Nucleic Acids Research, Vol. 18, No. 5 Ne) 1990 Oxford University Press 1115

The cyanelle genome of Cyanophora paradoxa, unlike the chloroplast genome, codes for the ribosomal L3 protein Jean-Luc Evrard, Clayton Johnson', Ines Janssen2, Wolfgang Loffelhardt2, Jacques-Henry Weil and Marcel Kuntz* Institut de Biologie Moleculaire des Plantes et Universite Louis Pasteur, 12 rue du General Zimmer, 67084 Strasbourg Cedex, France, 1Max-Planck-Institut fOr Molekulare Genetik, Abt. Wittmann, Ihnestrasse 73, 1000 Berlin 33, FRG and 2lnstitut fOr Aligemeine Biochemie, Universitat Wien, Wahringerstrasse 38, 1090 Wien, Austria Received December 20, 1989; Revised and Accepted January 30, 1990

ABSTRACT We describe a 1132 bp sequence of the cyanelle genome of Cyanophora paradoxa containing the rpI3 gene. This gene, which is not chloroplast encoded in plants, is the first of a long cyanelle ribosomal operon whose organization resembles that of the S10 operon of E. coli. We have shown that the rpI3 gene is transcribed in cyanelles as a 7500 nucleotide precursor and that the 5'-end of the mRNA starts approximately 90 nucleotides upstream from the initiation codon. However, no typical procaryotic promoter could be found for this gene. We have detected, using anti E. coli L3 antibodies, the cyanelle L3 protein in cyanelle extracts and in E. coli cells transformed with the cyanelle rpI3 gene.

INTRODUCTION Cyanophora paradoxa is a biflagellated protist containing photosynthetic organelles (cyanelles) resembling endosymbiotic cyanobacteria. Cyaneiles possess cyanobacterial features such as a lysozyme-sensitive peptidoglycan cell wall, an unstacked thylakoid membrane structure and phycobiliproteins and chlorophyll a, but not chlorophyll b. On the other hand, cyanelles have common characteristics with chloroplasts. The circular cyanelle genome has a size (about 130 kbp) similar to that of typical chloroplast genomes and is therefore much smaller than cyanobacterial genomes. The cyanelle genome encodes tRNAs [1, 2, 3, 4, D. Bryant pers. comm.], two sets of rRNA genes, located as in most chloroplasts, on large inverted-repeated sequences [5], and a number of protein genes which are also common to chloroplast genomes [6, 7, 8, D. Bryant pers. comm., Loffelhardt et al. unpublished results]. As in land plant chloroplasts, the cyanelle tRNALeuuAA gene is split by a class-I structured intron [4]. However class-Il structured introns, which are found in land plant and green algae chloroplasts, have not been identified in the cyanelle genome. The cyanelle genome also contains the gene coding for the small subunit of the *

To whom correspondence should be addressed

EMBL accession no. X17498

ribulose-1,5-bisphosphate carboxylase [9], which is nuclear encoded in higher plants or green algae. Other genes present in the cyanelle genome code for the subunits of the phycobiliproteins allophycocyanin [10, 11], phycocyanin [11, 12] and the large anchor phycobiliprotein LCM 100 [13], all of which do not exist in higher plants or green algae. We have previously described the organization of two ribosomal protein gene clusters on the cyanelle genome of Cyanophora paradoxa [ 14]. One of the two clusters contains the rpl3, rpl2, rpsl9 and rp122 genes [14]. The second gene cluster, which is located upstream of the first one and oriented divergently, contains the rp133 and rpsl8 genes. Of particular interest is the finding that, unlike the other above-mentioned ribosomal protein genes, the rpl3 gene is not present in the chloroplast genomes studied to date. Here we report the nucleotide sequence of the cyanelle rpl3 gene and of the intergenic region between the two ribosomal protein gene clusters. The expression of these two gene clusters has been studied by Northern blot analysis and SI nuclease mapping. In addition, immunological characterization by Western blot immunostaining of the protein L3 encoded by the cyanelle genome is presented.

MATERIAL AND METHODS Cyanophora paradoxa strain LB555 (UTEX) was used in these studies. Partial nucleotide sequence of the restriction fragment PstI-9 (5.1 kb) of cyanelle DNA from Cyanophora paradoxa has been reported previously [3, 14] and was shown to contain several tRNA and ribosomal protein genes. Nucleotide sequences were determined for both strands using the 'Sequenase' kit from United States Biochemical Corp. (USB) and a-[32P]dATP (400 Ci/mmol). Either the double strand sequencing technique [15] or the single strand sequencing technique after subcloning into M13 derived vectors were used. Oligodeoxyribonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer by A. Hoeft (Strasbourg).

1116 Nucleic Acids Research 1

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Figure 1. Sequence of the rpl3 gene and its 5' flanking region. Genes are in uppercase. Putative -35' and '- 10' boxes are indicated with arrows showing the direction of transcription, SI signals are indicated by vertical arrows, the Shine and Dalgarno sequence of the rpl3 gene is shadowed and the sequence complementary to the 28-mer oligodeoxyribonucleotide which served as ORF102 specific probe is underlined.

Cyanelles were isolated as described [6]. Cyanelle RNA was purified, glyoxylated, fractionated on 1.5% agarose gels and transferred onto Hybond-N (Amersham) membranes as already published [8]. The BRL 0.24-9.5 kb RNA ladder was used to estimate RNA sizes. Marker RNA bands were visualized on the blot by staining with methylene blue. Gene-specific probes were obtained by isolation of subfragments of the cloned cyanelle DNA fragment PstI-9 from preparative low-melting agarose gels and their purification on NACS columns (BRL). These gene-specific probes were radiolabelled using the 'random priming' method [16] in the presence of a-[32P]dATP (3000 Ci/mmol) and hybridized to the filter-immobilized RNAs at 37°C, in the presence of 50% formamide, as recommended by Amersham. Alternatively, radioactive gene-specific probes were obtained by labelling synthetic single strand oligodeoxyribonucleotides in the presence of 100 MCi of -y-[32P]ATP (3000 Ci/mmol) and T4-polynucleotide kinase (BRL). All radioactive compounds were purchased from Amersham. SI nuclease analysis was performed as described in Current Protocols in Molecular Biology [17] with the following modifications: 15 Ag of cyanelle RNA were used, the overnight hybridization with labelled DNA was done at 37°C and the S1 digestion was for one hour, using 75 units of enzyme.

Cyanelle protein extracts were produced by boiling purified cyanelles in loading buffer (0.08 M Tris-HCl, 0.1 M DTT, 4 M urea, 2% SDS, 10% glycerol) for two minutes. Proteins were separated by SDS-PAGE with a gradient of acrylamide ranging from 20 to 15% [18, 19] and then electroblotted onto nitrocellulose in Tris-glycine buffer (150 mM glycine, 20 mM Tris) for one hour at 65 volts at room temperature. The cyanelle rp13 gene was expressed in E. coli under the control of the lac promoter as follows: E. coli cells were grown in rich medium up to OD6Wjnm= 0.3 before addition of 1/2000 culture volume of IPTG (24 mg/mi) and then further grown for 2 hours; cells were then pelleted by centrifugation in an Eppendorf tube and proteins were extracted by boiling cells in loading buffer. Immunodetection of cyanelle ribosomal protein L3 was done by adsorbing rabbit E. coli L3 antiserum on Western blots of cyanelle and other control extracts separated by SDS-PAGE. The bound rabbit IgGs were detected with affinity-purified, peroxidase-conjugated goat anti-rabbit IgG (Dianova GmbH, Hamburg, F.R.G.). The immunostaining procedure used was that described by Towbin and collaborators [20] and Bartsch [21], with the following modifications: Tris-buffered saline was replaced with phosphate buffered saline (PBS- 10 mM NaH2PO4, pH 7.2,

Nucleic Acids Research 1117 Cyanelle

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RESULTS Nucleotide sequence of the cyanelie rpB3 gene and its 5' flanking region The ribosomal protein genes of interest here are organized in two gene clusters which are oriented divergently. One of these gene clusters contains the rp12, rpsl9 and rp122 genes [14] as well as a number of other ribosomal protein genes located downstream [22]. The nucleotide sequence of the 5'-end of this gene cluster reveals an open reading frame (Fig. 1). The 209 amino acid long deduced protein sequence of the ORF shows more than 44% homology to the E. coli L3 ribosomal protein (Fig. 2). The identical amino acids are distributed throughout the protein sequence and no particular well conserved domain could be identified. The proposed rp13 gene possesses a ShineDalgarno like sequence (AAAGGA) which is complementary to the 3' end of the cyanelle 16S RNA [2] and the initiation codon is GTG instead of ATG. The proposed cyanelle rpl3 gene is separated from the downstream rpl2 gene by 61 bp. The nucleotide sequence of the upstream region of rpl3 terminating at the gene cluster containing the rp133 and rpsl8 genes (see upper part of Fig. 3), contains an ORF terminating 124 bp upstream of the rpl3 gene (Fig. 1). This ORF does not show any similarity to any known proteins and possesses an atypical codon usage. It was previously called ORF93 [14], however, since nucleotide sequence has revealed that it can potentially encode a 102 amino acid long polypeptide, we now propose to call it ORF 102.

Transcription of the rp13 and rp133/rpsl8 operons Hybridization of specific probes to Northern blots has shown that the cyanelle rpl3 gene, as well as the other previously published ribosomal protein genes, are transcribed. In Fig. 3, the rp13 or rp12 probes show a single messenger RNA of about 7500 nucleotides (nt.). No hybridization signal is obtained with an ORF102 probe (a 28-mer oligodeoxyribonucleotide, see Fig. 3), indicating that this ORF is not transcribed in cyanelles (or very weakly). Thus, the 7500 nt. transcript begins shortly before the rp13 coding sequence. S 1 nuclease mapping shows four possible 5'-ends for the 7500 nt. transcript (not shown), either 88, 89, 90 or 91 nt. before the GTG

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initiation codon (see Fig. 1). A prokaryotic-type '-10' promoter element is found 7-10 nt. (depending on which end is considered) upstream of the four 5'-ends of the 7500 nt. transcript, but no '-35' promoter element is present. Yet, a sequence (TTGCAA) resembling a '-35' element is found 84 bp upstream of the start codon. An rp133IrpsJ8 probe shows two different transcripts. One transcript is about 700 nt., long enough to represent a dicistronic mRNA for rp133 and rps18, and a second transcript of about 1100 nt. (Fig.3). SI nuclease mapping experiments show a single 5'-end for these transcripts 328 nt. before the initiation codon of rp133 (not shown). However, since the 1100 nt. transcript is much weaker than the 700 nt. transcript, the 5'-end of the 1100 nt. transcript may be undetectable in these experiments. If the end of the transcript(s) occurs after a long inverted repeated sequence previously described [3, 14], the size of the major RNA would be 845 nucleotides, which is compatible with the size estimated on gels (about 700 nt.). If the 700 nt. and the 1100 nt. transcripts share the same 3'-end, then initiation of transcription of the larger transcript takes place in the rpl3 coding region. However, since the 1100 nt. transcript was not detected with the rpl3 probe, it is likely that the difference in size between

1118 Nucleic Acids Research

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the two transcripts is due to a 3'-extension of the l100 nt. transcript. A '-10' like element (TAAATT) is located 7 bp upstream of the 5'-end of the rpl331rps]8 transcript(s) (see the sequence of the complementary strand in Fig. 1, position 264). This '-10' like element is separated from a '-35' like element (TTGCAA) located 19 bp upstream of the 5'-end of this same operon (position 289 in Fig. 1). The complementary sequence of this '-35' like sequence (which also reads TTGCAA) is also the abovementioned '-35' like element located upstream of the 5'-end of the rpl3 transcript.

Protein product of the cyanelle rp13 gene in cyanelles Individual cyanelle ribosomal proteins have not been identified so far. Therefore, in order to determine if an L3 protein is present in cyanelles, a Western blot of total cyanelle protein extract, separated by SDS-PAGE, was adsorbed with E. coli L3 antiserum (Fig.5). Antisera to E. coli L3 has previously been shown to cross-react with a homologous protein in spinach chloroplast ribosomes [21]. Cyanelle extract (Fig.5) showed a distinct crossreacting band. The molecular mass of this protein (determined from a plot against Mr standards) is approximately 22,500 daltons, which agrees with that predicted from the nucleotide sequence (22,462 daltons). An extract of E. coli transformed with the cyanelle rpl3 coding region under the control of the lac promoter also showed a cross-reacting band at the same position as the cyanelle band (as expected), while this band was absent in the control extract (E. coli+vector). The origin of the additional cross-reacting bands in the control extract, e.g. protein degradation, has not been determined. These results show that an L3 protein is present in cyanelles and that the rpl3 gene isolated and sequenced here is expressed in the cyanelles.

DISCUSSION We report here the first nucleotide sequence of an rp13 gene from a photosynthetic organism, namely C. paradoxa. The rpl3 gene is present on the cyanelle genome of C. paradoxa and the product of the gene, the ribosomal protein L3, was detected by Western blot analysis. The rp13 gene is located at the 5'-end of a long gene cluster and co-transcribed with the other ribosomal protein genes present downstream. Such a ribosomal protein operon also exists in the chloroplast genome of land plants and Euglena gracilis. However, the cyanelle ribosomal protein operon

Figure 5. Immunostaining of Western blots with E. coli L3 antiserum. The SDS gels blotted contained extracts of E. coli transformed with pUCl9 plasmid (E. coli + vector), pUC l9 plasmid with cyanelle L3 insert (E. coli + L3 insert), and a total cyanelle extract. The bands corresponding to E. (oli L3, cyanelle L3, and the positions of Mr markers used are indicated. The presence of one of these bands, as well as E. coli L3, in the cyanelle lane is due to overflow from an adjacent E. coli extract lane. The E. coli L3 band in these experiments apparently migrated with a higher Mr (-22,900) than the known Mr of 22,232 [36].

possesses some unique features. First, in contrast to cyanelles, the rpl3 gene has not been found in the chloroplast genomes studied so far and is most likely nuclear-encoded in these organisms. However, no nuclear rpl3 gene has presently been identified. Second, the chloroplast ribosomal protein operon contains an rp123 gene at the 5'-end, not found in the cyanelle rpl3 operon. However, it should be noted that rp123 is not always functional in chloroplasts [23]. Third, the cyanelle rpl3 operon is transcribed into a unique long messenger RNA, whereas in tobacco chloroplasts multiple smaller mRNAs have been detected [24]. On the other hand, in spinach chloroplasts predominant large transcripts could be detected [25]. In Euglena gracilis both large transcripts and processing products have been detected [26]. The cyanelle rpl3 and the chloroplast rp123 operons show a gene arrangement similar but not identical to that of the Sl0 and spc ribosomal operons of E. coli [24, 27, 14]. In E. coli more genes are present and it is likely that the corresponding genes from cyanelles and chloroplasts have been transferred to the nucleus of the eucaryotic cells [28, 29, 30, P. M. Smooker (L35), C. Johnson (PSrpl) and A. R. Subramanian-in progress]. Since regulation of the E. coli SlO operon occurs in part at the transcriptional level [31], we have attempted to define the promoter region of the cyanelle rpl3 operon as well as that of the rp1331rpsl8 operon which is located upstream of the rpl3 operon and transcribed divergently. S1 nuclease mapping experiments indicate that a 130 bp region (position 260 to 390 in Fig. 1) between these operons contains two divergent promoters. Such close promoters have been shown to interact with each other in the maize chloroplast genome in the case of the rbcL and atpB genes [32] or to be a target for regulatory proteins in E. coli when the genes are involved in an identical function [33]. Since in E. coli the 5 '-untranslated leader of the Sl0 transcript is involved in the expression of the operon and its regulation [3 1 ], we have compared the untranslated leader of the cyanelle rpl3

Nucleic Acids Research 1119 transcript to that of the E. coliS50 transcript. The 5'-end of the cyanelle rp13 transcript can be folded into a highly structured stem and loop structure which maintains an AAUCGUA sequence within theeloop (Fig. 4). This sequence is also found in the HE loop of the leader of the E. coli S1O operon [34], although the secondary structure of the latter leader sequence appears more complex. Moreover, this sequence exists also in the E. coli 23S rRNA (near position 1600) but not in the other E. coli rRNAs. Unfortunately, the complete sequence of the cyanelle 23S rRNA is not available, but this sequence does not exist in the published chloroplast 23S RNAs sequences. Shen et al. [34] proposed that this sequence has a stabilizing effect on the HE stem and loop structure. It is not known whether this stem and loop structure also plays a role in the regulation of the rpl3 operon in cyanelles. The cyanelle rp13 coding sequence is preceded by a typical Shine-Dalgarno sequence, whereas the three ribosomal protein genes located immediately downstream [14] possess a shortened version of this sequence, usually only a few As. This may be due to the fact that rp13 is the first gene of a long operon and it has been suggested that in highly regulated operons in E. coli the ribosomes can pass through the spacers between adjacent cistrons [35]. Therefore, one might speculate that in cyanelles also, ribosomes can pass in some cases through the spacers of polycistronic mRNAs. Alternatively, since the initiation codon of rp13 is GUG instead of AUG, it is possible that a better Shine and Dalgarno sequence is required to interact with the 16S rRNA and to allow initiation at the GUG codon.

ACKNOWLEDGEMENTS Part of this work was done in Dr. A. R. Subramanian's laboratory. We thank him for his interest in this work. We are grateful to Dr. R. Schantz for helpful suggestions.

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