The two genes for the small subunit of RuBP Carboxylase/oxygenase are closely linked in <i>Chlamydomonas reinhardtii</i>

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The two genes for the small subunit of RuBP Carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii

GOLDSCHMIDT-CLERMONT, Michel P.

Abstract

Ribulose bisphosphate carboxylase-oxygenase (Rubisco) is a key enzyme in the photosynthetic fixation of CO₂ by the chloroplast. The synthesis of the enzyme is an example of the cooperation between the chloroplast and the nucleocytoplasmic compartments, as it is assembled from subunits encoded in the two respective genomes. I have used a synthetic oligonucleotide probe to isolate the nuclear Rubisco small subunit genes (rbcS) directly from a genomic library of Chlamydomonas reinhardtii DNA. They constitute only a small family: there are two rbcS genes, and an additional related sequence, in the C. reinhardtii genome. All three are clustered within 11kb at a single locus, and should thus be particularly well suited for genetic manipulation. The pattern of expression of rbcS RNA is dependent on the growth conditions.

GOLDSCHMIDT-CLERMONT, Michel P. The two genes for the small subunit of RuBP

Carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii . Plant Molecular Biology , 1986, vol. 6, no. 1, p. 13-21

DOI : 10.1007/BF00021302

Available at:

http://archive-ouverte.unige.ch/unige:151544

Disclaimer: layout of this document may differ from the published version.

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The two genes for the small subunit of RuBP Carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii

Michel Goldschmidt-Clermont

Department of Molecular Biology, University of Geneva, Sciences II, 30 quai Ernest-Ansermet, CH 1211 Gen~ve 4, Switzerland; Telephone: 41.22. 21.93.55 extension 2188

Keywords: rbcS, ribulose bisphosphate carboxylase-oxygenase, gene organization, light regulation, synthetic oligonucleotide

Summary

Ribulose bisphosphate carboxylase-oxygenase (Rubisco) is a key enzyme in the photosynthetic fixation of CO2 by the chloroplast. The synthesis of the enzyme is an example of the cooperation between the chloroplast and the nucleocytoplasmic compartments, as it is assembled from subunits encoded in the two respective genomes. I have used a synthetic oligonucleotide probe to isolate the nuclear Rubisco small subunit genes (rbcS) directly from a genomic library of Chlamydomonas reinhardtii DNA. They constitute only a small family: there are two rbcS genes, and an additional related sequence, in the C. reinhardtii genome. All three are clustered within llkb at a single locus, and should thus be particularly well suited for genetic manipulation. The pattern of expression of rbcS RNA is dependent on the growth conditions.

Introduction

Although the chloroplast is equipped with a fully competent genetic apparatus which produces many of the organelle's macromolecules, numerous polypeptides are encoded in the nucleus, translated as precursors in the cytoplasm, and imported into the chloroplast (reviewed in ref. 13). Thus two genetic systems cooperate in the assembly of the photosynthetic machinery. We have chosen to study how this coordination is achieved and regulated in Chlamydomonas reinhardtii. This unicellular alga is a facultative phototroph which can also be grown on a reduced carbon source (acetate) in the dark.

Heterotrophic growth allows the isolation of many photosynthetic mutations which have been mapped to the nuclear and the chloroplast genomes (15, 17, 29). Such mutations in one or the other compartment introduce perturbations that should aid in understanding the mechanisms that are involved in the coordinate expression of the two genetic systems.

The components of the photosynthetic appara-

tus, and in particular the membrane bound com- plexes, are highly integrated multimolecular systems. Mutations that affect one subunit often have pleiotropic effects on many others. The situation is simpler in the case of ribulose bisphosphate carboxylase-oxygenase (Rubisco). This soluble chloroplast enzyme is composed of only two kinds of subunits (reviewed in refs. 21, 24). The large subunit (LS) is coded and synthetized in the chloroplast, while the small subunit (SS) is coded and translated as a precursor (pSS) in the nucleocytoplasmic compartment and imported into the chloroplast. The enzyme is of interest because of its involvement in the initial step of photosynthetic CO 2 fixation. The holoenzyme, composed of eight LS and eight SS, is quite large (MW around 500000) and is also very abundant, which might compensate for its rather slow enzyme kinet- ics. In addition to CO2 fixation, Rubisco curiously also catalyzes the oxygenation of its substrate ribulose bisphosphate by 02, a reaction which initiates photo-respiration and results in a net loss of CO2.

This reaction is believed to reduce the overall pho-

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tosynthetic yield of many plants and there is therefore much interest in understanding and possibly modifying the properties of the enzyme.

The chloroplast LS gene (rbcL) of C. reinhardtii has been thoroughly characterized in the wild type strain (10, 22) and in several rbcL- mutants lacking Rubisco (11, 36, 37). I report here the initital characterization of the other components of this dual genetic system, the nuclear SS genes (rbcS), which were isolated directly from a C. reinhardtii genomic library using a synthetic oligodeoxynucleotide hybridization probe.

Materials and methods Strains and media

Chlamydomonas reinhardtii wild type 2137 A(+) was obtained from Dr R. S. Spreitzer and cwl5(-) from Dr. J.-D. Rochaix. Bacteriophage lambda EMBL3 and the E. coli host NM534 (rk- (P2 cox3)) were provided by Dr. N. Murray. C. rein- hardtii was grown in Tris-Acetate Phosphate (TAP) medium (16) or in the same medium lacking acetate and adjusted to pH 7.0 with HCI (Minimal medium).

DNA preparation and analysis

Phage were grown and phage DNA purified using standard procedures (9). DNA from C. rein- hardtii cwl5(-) was prepared as described (27) and purified by two successive CsCI density gradient centrifugations in the Ti50 rotor (Beckman) at 35000 rpm for 60 h. The first was in 4.7 M CsC1, 80/~g/ml ethidium bromide, 10 mM Tris pH 7.5, 1 mM EDTA and the second in 5.6 M CsC1, 10 mM Tris pH 7.5, 1 mM EDTA. The main band (enriched for nuclear DNA) was used to construct the EMBL3 library. The second gradient centrifugation was omitted for the DNA used in the restriction digest experiments,

DNA restriction fragments were separated on 0.8% agarose gels containing 0.5 #g/ml ethidium bromide and transferred to nitrocellulose (Schleicher and Schuell BA85) (44). HindIII and EcoRI cut lambda DNA was used as a molecular weight standard (18).

Genomic library in lambda EMBL3

20/~g of C. reinhardtii cwl5(-) DNA were partially digested with 12 units of MboI in a volume of 500/~1. Aliquots (165/~1) were removed after 15, 30 and 60 min and added to a tube containing 20 #1 of 0.4 M EDTA on ice. The pooled aliquots were heated to 68 °C for 5 min and loaded on a 10 to 40%

sucrose gradient in 1 M NaCI, 20 mM Tris pH 7.5, 10 mM EDTA and centrifuged in the SW 40 rotor (Beckman) at 30000 rpm for 18 h (23). Fractions (500 #I) were collected from the bottom of the tube and aliquots (100/~1) were run on a 0.4% agarose gel calibrated with undigested and HindlII digested lambda DNA. Fraction 9 contained approximately 0.3/zg of DNA fragments larger than 15kb with a mean size around 20kb. This DNA was mixed with 3/~g of EcoRI and BamHI digested lambda EMBL3, ethanol precipitated and washed, dried briefly, resuspended in 8/~I 10 mM Tris 7.5, 1 mM EDTA and heated to 70°C for 3 min. The cohe- sive ends of lambda were annealed by adding 1/zl of 0.25 M NaC1, 0.1 M MgCI2 and heating to 50°C for 15 min. 1 #1 of 0.1 M DTT, 0.01 M ATP was then added and the DNAs were ligated with 10 units of T4 ligase at 12 °C for 16 h. Two 3 #1 aliquots of the DNA were packaged-in vitro (23) yielding approximately 2×105 recombinants on NM534 (20% of total phage). These were pooled in five separate fractions that constituted the genomic library (available on request). Phage plaques grown on E. coli C600 were screened by making amplified replicas on Millipore HATF filters (Woo, 1979). Af- ter baking, the filters were treated with 50/~l/ml Proteinase K in 0.1% SDS, 10 mM Tris, 1 mM EDTA with gentle rocking and manual scrubbing with gloved fingers.

Labelling and hybridization of nucleic acids The synthetic oligonucleotide mixture was kindly provided by A. Chollais (Biogen SA, Geneva). It was labelled with ot-32P ATP (Amersham, 5000 Ci/Mmole) using T4 poly-nucleotide kinase, purified by loading on DE 52 (Whatman), washing with 0.15 M NaC1, 20 mM Tris pH 7.5, 2 mM EDTA, 0.1%0 SDS and eluting by increasing the NaC1 to 1 M (45). The filters were pretreated at 50°C and hybridized at 37°C in 6xSSC, 1 mM EDTA, 0.1%0 SDS, 25/~g/ml E. coli DNA

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oligonucleotides (20- 30 bases, prepared by limited DNaseI treatment), 5 ×BFP (SSC is 0.15 M NaC1, 0.015 M Na citrate, pH 7.5; BFP is 0.02% each of Ficoll 400, Bovine Serum Albumin and Polivinyl- pyrrolidone). The filters were washed in 6x SSC, 0.1% SDS at 37 °C (39).

Total RNA was reverse-transcribed to produce 32p-labelled cDNA: a 25/zl reaction containing 50/zg RNA, 40 ng oligodT, 50 mM Tris pH 8, 10 mM MgCI2, 50mM KC1, 20mM Dithri- othreitol, 0.3 mM each of dATP, dGTP, TTP, 2 #m dCTP, 40/~Ci ofl2pdCTP, 1 unit of RNAse in- hibitior (Biotec) and 10 units of AMV reverse tran- scriptase (Boehringer) was incubated for 1 h at 42 °C. The reaction was terminated by adjusting to 0.1% SDS, 10 mM EDTA, 0.1 mg/ml carrier salmon DNA, 200 #g/ml proteinase K in a total volume of 50/zl and incubation at 42°C for 15 min.

The RNA template was hydrolyzed with 0.25 M NaOH at 42°C for 30 min and after neutraliz- ing with 0.25 M HC1, the cDNA was recovered by gel filtration on Sephadex G50 (Pharmacia).

Plasmid DNA and restriction fragments were prepared and labelled by nick-translation following standard procedures (9).

Nitrocellulose filters with bound DNA or RNA were pretreated for 4 - 6 h at 42°C in 5×SSPE, 50% formamide, 5×BFP, 0.2 mg/ml denatured salmon DNA (1 ×SSPE is 0.18 M NaC1, 10 mM Na phosphate, 1 mM EDTA pH 7.0), hybridized with 0.2 to 0.5×106 cpm/ml radioactive probe in the same buffer at 42 °C for 16-18 h and washed several times at 50°C in 0.5×SSPE, 0.25% SDS.

Nucleotide sequencing

The BamHI SalI 1.3kb fragment (from lambda 32) and 1.35kb fragment (from lambda 36) and the SalI 0.6kb fragment (from lambda 23) were purified by agarose gel electrophoresis and eluted on DEAE nitrocellulose (Schleicher and Schuell NA45). The fragments (approximately 0.5/zg) and the oligonucleotide mixture (40 ng) were annealed in 15 #1 of 3 mM Tris, pH 7.5, 0.3 mM EDTA by boil- ing for 5 min and cooling quickly on ice, and then aliquoted for the four dideoxynucleotide sequencing reactions (30).

RNA purification and analysis

C. reinhardtii 2137A (+) were grown to a density of 1-2×106 cells/ml (under 4000 lux from fluorescent lights when appropriate), chilled on ice, harvested and washed by centrifugation and lyzed by extensive vortexing in 6 M guanidium hydrochloride, 0.1 M sodium acetate pH 5 with acid- washed glass beads (450-500 micron diameter, Sigma). RNA was prepared by centrifugation through CsCI and ethanol precipitation from guanidium hydrochloride (7) and then from 0.1 M NaCI, 1 mM EDTA.

RNA samples were heated at 50°C f6r 15 min in 50% formamide, 6% formaldehyde, 10 mM Na phosphate pH 6.8, 5 mM Na acetate, 1 mM EDTA and separated by agarose gel electrophoresis in the same buffer lacking formamide (20) and blotted to nitrocellulose filters (41). DNA standards (HinfI cut pBr322 (40)) used to calibrate the gels were treated similarly but denatured at 65 °C for 15 min.

Results

Cloning strategy

The protein sequence of the mature SS of C.

reinhardtii begins with the residues Met Met Val Trp Thr... (31). Because there are only single codons for Met and Trp in the genetic code, the corresponding gene sequence can be predicted for fourteen nucleotide residues with ambiguity only in the third position of the codon for valine (Fig. la).

The complementary mixture of four fourteen-mer oligodeoxynucleotides, synthetized and kindly provided by A. Chollais (Biogen SA, Geneva) was used as a hybridization probe to isolate the rbcS genes.

A sequence of 13 defined bases is expected on a statistical basis to occur with a frequency of (1/4) 13 in a random sequence of four possible nucleotides, that is once very 0.7×108 bases. (Correction for the GC content of the probe (6/13) and of the C.

reinhardtii genome (65%) gives a value of one per 1.7 × 108 bases). The C. reinhardtii genome consists of approximately 1×108 bases. It was therefore theoretically possible to isolate rbcS genes directly from a genomic DNA library by screening with the oligonucleotide hybridization probe at high strin- gency. On a statistical basis however one might also

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expect to isolate spurious sequences with a similar probability.

Isolation of rbcS genes

A genomic library of C. reinhardtii DNA was constructed in the bacteriophage lambda vector EMBL3 (25). The library was prepared by partially digesting DNA with Mbo I, isolating large fragments and ligating them to BamHI digested vector DNA. After in vitro packaging into phage parti- cles, the recombinants were selected on an appropriate host and the library was screened using the 32p labelled mixture of oligonucleotides as hybridization .probe. Six independent positive iso- lates were arbitrarily chosen for further examina- tion.

Phage DNAs were digested with BamHI and Sail, and the resulting fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose filters. Fragments of three sizes were labelled by hybridization to the oligonucleotide:

0.6, 1.3 and 1.35kb (Fig. 2, panels a and c, lanes 3).

Five phages (clones 23, 31, 32, 33, 34) had a 1.3kb positive fragment and three of them (clones 31, 32, 33) also had a 0.6kb fragment (see Figure 2, panel a, lane 3 for phage 32). The five phages also shared other unlabelled restriction fragments of identical mobility, suggesting that they might form a group with overlapping cloned sequences. This was con- firmed by their restriction maps as analyzed with additional enzymes (Fig. 3).

In order to ascertain whether the sequences that hybridized to the oligonucleotide were indeed part of rbcS genes, and not spurious occurences of the oligonucleotide, the adjacent sequences were determined. The three BamHI Sail fragments were isolated on a preparative scale and partly sequenced by the dideoxynucleotide chain termination method (30) using the oligonucleotide mixture as a primer for elongation. The sequences upstream of the oligonucleotide in the 0.6kb and the 1.3kb fragments did encode the protein sequence of the transit peptide of the SS (Fig. lb).

s~

(5") (3")

M e t M e t V o l T r p ThP P~o . . . ATG ATG GTN TGG RCN CCN ( 3 " ) ITAC TAC CAN ACC TC~N GGN ( 5 " )

b

S¢II 0.6 (3") Bali BomHI 1o3 (3") pSS

SolI 0.6 SQII BamHI 1.3

pSS

CAC GCG GGG TAC CGG CGC GRC TTC GGG CGG G GGG TRC CGG C-C GRC TTC GGG CGG V o l R r g P r o M e t A l o A l a L e u L y s P r o A l o

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CAG TTC CGA CGG GGG CRC CGA CGG GGC CGA $ CAG TTC CGG CGG GGG CAC CGA CGG GGC CGA Vo1 L y s A I o A l e P r o V o l ~ l o A l o P r o ~ i o

31

SolI 0.6 GTC CGG (5")

S o l I 8omHI 1 . 3 GTC CGG TTG ( 5 " ) pSS G I o RIG RSn

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Fig. 1. (a) The oligonucleotide probe. The amino-terminal sequence of the mature small subunit of Rubisco (31) allows the prediction of the corresponding nucleotide sequence in the rbcS gene. Because of the degeneracy in the genetic code, some positions remain uncer- tain (N =A, G, C or T). The mixture of four tetradecamer oligodeoxynucleotides corresponding to the sequence that is boxed was used to isolate the rbcS genes.

(b) Identification of rbcS sequences. To establish whether cloned fragments did encode the SS, partial nucleotide sequences adjacent to the sites of oligonucleotide hybridization were determined. In two cases, the sequences shown (or strictly, their complementary strands) encode the sequence of the transit peptide of the Rubisco SS except at one position (residue 41) where glutamine is predicted in place of glutamic acid (31). The sequences in the 1.3kb BamHl SalI fragment and the 0.6kb SalI fragment differ by a silent substitu- tion (*).

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Fig. 2. Mapping of rbcS sequences in cloned DNA. Restriction fragments of X recombinants 32 (panels a and b) or 36 (panels c and d) were separated by agarose gel electrophoresis, transferred to a nitrocellulose filter and hybridized with the oligonucleotide probe (panels a and c, labelled 'oligo'). The probe was removed by denaturation and the same filter was hybridized with cDNA prepared by reverse transcription of total RNA (panels b and d, labelled 'cDNA'). Lanes 1: BamHI EcoRI; 2: BamHl, 3: BamHI Sail; 4: SalI; 5:

Sail HindlII; 6: Sail EcoRl.

However the sequence of the 1.35kb fragment from the sixth phage (clone 36) was unrelated, suggesting this phage did not contain a bona fide rbcS gene. This conclusion was also supported by the failure of 36 to hybridize with the 1.3kb fragment containing SS transit peptide sequences (not shown) or to the cDNA probe described in the next section (Fig. 2, panel d). Recombinant 36 probably

contains a random occurence of the oligonucleotide sequence; it was not studied any further.

Two closely linked rbcS genes

The restriction maps of the five remaining phages were derived. They form an overlapping set and span about 31kb (Fig. 3). Within this region,

7,EMBL3 recombinonts : _lkb

33 32 31

Map: ~ B SH H B SB B S~SfB R SSSHB

B S S S

Hybridization : oLigonucteotide(5') ~ 8 R

eDNA (3') ~ B~

i

~ ~, y,~

5' fragment ~, ~'

3' fragment ~ ~, s ~6 i R

rbcS sequences : - - ' - - ~ = 5'3' ~ ,

Fig. 3. Physical map of the rbcS locus. The restriction map was derived from the single and multiple digest patterns of five X recombinants containing overlapping fragments of the locus, as indicated by the lines at the top. (B: BamHl, H: HindlII, R: EcoRl, S: Sail).

The approximate location of the rbcS sequences was determined by identifying the cloned DNA restriction fragments that hybridized to: (i) the mixture of four oligonucleotides, a probe specific for the amino-terminal, 5' part of the genes ('oligonucleotide', see Fig. 2a);

(ii) to oligodT-primed cDNA reverse-transcribed from total RNA ('cDNA', see Figure 2b), a probe specific for the 3, part of the genes;

and (iii) to the 0.6kb Sail fragment and the 1.5kb Sail EcoRl fragments (black bars), respectively containing the 5' part and the 3' part of a rbcS gene. Two putative genes (arrows at the bottom of the fig.) and additional related sequences (bar) were thus identified.

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the oligonucleotide probe hybridizes to the 5' (amino terminal) part of rbcS genes in two segments (the 0.6kb Sail fragment and the 1.3kb BamHI SalI fragment). A 32p-labelled probe specific for the 3' part of genes coding for abundant polyadenylated RNA was prepared by oligodT-primed reverse transcription of total Chlamydomonas RNA. Within the cloned 31kb, this 3'-specific probe also hybridizes to two segments (the BamHI EcoRI 1.35 and 1.5kb fragments; Fig. 2, panel b, lane 1) that are in close proximity to the fragments that contain the amino-terminal SS sequences (Fig. 3). Two putative rbcS genes, spaced approximately 3 to 4kb apart in tandem (head to tail) orientation are thus identified.

Another region in the locus shares sequence homology with the fragments containing rbcS genes:

the 2.6kb BamHI fragment crosshybridizes to fragments that contain the 5' part (Sall 0.6kb) or the 3' part (Sail EcoRI 1.6kb) of the gene and flanking sequences (Fig. 3; data not shown but see Fig. 4, lane D). However this region does not hybridize with the synthetic oligonucleotide (Fig. 2, panel a;

note for example the absence of a 2.6kb BamHI band in lanes 1- 3). It only hybridizes to a very mi- nor extent with the 3' specific cDNA probe. This suggests that the 2.6kb Barn HI fragment contains only an incomplete rbcS pseudogene, or repeats of adjacent sequences. A preliminary nucleotide sequence analysis (unpublished) confirms this in- terpretation: the homology is due to a short trun- cated rbcS sequence and to the repetition of a flanking sequence (located distally to the rbcS gene in the 1.3kb EcoRI Sail fragment),

Organ&ation of rbcS genes in the Chlamydomonas reinhardtii genome

The rbcS sequences that were cloned are closely linked at a single locus. In order to determine whether there are additional rbcS genes in the C.

reinhardtii genome, total DNA was digested with various restriction enzymes, transferred to nitrocellulose and hybridized with a cloned rbcS fragment (Fig. 4). The pattern that is obtained is completely consistent with the restriction map of the cloned DNA. This is examplified by BamHI Sail digest (Fig. 4, lane B) which gives three labelled fragments that comigrate with those produced by digestion of the lambda clone 32 (lane D). Digests with HindlII

Fig. 4. Mapping of the rbcS sequences in the C. reinhardtii genome. Restriction fragments of C. reinhardtii (lanes A, B and C; 2.5 t~g/lane) or lambda recombinant 32 (lane D; 2.5 #g/lane) were separated by agarose gel electrophoresis, blotted to nitrocellulose and probed with a 32p labelled fragment encoding the 55 amino-terminal residues of the small subunit precursor (a 194 bp Taq I fragment of the 0.6kb Sail fragment, unpublished data). Lane A" BamHI EcoRI (3.8, 2.6 and 1.9kb); B and D: BamHl SalI (2.6, 1.3 and 0.6kb); C: SalI (4.3 and 0.6kb).

and HindIII EcoRI also give the expected patterns (not shown). No additional fragments are detected and there is therefore only a single locus in the C reinhardtii genome that contains rbcS genes.

Expression of the rbcS genes

The cloned rbcS DNA provides hybridization probes to monitor the levels of rbcS RNA tran- scripts. Total RNA was separated by agarose gel electrophoresis in the presence of formaldehyde and transferred to a nitrocellulose filter. Two main RNA bands (approximately 0.8 and 1.1kb) hybridize to the rbcS probe (Fig. 5). Several faint larger

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amount of RNA in the upper band is greater in cells grown mixotrophically on acetate in the light (Fig. 5, lane 2). The change in the relative intensity of the two bands is also apparent when dark-grown cells are transferred to the light for six h (Fig. 5, lane 6) but not if dark-grown cells are illuminated for one h and then returned to the dark for five h.

Discussion

Fig. 5. Expression of rbcS RNA. RNA samples were denatured and electrophoresed in the presence of formaldehyde in an agarose gel, transferred to a nitrocellulose filter and hybridized with a plasmid subclone containing the 0.6kb SalI fragment.

The cells were grown in minimal medium ('Min', lane 3) or in acetate medium ('Ac', all other lanes). In the experiment shown in panel a, the cells were maintained in continuous darkness (CD) or in continuous light (CL). In panel b the cells were grown in the dark and either kept in the dark (CD, lane 4), transferred to the light for 1 h and returned to the dark for five h (IL-5D, lane 5) or transferred to the light for six h (6L, lane 6).

bands are also apparent and could be due to hybridization artefacts or could represent precursors or splicing intermediates.

The relative amount of RNA in the two main bands is dependent on the growth conditions. The lower band is more prevalent in RNA from cells grown heterotrophically on acetate in the dark (Fig. 5, lanes 1 and 4) while the upper band is a mi- nor and somewhat variable component. The lower band is also more intense than the upper one in cells grown phototrophically in minimal medium in the light (Fig. 5, lane 3). In contrast, the relative

The results described here show that synthetic oligodeoxynucleotide probes can be used to isolate genes directly from the nuclear genome of Chlamydomonas reinhardtii. The probe that was designed, with 13 defined bases, is probably the smallest that can be used singly with sufficient specificity for the direct screening of a genomic library from an organism with DNA of this com- plexity. With the progress in protein micro- sequencing the approach could become quite generally useful.

In vascular plants, the rbcS gene family com- prises up to a dozen members, scattered at a number of different loci. (2, 3, 5, 6, 12, 46). The situation is simpler in C. reinhardtii, where the small size of the rbcS locus may make it amenable to genetic manipulation. Because the genes are clustered at a single locus, it might be possible to isolate deletion mutants that lack the SS completely. With the use of DNA transformation (28), in vitro mutated genes could then be re-introduced and their phenotypes analyzed.

In higher plants, the transcription and accumulation of SS RNA is induced by light (1, 14, 26, 33, 35) under the control of the phytochrome system (19, 34, 38, 42, 43). The effect of light is also seen when heterologous rbcS genes or gene chimaeras are reintroduced into the genome by transformation (4, 18, 32).

C. reinhardtii maintains a green, photosyntheti- cally competent chloroplast in the dark and it is perhaps not surprising that there is no overall induction in the rbcS RNA levels when cells grown in the dark on acetate are shifted to the light. Howev- er, a change in the relative distribution of RNA in the two major bands does occur. This change probably reflects an indirect effect of light on the overall metabolism of the cells rather than a direct light induction, because the pattern of RNA in cells grown

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in the light, but in minimal medium, resembles the pattern of dark grown rather than light grown cells from acetate medium. It will be of interest to determine the molecular nature of the two main RNA bands: they could be the products of two different genes, or different processing products of the same gene. If two RNAs were to code for variant polypeptides, then the relative amount of variants in the composition of the eight small subunits of the Rubisco holoenzyme would change with different growth conditions, perhaps adapting its enzymatic properties.

The isolation of the rbcS genes now provides us with a complete set of hybridization probes to in- vestigate the coordination of nuclear and chloroplast Rubisco gene expression.

Acknowledgements

I wish to thank Dr J.-D. Rochaix for his support, critical advice and friendly encouragement, M. Ra- hire for her excellent technical assistance, Dr A.

Chollais for kindly providing the synthetic oligonucleotide, Dr M. M. Cordonnier for access to darkroom facilities and Drs J. Erickson, S. May- field and J.-D. Rochaix for their helpful comments on this manuscript. I also acknowledge the efficient help of J. Snowden, Y. Epprecht and O. Jenni in preparing this manuscript. This work was supported by grants 3.258.082 and 3.587.084 from the Swiss Nation Science Fund.

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Received 4 July 1985; in revised form 7 October 1985; accepted 14 October 1985.