Mol Gen Genet (1987) 208:377-383

© Springer-Verlag 1987

Splitting of the ribosomal RNA operon on chloroplast D N A from Chlorella elh'psoidea Takashi Yamada* and Miyuki Shimajii Department of Cell Biology, Mitusbishi-Kasei Institute of Life Sciences, 11 Minarniooya, Machida-shi, Tokyo 194, Japan

Summary. We have found in the chloroplast rRNA genes of the unicellular green alga Chlorella ellipsoidea an ex- tended rearrangement, an inversion of a 5.0 kb region which includes the genes for Ala tRNAuG o 23 S rRNA and 5 S rRNA, that results in the rRNA operon being split into two oper- ons: namely, operon 1, 16 S r R N A - t R N A ~ , v and operon 2, tRNAA~c--23 S r R N A - 5 S rRNA. $1 mapping using in vitro transcribed rRNAs showed that transcription of the 16 S rRNA gene initiated at 202 and 204 bp upstream from the 5' end of the 16 S rRNA gene, where the - 1 0 region and the - 3 5 region corresponded to TATCGT and T T G A C G , respectively. On the other hand, the genes for

Ala tRNAu~ c and 23 S rRNA were transcribed in the opposite direction from a point 593 bp upstream from the 5' end of the 23 S rRNA gene; the - 1 0 and - 3 5 regions were TATTCT and TTGACG, respectively. Sequences around these two putative promoter regions are very similar to each other; this suggests that the two promoters could po- tentially be transcribed by the same RNA polymerase at the same efficiency. In addition, the second promoter (operon 2) is flanked by inverted repeat sequences of 45 bp, next to which a 12 bp unique sequence is repeated tandemly, giving a possible secondary structure very similar to that of transposable elements. A mechanism through which one prokaryotic operon consisting of several genes splits into two or more smaller operons is discussed in the light of these observations.

Key words: Chlorella - Chloroplast DNA - Gene inversion - rRNA genes - Split operon

Introduction

The rRNA operons of chloroplasts (cp) are thought to be arranged like those of Escherichia coli and the blue-green alga Anacystis nidulans in the order 5', 16 S-- tR~TAne t~,TAA~, ~ S--5 S, 3' (Whitfeld and Bot- x , , L G A U - - ~ ± ~ x U G C - - ~

tomley 1983). In spite of some structural variations such as in the 4.5 S rRNA gene in higher plants (Whitfeld and Bottomley 1983), the 3 S and 7 S rRNA genes (Rochaix and Darlix 1982) and the intron in the 23 S rRNA gene (Allet and Rochaix 1979) in Chlamydomonas reinhardii, and the introns in the genes for tRNAGA uIle and tRNAuc cAla in

Offprint requests to ." T. Yamada

* Present address. University of California, Dept. of Biology, 405 Hilgard Ave., Los Angeles, CA 90024, USA

higher plants (Koch et al. 1981; Takaiwa and Sugiura 1982b), all cp rRNA operons so far studied are transcribed from the promoters located upstream from the 16 S rRNA gene (Whitfeld and Bottomley 1983; Strittmatter etal. 1985). The primary transcripts are then processed post- transcriptionally to mature rRNAs, which ensures that a set of rRNAs will be made in equal quantities.

The cp rRNA genes of the unicellular green alga Chlor- ella ellipsoidea are located in the inverted repeat region (22.5 kb) of the cp DNA (Yamada 1983), and there are consequently two sets per genome. Recently, peculiar fea- tures of the Chlorella cp rRNA genes have been revealed (Yamada and Shimaji 1986a): (i) the 16 S-23 S spacer re- gion is extraordinarily large, ca. 4.8 kb, almost twice as large as that of most higher plants; (ii) the Ala tRNAuac gene is absent from the 16S 23 S spacer region but the

I l e tRNAGA u gene is present intact; (iii) there are four open reading frames (ORFs) flanked by some repetitive se- quences in the spacer region; (iv) the 23 S rRNA gene is coded on the opposite strand in the reverse order; (v) the 4.5 S rRNA gene is not separated from the 23 S rRNA gene. In addition to these features, the 5 S rRNA gene has been found to be close to the 3' end of the 23 S rRNA gene in the same orientation (Yamada and Shimaji 1986b) and a new type of intron 243 bp long has been found in the 23 S rRNA gene (Yamada and Shimaji 1987).

Three major questions arise here: (i) through what mechanism did these rearrangements occur?; (ii) from which promoter are the genes for tRNAA~c and 23 S rRNA transcribed? and (iii) how is the synthesis of 16 S and 23 S rRNAs coordinated? In order to answer these questions we determined the nucleotide sequences of the upstream regions of both 16 S and 23 S rRNA genes and also the transcriptional initiation sites for these genes. It was found that the cp rRNA operon of Chlorella was split into two back-to-back operons.

Materials and methods

Strains. C. ellipsoidea C-87 was obtained from the algal culture collection of the Institute of Applied Microbiology, University of Tokyo. E. coli HBI01, JM101 and JM109 (Yanisch-Perron et al. 1985) were used for bacterial trans- formation and propagation of plasmids.

DNA and RNA. Chloroplast DNA from C. ellipsoidea was prepared as described previously (Yamada 1982). Plasmid DNAs were prepared according to Maniatis et al. (1982).

378

I

a

55 P1 I 6S rRNA ORF1 ORF2ORF3 tR RF4 3"~][

• I I "~ IBm , I-

2 3 S r R N A

L--

b

P1 1 ~ S rRNA

* I I

tRNAIletRNA ALa ORF1 OI~20RF3 ~ 2 3 $ r R N .~ ~ ni

m o ~<

I 1 . 6 5 Io85 I 1.75 10.60 1111.15 I[ 2.15 II 0.1 02 015 015

P C C S 1 2 3

1.60

C

pCCEX302

PCCEI02 pCCSEI07 pCCElO5 pCCEI01

PCCEI03 PCCEIOO

t ~ N A Ser

II [

10.65 I 1.45 I

PCCE1B4 pCCSEIO0

Fig. 1. a Map of the rRNA genes of Chlorella ellipsoidea chloroplast (cp) DNA. Orientations of transcription are shown by arrows. Positions of rRNA genes and open reading frames (ORFs) are indicated by boxes, b A possible organization of the Chlorella rRNA genes of the normal type (Whitfeld and Bottomley 1983), before the inversional event. Inversion occurred in the region indicated by the thick arrow. * 1, 2 indicate the promoter regions. <~ indicates repetitive sequences found in this region (Yamada and Shim@ 1986a). e Physical map and clones covering this region. Sizes are shown in kb

._.=_.=_ ._ ~ ._ ~g ~

10.151 I II 03 10.2110-2310-2 110.15[ il 0.05 0.05 0.02 0.02 0.050.02

2 3 S rRNA]

i

x,,~:~:: ~ ,q:=='-"- v.~:z::~ xv~:z: "7-

110.11 0.76 1 0.02 10.2.5 [llo.lil[ 0.5 10.,I 0.01 001001 0050£15

I 1 65 rRNA

] b ~

Fig. 2a, b. Sequencing strategies. A J.45 kb insert of pCCSEJ06 was used for the 23 S rDNA leader sequence (a) and a 2.5 kb insert of pCCEX302 was used for the 16 S rDNA leader sequence (b). Sizes are shown in kb

A gene l ibrary of C. ellipsoidea cp D N A was constructed by l igation of SstI-digested cp D N A fragments and SstI- digested pUC13 (Yamada et al. 1986; Y a m a d a and Shimaji 1986a) and t ransformed into E. eoli HB101 and JM109. Subclonings were carried out using the same vector and

hosts (Yamada and Shimaji 1986a). Ribosomal RNAs were prepared as described previously (Yamada 1983).

Sequencing o f DNA fragments. Restrict ion fragments con- taining the Chlorella cp r D N A were cloned into M13 m p l 8 and 19 (Horrander et al. 1983) in both directions. Single- s t randed D N A was sequenced using the chain terminat ion procedure (Sanger et al. 1977), using ~[32P]dCTP (800 Ci/ mmol, Amersham) and a sequencing kit (Takara Shuzo Co.). Both D N A strands were sequenced at least twice, and overlaps were obtained at each restriction site.

In vitro RN A synthesis. In order to obta in sufficient amounts of r R N A precursors for $1 mapping, in vitro tran- scription with E. coli R N A polymerase (Pharmacia) was carried out according to Str i t tmat ter et al. (1985): Fou r micrograms of D N A template (plasmid D N A s of pCCEX302 and of pCCSE106 for 16 S and 23 S r R N A regions, respectively) were t ranscribed with 1.2 units E. coli R N A polymerase in a 100 pl reaction mixture containing 40 m M Tris-HC1, pH 7.9, 10 m M MgCI2, 0.1 m M EDTA, 0.1 m M DTT, 0.8 m M potass ium phosphate, 80 m M KC1 and 0.2 m M each of NTPs. After 30 min incubat ion at 37 ° C, the D N A template was degraded by addi t ion o f RNase-free DNase (Worthington) to a final concentrat ion of 20 units/ml. After further incubat ion for 30 min at 37 ° C, the react ion was s topped by addi t ion of 12 pl 10% SDS and 12 pl 3 M sodium acetate, p H 4.8. The resulting solu- t ion was extracted twice with phenol /chloroform and finally R N A was precipi tated with ethanol. The total R N A from one in vitro t ranscript ion assay was used for hybr idizat ion in SJ mapping.

Fig. 3a--e. Nucleotide sequence of the upstream regions of the 23 S (a) and the 16 S rRNA genes (b), showing the strand corresponding to the transcribed RNA. Coding regions are boxed and compared with those of tobacco (N.t.) 16 S (Tohdoh and Sugiura 1982) and 23 S rRNA genes (Takaiwa and Sugiura 1982a) and of Escherichia coli (E.c.) 16 S (Brosius et al. 1978) and 23 S rRNA genes (Brosius et al. 1980), showing strong similarity to each other. Pribnow box ( - 1 0 region) and - 3 5 region are also boxed. Vertical thick arrows indicate the transcriptional initiation sites determined by S1 mapping. Horizontal arrows indicate tandem and inverted repeat sequences. ~- and a-elements found repetitively in the 16 S 23 S spacer region (Yarnada and Shimaji 1986a) are also indicated, c Both promoter sequences (16 S and 23 S rRNA genes) are aligned and compared with each other

Sstl t RNASe~©cI" Sinai GAGC1~CGATTGCTAATCGAG1~TACAGCTCCCTGTACCGAGGGTTCGAATCCCTCTCACTCCG~ACTTGTAACCC~GGGAGCCTAAGTATAAATCACACTC

TCCATTGTTTGAATACAAAGTATTCAAAATGTGAAAATATTTATACAAAGTTGTGTGCTTCGCCCTTTGTACCAAAGGGTAAAGACACAAAGCCTAGGTT

Accl Hincll CCTAACTATTGTTG~CAAGGG~jATA~GAACAAAGTTCGTCjAA~TTACTAC~AA~G~AGAAGACTTTT~TT~TGCGCTGCGCTACGTTGGAGTTTAGGcA

100

200

300

ACAA T A T AAAGTTTT AACAAGCT ACGAGCCCCCTT GCCA TT GCTTTCT AAGTTT T GCA A A GCA C A..A_A C_T T A_GA A A.~A CT T GGT A T AAA T AAAAAC _~_CTTT~

Hindlll TGTTTTTATTTATACTTAGCAAGGGCAATGGGGACTACGGGTTAGCA~AGCTTCGC~TGCTCACcAAGGGGTATGCGAGT~AGTAATTTATTTATAAATTA

400

500

CTTACTCGCCAACCTTACTACCAACAAAGTTGGTAGTTTAGGCAACCAATACTCCAAGGTTACCAAACGAAGTTTGGTAACCAAGGTCTTCCACGAAGTT 600

-3 5 -1 0 ] Dral Sma~ 0 AAACAAAGTAGAATTAGAGAGAGGCGGACTGGTi~-G~C~TAAATAAGCT~TGTAGG~F~C~GAA~GGAAGATAAAAAa~AAAA~AATTTGAAACC~

-IF

GGGGTCGAGAGAAGACTTCCCTAAAACTTTGTTCCTAGCGAATCTTGTTCAAGTTTTCCTAAGTACAAA~AAAAAAcGTTTTTG~TTTTTATT~ATACA~ 800

Hindlll ACT-TZGG-A-AA.GCCTAAATATTTTGCGTAAGCAGAAAAATTGGCTTCGTTTGTAATTTAGAAATCCTTTA~AGCTT~GCCCAT~G~TT~AC AACGTAGTT 9 0 0

• I000 TGTAAAACTTCATTATAAACGCAGTTTATAATTTA~G~TTATG~ATTCGcATAAACTTTAAGACTATTCCAAAGTTACTAAACGTAGTTTAGTCACCAAG

tRNA Ala , 1100 GTTTTACAAGCGTAG~TTGTAAAACGTC~TACGGACAAATT~T~ATTAAGCAA~GGAGTATAGCTCAGTTGGTAGAGCGCTG~CTTTGcAAG~CAGATGT

Hindlll Hindlll ~rrcGA~Tcc~cTT~qTTcc~ccAGcTTcTTAArTA~rA~c~G~T~Tc~A~crAAAr~TTATT~^~TA~AG~T~AAA~AGcT~TGcTT~A~ ~2oo

Hindlll 2 3 S r RN A Xba=

N.L. [rTCAAACGAGGAAAGGCTTACGGTGGATACCTAGGCACCC E,c, I~'TAAGCGACTAAGCGTACACGGTGGATGCCCTGGCAGTCA

C.e. AG.AGACGATGAAGGGCGTGGAAAC~AACGAAATGCTTCGAGGAGCTGGAAACAAGCTATGATTCGGAGATTCCCGAATAGGGCAACC N.t, AGAGACGAGGAAGGGCGTAGTAATCGACGAAATGCTTCGGGGAGTTGAAAATAAGCATAGATCCGGAGATTCCCGAATAGGGCAACC E.c. GAGGCGATGAAGGACGTGCTAATCTGCGATAAGCGTCGGTAAGGTGATATGAACCGTTATAAC~GGCGATTTCCGAATGGGGAAACC a

• 1 4 0 0

Bgl II Asu II

G~AATAAGT~cGATTCcAT~T~rA~G~cAAGCc ~TAA~T~G~CTG cc~TG~TTT~GATTATTTAc~ ~TT~TAGG~ Ac~GGcAA~TTG~A~GTT Hael

T~GTTAGGA~c~GTGGT~AGTcTTTc~ccTGTGGTTcTT~TcAAATTA~TcTc~TG~GA~A~TG~cGTTTAGG~TG~GAA~ATATTc~G~TG

2110

300

GAGAAGcTG~TcTGTTTTT~'7AGATTAATAcA~TcTTATGTT~TTcE~G~cTAAcGAAATTAAcTcT~TATTTTAT~TGG~TTT~GAATG~GA~AA Oral

AAAGAAAAT~GGTGAcTTT+~AAAATAAGTTAGTTTATAAArAArTA~T~G~TATAA~TGTTGAAmTG~AcTTAAA~A~ATAATGTTTAGGcT~A~GG Hincll Oral Dral Oral

- 3 5 - 1 0 !J~ T T ~ A A A T T T T A T A T T = f C A T T T T T T T A G T C C T C T A A A G A G A G ~ G A C T G G ~ A ~ A T A T A A A T T G T A G G T ~ T A A A A G ~ A T G A A T ~ G T T A

Sstl Bindlll Bindlll

Bglll Xbal c.~. TcAAATTTA~AAGTATAGcTTTT~AAcTT~AT~TT~TGcT~cccAGAA~ccAGGA~c~T~G~TT~GGcTAcGGT~GA~mAA~TmTT~ATGG N . t . ~ " m = " - - ITCTCATGG E ,c . ~-AAATTGA

1 6 S rRNA Sphl Hindlll C . e , AGAGTTTCGATC~TGGCTCAGGATGAAC~CTGGCGGCATC~CTT~ACA~ATGCAAGT~GAAC~AGGTTTGATTTTTTTAA~CTT N . L , AGAGTT-CGATCCTGGCTCAGGATGAACGCTGGCGGCATGCTTAACACATGCAAGTCGGACGGGAAGTGG . . . . . . . . . . . . TG ~.c. A~AGTTT-~ATCATGGCTCAGATT~AACGCTGGCGGCA~GCCTAACACATGCA~GTCGAAC(;GTG(]CAGGAA~AAGCTTGCTTC

400

500

600

?00

800

900

• I000

b

C

380

S1 mapping. $1 mapping with in vitro synthesized RNA, was carried out according to Berk and Sharp (1977) with minor modifications: 0.1 gg of DNA labeled at the 5' end with T4 polynucleotide kinase (Toyobo Biochemicals) and 7132p]ATP (3000 Ci/mmol, Amersham) was coprecipitated with in vitro synthesized RNA and dissolved in 50 gl of 80% deionized formamide containing 0.4 M NaC1, I mM EDTA and 40 mM Mops (3-[N-morpholino]propanesul- fonic acid, Sigma), pH 6.7. After denaturation at 95 ° C for 5 rain, the mixture was incubated at 50 ° C for 6 h for RNA/ DNA annealing. The hybrids were then rapidly chilled by mixing with 450 gl of ice-cold $1 buffer (30 mM sodium acetate, pH 4.5, 50 mM NaC1, 1 mM ZnSO4 and 5% gly- cerol); single-stranded regions were then digested with 1000 units of nuclease S1 (Pharmacia) for 1 h at 37 ° C. After phenol/chloroform extraction, the resulting fragments of the coding strand DNA were analyzed on a sequencing gel.

Results

Nucleotide sequence of the upstream regions of 16 S and 23 S rRNA genes

The clone containing the cp rRNA genes (pCCS123) se- lected from the gene library of Chlorella cp DNA (Yamada and Shimaji 1986a) was used for subsequent analyses. Re- striction mapping and hybridization with 32p-labeled cp rRNA probes allowed location of coding regions as shown in Fig, la . In order to determine the nucleotide sequence of the upstream regions of both 16 S and 23 S rRNA genes, subclones of pCCS123 were obtained as shown in Fig. 1 c. The nucleotide sequence of the 23 S upstream region was determined using pCCSEI06 by the strategy shown in Fig. 2a. Since pCCS123 did not contain enough of the 16 S upstream region, another clone covering this region (pCCEX302) was obtained by digestion of the cp DNA with EcoRI and XhoI and ligation to EcoRI- and SalI- digested pUC13 (Fig. 1 c). About 1.0 kb of pCCEX302 se- quence was determined for the 16 S upstream region by the strategy shown in Fig. 2b.

Figure 3 shows the 1387 bp sequence including 127 bp at the 5' end of the 23 S rRNA gene (a) and the 984 bp sequence including 92 bp at the 5' end of the 16 S rRNA gene (b). Ala The tRNAuG c gene lost from the 16 S-23 S spacer (Yamada and Shimaji 1986a) was found on the opposite strand, 135 bp upstream from the 5' end of the 23 S rRNA gene in the same orientation (Fig. 3 a, positions 1054-1126). Neither this gene nor the tRNA~,YAu gene in the 16 S-23 S spacer does not contain any intron (Yamada and Shimaji 1986a). Figure 4 shows the sequence and clover leaf struc- ture that can be deduced for Ala tRNAuGc based on the DNA sequence. This sequence shows 86.3%, 93.2%, 94.6%, 86.3% and 86.3% homology to that of the blue-green alga A. nidulans, the unicellular green alga C. reinhardii, EugIena gracilis, tobacco and maize, respectively (Sprinzl et al. 1985 ; Schneider and Rochaix 1986). Another gene coding for

S e r tRNAGc v was found 1198 bp upstream from the 5' end of the 23 S rRNA gene. On the other hand, the gene for tRN Aval known to be located in the upstream region of • X G A C

the 16 S rRNA gene of most higher plants was absent from the corresponding region of Chlorella (Fig. 2b). This is also the case for the cp rRNA operon of another unicellular green alga, C. reinhardii (Schneider et al. 1985).

G U A

U

G

G A U

OH

A

C

C

A

~ - c G - C

G - U

A - U

G - C

U - A

A - U G U A

U U C G C C

A l G l I I [

C U C G A G C G G I I I I UuC

G A G C C U

G G

U C - G A

U - A

G - C

C - G

C - G U A

U A

U C G

f R N T A A l a Fig. 4. Clover leaf structure of Chlorella chloroplast . . . . . uGc that is predicted from the DNA sequence

SI mapping of 5' termini of I6 S and23 S rRNAs

The transcriptional initiation sites for 16 S and 23 S rRNA genes were determined by in vitro transcription and S1 map- ping. For the 23 S rRNA gene, an S1 nuclease-resistant DNA fragment was obtained with the Y-terminally labeled coding strand of the 1.45 kb XbaI-XbaI fragment of pCCSE106 (including the 42 bp coding region of the 23 S rRNA gene and the whole tuV,TA Ala gene) annealed with ~ • U G C

transcripts formed by in vitro RNA synthesis. Schematic presentation of the procedure is shown in the lower part of Fig. 5a. A fragment of 636 bp appeared on the gel (Fig. 5 a); this meant that transcription initiated at 593 bp upstream from the 5' end of the 23 S rRNA gene and 386 bp upstream from the 5' end of the A~a tRNAuG c gene, that is both genes were cotranscribed (Fig. 3 a). In the same way, for the 16 S rRNA gene, Sl-resistant fragments were ob- tained with the Y-terminally labeled coding strand of the 426 bp HincII-SphI fragment of pCCEX302 (including the 44 bp coding region) annealed with in vitro transcripts (Fig. 5 b). Two fragments of 247 bp and 249 bp were de- tected; the initiation occurred at 202 and 204 bp upstream from the 16 S rRNA gene. The transcriptional initiation points for the genes for 16 S rRNA and tRNAA~c-23 S rRNA are indicated by arrows in Fig. 3 a, b.

Discussion

Bidirectional transcription of the rRNA genes

Since our preliminary $1 mapping experiments using rRNAs isolated from Chlorella chloroplasts gave only faint signals for rRNA precursors, we decided to use rRNA pre- cursors synthesized in vitro with E. coli RNA polymerase in order to determine putative promoter regions. Strittmat- ter et al. (1985) have pointed out a potential problem in using a heterologous system for in vitro transcription of chloroplast rRNA genes from higher plants where multiple promoter sequences are involved. However, $1 mapping with in vitro rRNA precursors in this study gave unique transcriptional initiation sites for the 16 S rRNA gene and for the t R N A ~ c and 23 S rRNA genes as shown in Fig. 3 ;

a

Xb a I

23s'D

1 , 4 5 kbp

636

b~

Xbal Hincll

0 ' PUC

!!ii~i, "

$Ph I

6s

44,bp 4 2 6 b p

249 247

Fig. 5 a, 6. Determination of the transcriptional initiation sites by $1 mapping, a A 636 bp St-resistant fragment was obtained for the 23 S rRNA region. 6 Two bands (247 bp and 249 bp) were obtained for the 16 S rRNA region. The sequence ladders are given as size markers. Schematic presentation of the procedure is shown in the lower part. Experimental details are described in the text

381

those sites exactly corresponded to the faint signals found with homologous rRNAs. Before the transcriptional initia- tion point for the 23 S r R N A gene determined by S1 map- ping, there are a 'Pr ibnow box ' (or - 1 0 region) TATTCT sequence (Fig. 3a, positions 658-663) and a - 3 5 region T T G A C G sequence (Fig. 3a, positions 634-639). For the 16 S r R N A gene, the - 1 0 region and - 3 5 region corre- spond to the sequences T A T C G T (Fig. 3b, positions 676-681) and T T G A C G (Fig. 3b, positions 652-657), re- spectively. Sequences around these promoter regions (16 S and 23 S) are very similar not only to each other as aligned in Fig. 3c but also to those of higher plants (Strittmatter

et al. 1985). There is no other promoter-like sequence at least 1.0 kb upstream of either the 16 S or the 23 S r R N A gene as shown in Fig. 3 and the val tRNAcAc gene is absent from the 16 S upstream region. Therefore, it is reasonable to assume that the two promoters are transcribed by the same ribosomal R N A polymerase at the same efficiency, even in the opposite direction as shown in Fig. 1 a. This ensures that a set of rRNAs will be made in equal quantities as they are when derived from the same primary transcript. In order to confirm this, it would be interesting to make an in vitro transcription system using the homologous R N A polymerase isolated from Chlorella chloroplasts.

382

A A A C

CG G .TG A

G.T T A-T T T A-T C-G

C C A-T G-C C-G A-T A-T 'T-A A-T T-A A-T C-G A-T A-T. A-T T-A T-A

A C A-T T-A A-T T-A A-T T-A A-T AAAGCACAAACTTAGAAAA~ T.TA A T-A . . . . . . . . :-'TT r.T- A r ~ GA T-A ".~6-CTTAG~AAGG ~ T-A "" I I I I I 1 . . 321 bp I ~ - 3 5 T-A TTATAAATCCGAAAGGATTCAAAC- GAATCcTTTT G _/ T-A "A-T A A ~ll, -lO

GT-A T-A C ~ G-C A C T.G T T-A

T T T-A Tc_GTT T T-A A-T A-T

CA_ T T-A A-T T-A A-T I-A A-T T-A A-T T-A T-A T-A A-T G-C A-T T.G A-T T T T-A TT A-T T-A

AAAGCACAAACTTAGAAAACTTGG- CAAACTTAGGAAAGC CTAAATATT

Fig. 6. Possible secondary structures for the transposon-like 464 bp sequence that includes the second promoter (P2) of the Chlorella chloroplast rRNA operon (positions 358-821, Fig. 3a). Arrow indicates a 12 bp tandem repeat sequence. Dotted line indicates a 14 bp sequence that is similar to the repetitive sequence (a-element) found in the 16 S-23 S rRNA spacer region (Yamada and Shim@ 1986a)

tRNAvoc Inversion of genes for 23 S rRNA and Ala

We have found here that the rRNA operon of Chlorella chloroplasts is split into two operons: operon 1, 16 S r R N A - tRNAn~u and operon 2, ~.,~*PT"TA Ala , ucc -- 23 S r R N A - 5 S rRNA as shown in Fig. la . For comparison, an ar- rangement of rRNA genes typical of those of higher plant chloroplasts is shown in Fig. 1 b. The rearrangement found in the cp rRNA genes of C. ellipsoidea must have occurred through an inversion, one endpoint of which was confined

• • Ile tRNAAI. within the tRNAGA U - uoc spacer region, since in the t D N I A A l a ChloreIla 16 S-23 S spacer, the . . . . . . UGC gene is lost,

whereas the gene for t R N A ~ u is present intact• The other point of inversion must be confined to between the

Ala tRNAuG c gene and the promoter sequence. Thus, the origi- nal sequence of ca. 5.0kb containing the genes for

Ala tRNAuGc, 23 S rRNA, 5 S rRNA and ORF4 as shown in Fig. 1 b must have been inverted.

Mechanism of the inversion

This example of rDNA rearrangement in Chlorella cp DNA shows a mechanism through which one prokaryotic operon comprising several genes can split into two or more smaller operons, like those of eukaryotic operons. A prerequisite for this inversion to be viable is the presence of other ade- quate promoter sequences in the reverse orientation. In the case of Chlorella rRNA genes, the second promoter (P2) that closely resembles the primary one (P1) (Fig. 3 c) might

have been present by chance in a suitable position to induce the inversion. Otherwise, some promoter sequences might behave as mobile genetic elements and promote the rearran- gement of the cp DNA. Indeed, the promoter sequence upstream from the 23 S rRNA gene (P2) is flanked by in- verted repeat sequences (positions 381-415 and 763-798, Fig. 3 a) just like prokaryotic transposable elements (Iida et al. 1983) and yeast Ty elements (Roeder and Fink 1983). Two possible secondary structures for the 464 bp sequence around the P2 promoter (positions 358-821, Fig. 3a) are shown in Fig. 6. Furthermore, a 12 bp sequence AAACT- TAGA(G)AAA is repeated tandemly next to the inverted repeats (positions 365-376 and 799-810, Figs. 3a and 6); a part of this sequence (positions 368-381) resembles the s-elements found repeated in the 16 S-23 S spacer region (Yamada and Shimaji 1986a). It is worth noting that such an a-element is located between ORF4 and the tRNAn~u gene (Fig. 1 a), where one endpoint of the inversion exists. These structures strongly suggest that the unit including the promoter sequence and its flanking inverted repeats has a direct connection with the inversional event.

Acknowledgements. We thank Drs. M. Kageyama, A. Kikuchi, Y. Kikuchi and H. Mizutani for helpful criticism of the manuscript.

References

Al le t B, R o c h a i x J D (1979) S t r u c t u r e ana lys i s a t t h e e n d s o f the intervening DNA sequence in the chloroplast 23S ribosomal genes of C. reinhardii. Cell 18:55-60

383

Berk A J, Sharp PA (1977) Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hy- brids. Cell/2:721-732

Brosius J, Palmer ML, Kennedy PJ, Noller HF (1978) Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci USA 75:4801-4805

Brosius J, Dull T J, Noller HF (1980) Gene organization and prima- ry structure of a ribosomal RNA operon from Escherichia coli. Proc Natl Acad Sci USA 77:201-204

Horrander J, Kempe T, Messing J (1983) Construction of improved M13 vector using oligonucleotide-directed mutagenesis. Gene 26:101-106

Iida S, Meyer J, Arber W (1983) Prokaryotic IS elements. In: Shapiro JA (ed) Mobile genetic elements. Academic Press, New York, pp 159-221

Koch W, Edwards K, K6ssel H (1981) Sequencing of the 16S-23S spacer in a ribosomal RNA operon of Zea mays chloroplast DNA reveals two split tRNA genes. Cell 25:203-2~3

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 363-402

Rochaix JD, Darlix JL (1982) Composite structure of the chloro- plast 23S rRNA gene of Chlamydomonas reinhardii. J Mol Biol 159: 383-395

Roeder GS, Fink GR (1983) Transposable elements in yeast. In: Shapiro JA (ed) Mobile genetic elements. Academic Press, New York, pp 299-328

Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467

Schneider M, Rochaix JD (1986) Sequence organization of the chloroplast ribosomal spacer of Chlamydomonas reinhardii: un- interupted tRNA lie and tRNA "h genes and extensive secondary structure. Plant Mol Biol 6:265-270

Schneider M, Darlix JL, Erickson J, Rochaix JD (1985) Sequence organization of repetitive elements in lhe flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii. Nucleic Acids Res 23:8531-8541

Sprinzl M, Vorderwiilbecke T, Hartmann T (1985) Compilation of sequences of tRNA genes. Nucleic Acids Res 13 :r51-r104

Strittmatter G, Gozdzicka-Jozefiak A, K6ssel H (1985) Identifica-

tion of an rRNA operon promoter from Zea mays chloroplasts which excludes the proximal t ~'NTAv"I from the primary tran- script. EMBO J 4: 599-604

Takaiwa F, Sugiura M (1982a) The complete nucleotide sequence of a 23S rRNA gene from tobacco chloroplasts. Eur J Biochem 124:13-19

Takaiwa F, Sugiura M (1982b) Nucleotide sequence of the 16S- 23 S spacer region in an rRNA gene cluster from tobacco chlo- roplast DNA. Nucleic Acids Res 10: 2665-2676

Tobdob N, Sugiura M (1982) The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts. Gene 17:213-218

Whitfeld PR, Bottomley W (1983) Organization and structure of chloroplast genes. Annu Rev Plant Physiol 34:279-310

Yamada T (1982) Isolation and characterization of chloroplast DNA from Chlorella ellipsoidea. Plant Physiol 70: 92-96

Yamada T (1983) Characterization of inverted repeat sequences and ribosomal RNA genes of chloroplast DNA from Chlorella ellipsoidea. Curr Genet 7:481~t87

Yamada T, Shim@ M (1986 a) Peculiar feature of the organization of rRNA genes of the Chlorella chloroplast DNA. Nucleic Acids Res 14:3827 3839

Yamada T, Shimaji M (1986b) Nucleotide sequence of the 5S rRNA gene from the unicellular green alga Chlorella ellipsoidea. Nuclei Acids Res 14:9529

Yamada T, Shimaji M (1987) An intron in the 23S rRNA geue of the Chlorella chloroplasts: complete nucleotide sequence of the 23S rRNA gene. Curr Genet 11:347-352

Yamada T, Sbimaji M, Fukuda Y (1986) Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that pro- motes autonomous replication in yeast. Plant Mol Biol 6 : 245-252

Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp 18 and pUC 19 vectors. Gene 33:103-119

Communicated by K. Isono

Received December 8, 1986