45 and 37 kb resp.) both containing the whole inverted repeat. Both fragments were cloned in the cosmid pHC 79. The resulting clones were used to isolate SmH from the inverted repeat. This fragment lies in between two large Sma fragments and therefore is the only fragment to be cut out by Sma. In order to construct a Bam restriction map of the 16S-23S spacer region we have hybridized SmH, labeled by nicktranslation, to a Bam restriction digest of the cosmid containing PA. From Fig. 6 it is clear that SmH hybridizes to BH and BM corresponding to the coding regions from 23S and 16S rRNA respectively and also to two smaller Bam fragments. Since BH and BM contain a recognition site for Sma, the smaller Barn fragments must contain part of the spacer region between 16S and 23S coding regions. The order of the fragments was determined by a modified Smith-Birnstiel technique whereby an unlabeled partial digest of SmH

Table 1. Length of cloned fragments, loops and spacer in bp BM (cut out of pSpoc70)

BH (cut out of pSpoel8)

Cos PstA

short ds. 16S loop long ds.

short ds. 23S loop long ds.

spacer

149 -+18 1,531 ± 66 1,492 ± 66 N=17

Data derived from experiments as shown in Fig. 3 and Fig. 7

682 -+ 27 2,859 ± 182 2,843 ± 162 N=12

2,230 ± 184

N=15

R. J. A. Keus et al.: Chloroplast Ribosomal RNA Genes

11

Fig. 4a-e. Hybridization of labelled 16S rRNA (A - C) or the ColE-I fragment (D and E) to restriction fragments ofpSpoc70, a pSpoc70 digested with MspI; lane 1: marker DNA (pBR322) digested with MspI, lane 2: Ethidiumbromide stained gel, lane 3 autoradiogram. b As in A but digested with TaqI. c As in A but digested with sau3A, d Lanes 1 and 3: Ethidiumbromide stained gel ofpSpoc70 digested with MspI (lane 1) or MspI and BamHI (lane 3). Lanes 2 and 4: autoradiograms, e As in D but digested with TaqI instead of MspI

• • •

5OO SOL

pare

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.'/

I I

16S

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Fig. 5. Fine structure map of pSpoc70 showing the restriction sites for BamHI, MspI, Sau3A, TaqI and Bgll

was probed with [32p] labeled pSpoc70 (results not snown). They indicate that the spacer region between the two rRNA genes is 2,250 bp, containing three Bam restriction sites. The order o f the Bam sites is shown already in Fig. 2. The results were verified b y electronmicroscopic analysis o f R-loops formed between the cosmid containing PA and total cpRNA (Fig. 7). F r o m these data, a spacer length o f 2,230 bp -+ 100 bp was deduced. In several Rlooped molecules we found beside the expected loops o f 16S and 23S extra loops corresponding to transcripts o f the spacer region. F o r Nicotiana (Takaiwa and Sugiura 1982) and Zea mays (Koch et al. 1981) it is shown that

Fig. 6. Hybridization of labelled SmH to restriction fragments of CosPstA. CosPstA was digested with BamHI. Lanes 1 and 3: autoradiograms (lane 1: short and lane 3: long exposure time). Lane 2: ethidiumbromide stained gel

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R.J.A. Keus ct al.: Chloroplast Ribosomal RNA Genes

References

Fig. 7. Electron micrograph of R-loops formed between CosPstA and 16S plus 23S rRNA

in the spacer region two genes exist for tRNAs which are interrupted b y intervening sequences of 6 0 0 - 8 0 0 bp. If we assume a similar organization of this region in Spirodela oligorhiza cpDNA (cfr. Groot and van Harten-Loosbrock 1981) the transcripts we find could be either the unspliced precursors of the tRNAs or rather stable splicing remnants. We conclude that the organization of the ribosomal RNA transcription unit in Spirodela oligorhiza as highly similar to that of Nicotiana tabacum, Zea rnays and Spinacia oleracea. The 16S, 23S, 4.5S rRNA genes are of comparable size and similarly organized. The size of the spacer however (2,250 bp) is different. It is smaller than the spacers of Narcissus pseudo narcissus and Tropaeolum majus (resp. 2.47 and 2.73 kb) (Thompson et al. 1981) and Zea mays (2,408 bp) (Koch et al. 1981). It is larger, however, than the spacer in Nicotiana (2,080 bp) (Takaiwa and Sugiura 1982) and Spinacia (1,850 bp) Tomioka et al. 1981). It is evident, that relatively small differences in spacer size do not account for the relatively large size of the inverted repeat in Spirodela oligorhiza.

Acknowledgements. We like to thank Drs J. van Ee and M. Posno for the construction of the pSpocl8 and pSpoc70 respectively. We also appreciate the help of C. M. T. Molenaar and A. E. M. Jansen in the construction of the cosmid clones. The ColEI fragment of E. coli 16S rRNA was a kind gift of Dr. R. Charldorp (State University, Leiden).

Baan RA, Charldorp R van, Leerdam E van, Knippenberg PH van, Bosch L, Rooy IFM De, Boom JH van (1976) FEBS Letters 71:351-355 Bedbrook JR, Kolodner R, Bogorad L (1977) Cell 11:739-749 Bohnert I-IJ, Driesel AJ, Crouse EJ, Gordon K, Herrmann RG, Steinmetz A, Mubumbila M, Keller M, Burkhard G, Well JH (1979) FEBS Letters 103:52-56 Crouse EJ, Schmitt JM, Bohnert HJ, Gordon K, Driesel AJ, Herrmann RG (1978) In: Akoyunoglou G (ed) Chloroplast development. Elsevier/North-Holland Biochemical Press, Amsterdam New York Oxford, pp 565-580 Dagert M, Ehrlich SD (1979) Gene 6:23-28 Donis-Keller M, Maxam AM, Gilbert W (1977) Nucleic Acids Res 4:2527-2538 Dyer T, Bedbrook JR (1980) In: Leaver CJ (ed) Genome organization and expression in plants. Plenum Press, New York London, pp 305-311 Edwards K, K6ssel H (1981) Nucleic Acids Res 9:2853-2869 Fluhr R, Edelman M (1981) Mol Gen Genet 181:484-490 Groot GSP, Harten-Loosbroek N van (1981) Current Genetics 4:187-190 Grosveld FG, Dahl H-HM, Boer E de, Flavell RA (1981) Gene 13:227-237 Herrmann RG, Possingham JV (1980) In: Reinert J (ed) Chloroplasts. Springer, Berlin Heidelberg New York, pp 45-96 Jeffreys AJ, Flavell RA (1977) Cell 12:429-439 Koch W, Edwards K, K6ssel H (1981) Cell 25:203-213 Malnoe P, Rochaix JD (1978) Mol Gen Genet 166:269-275 Rochaix JD, Malnoe P (1978) Cell 15:661-670 Smith HO, Birnstiel ML (1976) Nucleic Acids Res 3:2387-2398 Southern EM (1975) Journ Mol Bio148:503-517 Takaiwa F, Sugiura M (1980) Mol Gen Genet 180:1-4 Takaiwa F, Sugiura M (1982) Nucleic Acids Res 10:2665 2676 Thompson JA, Hansman P, Knoth R, Link G, Falk H (1981) Current Genetics 4:25-28 Tomioka N, Shinozaki K, Sugiura M (1981) Mol Gen Genet 184: 359-363 Van Ee JH, Man in 't Veld WA, Planta RJ (1980) Plant Physiol 66:572-575 Van Ee JH, Vos YJ, Bohnert HJ, Planta RJ (1981) Plant Mol Biol 1:117-131 Van Ommen G-JB, Groot GSP, Borst P (1977) Mol Gen Genet

154:255-262 Veldman GM, Klootwijk J, Heerikhuizen H van, Planta RJ (1981) Nucleic Acids Res 9:4847-4862 Whitfield PR, Herrmann RG, Bottomley W (1978) Nucleic Acids Res 5:1741-1751 Wildeman AG (1980) J Biol Chem 255:11896-11900

Communicated by F. Kaudewitz Received December 2, 1982

Molecular cloning and characterization of the chloroplast ribosomal RNA genes from Spirodela oligorhiza.

The organization of the chloroplast ribosomal RNA genes in Spirodela oligorhiza has been determined. We have therefore characterized two cloned BamHI ...
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