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A large open reading frame ( orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein
BOUDREAU, Eric, et al.
Abstract
An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green alga Chlamydomonas reinhardtii. Besides having a short hydrophobic N-terminal domain with five putative transmembrane helices, the predicted orf1995 product is highly basic. orf1995 might be a homologue of the ycf1 gene in land plants, whose function has not yet been determined. Mutants of C. reinhardtii transformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that the orf1995 product is essential for cell survival.
BOUDREAU, Eric, et al . A large open reading frame ( orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein. Molecular and General Genetics , 1997, vol. 253, no. 5, p. 649-653
DOI : 10.1007/s004380050368
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S H O R T C O M M U N I C A T I O N
E. Boudreau·M. Turmel ·M. Goldschmidt-Clermont J.-D. Rochaix· S. Sivan· A. Michaels·S. Leu
A large open reading frame ( orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein
Received: 18 August 1996 / Accepted: 24 September 1996
Abstract An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green algaChlamydomo- nas reinhardtii. Besides having a short hydrophobic N- terminal domain with five putative transmembrane he- lices, the predicted orf1995 product is highly basic.
orf1995might be a homologue of theycf1 gene in land plants, whose function has not yet been determined.
Mutants ofC. reinhardtiitransformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that theorf1995 product is essential for cell survival.
Key words Green algae· Chlamydomonas reinhardtii· Chloroplast genome· Gene inactivation·
Transmembrane protein
Introduction
The chloroplast genomes of photosynthetic land plants are quite conservative in size (120–160 kb), organization and gene content (Palmer 1991). The DNA sequences of several land plant chloroplast DNAs (cpDNAs) (see Palmer 1991; Wakasugi et al. 1994; Maier et al. 1995) have revealed 110–118 often closely spaced genes whose products are mostly involved in mediating gene expres- sion and photosynthesis. Most land plant chloroplast
genes are arranged in oligocistronic operons, several of which resemble those found in cyanobacteria.
The cpDNAs of green algae have diverged more ex- tensively during evolution; they differ more in size (89–
400 kb) and organization (Palmer 1991). Their gene content appears to be similar to that of land plant chloroplasts but many of the ancestral bacterial operons that characterize the organization of land plant cpDNAs have been rearranged in green algal cpDNAs. About 75 genes have been localized on the cpDNAs of four species from the polyphyletic genus Chlamydomonas(C. gelati- nosa, C. moewusii, C. pitschmannii and C. reinhardtii) (Boudreau et al. 1994; Boudreau and Turmel 1995, 1996). Only six of these genes (tscA, tufA, rp15, orfA, orfB and orf715) were not previously reported in any land plant cpDNA. The absence oftufAfrom land plant cpDNAs has been attributed to its transfer to the nu- cleus in the green algal ancestor of land plants (Baldauf et al. 1990).
In this study, we report that theC. reinhardtiicoding sequences previously designated orf715 and orfA or orfS, respectively (Boudreau et al. 1994) are integral parts of an unusually large open reading frame,orf1995, that is essential for cell growth.
Materials and methods
DNA sequencing and RNA analysis
TheC. reinhardtii EcoRI fragment 23 was transferred from clone P- 581 into theEcoRI site of pBluescript KS+(Stratagene, La Jolla, Calif.) to produce the plasmid pBSE23. Nested deletion subclones from pBSE23 were generated in one direction using the Erase-a- Base System (Promega, Madison, Wis.). Subclones of the C.
reinhardtii EcoRI fragments 28 (clone P-582) and 33 (Herrin and Michaels, unpublished results) andBamHI fragment 10 (clone P- 17) were created using various restriction enzymes. DNA manip- ulations, cloning and sequencing were carried out using standard procedures. Sequence analysis was performed using the Genetics Computer Group Software (Version 8, Genetics Computer Group, University of Wisconsin, Madison, Wis.).
Mol Gen Genet (1997) 253:649 – 653 Springer-Verlag 1997
Communicated by R. G. Herrmann E. Boudreau·M. Turmel
De´partement de Biochimie, Faculte´ des sciences et de ge´nie, Universite´ Laval, Que´bec G1K 7P4, Canada
M. Goldschmidt-Clermont·J.-D. Rochaix
De´partement de Biologie Mole´culaire, Universite´ de Gene`ve, CH-1211, Gene`ve 4, Switzerland
S. Sivan·A. Michaels·S. Leu (&)
The Doris and Bertie Black Center of Bioenergetics in Life Sciences, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva, Israel
Total cellular RNA was isolated from light:dark synchronized cells of theC. reinhardtiistrain 137c, which were harvested 6 h after the onset of the light period, using the RNAgents Total RNA isolation system (Promega). RNA aliquots (5lg) were electro- phoresed and transferred to Nytran nylon membranes (Schleicher and Schuell, Keene, N.H.) as described. The blots were hybridized with a32P-labeled 779 bpTaqI fragment specific to the 5′part of orf1995(positions +77, +856 relative to the start of the ORF) and three 32P-labeled PCR-amplified probes that were specific to the central (positions +1017, +2086 and positions +2335, +4378) and the 3′part (positions +4717, +5982) of the gene.
Gene disruption, transformation and Southern blot analysis The plasmid pBSE23 was digested withNcoI and blunt ends were generated using Klenow DNA polymerase I. A 1.9 kb HindIII subfragment (positions 4712–6671 of the submitted sequence) from theBamHI fragment 10 was cloned into pBluescript SK+(Strata- gene) to create pBSH13. The plasmid pBSH13 was linearized with HpaI. TheaadA expression cassette conferring spectinomycin re- sistance was excised from pUC-atpX-AAD (Goldschmidt-Cler- mont 1991) by digestion withEcoRV andSmaI and ligated to the linearized plasmids pBSE23 or pBSH13. The resulting constructs used in our study were designated pBSE23-NcoI:aadA+/), having the aadA cassette inserted in both possible orientations, and pSBH13-Hpal:aadA+, having theaadAgene oriented in the same direction as that oforf1995transcription.C. reinhardtiiwild-type strains 137c and 4d (mt+) were transformed with these plasmids as described (Goldschmidt-Clermont 1991).
Transformants were selected on TAP agar plates containing 150lg/ml spectinomycin (Goldschmidt-Clermont 1991) and the transformants were recloned on TAP-spectinomycin plates three to four times. Total cellular DNAs were prepared fromC. reinhardtii strain 137c and pBSE23-NcoI:aadA+/) transformants, digested with EcoRI, fractionated by agarose gel electrophoresis and transferred onto Hybond-Nylon membranes (Amersham, Arling- ton Heights, Ill.). The blots were hybridized with theC. reinhardtii EcoRI fragment 23 as well as the 2.0 kb EcoRV-SmaI fragment containing theaadAcassette (Boudreau et al. 1994). Total cellular DNAs from the pSBH13-HpaI:aadA+ transformants were ex- tracted, digested withBamHI, fractionated by agarose gel electro- phoresis and transferred onto nitrocellulose membranes. These blots were hybridized with a 0.9 kb C. reinhardtii EcoRV-PstI fragment containing part of theC. reinhardtii atpBgene (Woessner et al. 1986) and with a 0.7 kbNcoI-PstI fragment containing part of theaadAcassette (Goldschmidt-Clermont 1991).
Results and discussion
Sequence analysis oforf1995
The C. reinhardtii cpDNA region spanning the EcoRI fragments 23, 28 and 33 (see Fig. 1) was sequenced en- tirely. Assembly of the resulting 6444 bp sequence (Genbank accession number X92726) with that of the 2.9 kbEcoRI-KpnI fragment (Woessner et al. 1986) re- vealed the presence of an open reading frame of 5988 bp encoding a putative protein of 1995 amino acids with a calculated molecular mass of 232179. This gene is lo- cated 675 bp upstream of atpB and in the same or- ientation (Fig. 1). The C-terminal domain of orf1995 was previously called ORF1 (Woessner et al. 1986). The orf1995 sequence lacks any structural features that re- semble RNA introns and the codon usage throughout this gene sequence is biased as in other C. reinhardtii chloroplast protein-coding genes. We identified a tran-
script of 7.4 kb by Northern hybridization using probes specific to different regions of orf1995, demonstrating that the full length of this gene is transcribed (data not shown).
The predicted orf1995 gene product is highly basic and heavily charged, with a calculated pI of 11.06 and a net charge of +178. The N-terminal portion of the protein, comprising the first 350 amino acids, is hydro- phobic and contains five putative transmembrane helices (Fig. 2). The remaining sequence of the protein is hy- drophilic and heavily charged with a net charge of +182.
Nearly 18% of the C-terminal region consist of lysine (178 amino acids) and arginine (118 amino acids).
Theorf1995sequence shows significant homology to unpublished chloroplast sequences of three other green algae: orf715 and orfA of Chlamydomonas eugametos (Boudreau et al. 1994; unpublished results of E. Bou- dreau and M. Turmel), orf956 ofNephroselmis olivacea (unpublished results of M. Turmel, C. Otis and C. Le- mieux) and orf819 of Chlorella vulgaris (M. Sugiura, personal communication). Although these ORFs differ by many insertions/deletions, several well conserved blocks of amino acids and the locations of the five pu- tative transmembrane domains are conserved (Fig. 2).
The N-terminal region of the orf1995-encoded protein shares 53% identity with the 715-amino acid protein sequence deduced from orf715 of C. eugametos, while the C-terminal region of theorf1995product shares 39%
identity with the 356-amino acid protein sequence de- duced from orfA. The C. eugametos orf715 and orfA, which are still not completely sequenced, are located on the same DNA strand and separated by only 5 kb. These ORFs are therefore probably integral parts of a single gene equivalent to the C. reinhardtii orf1995.
The orf1995 protein shares important structural si- milarities, but only low sequence homology, with the ycf1gene product of land plants. Likeorf1995, theycf1 genes of land plant cpDNAs encode large proteins which feature a hydrophobic N-terminal domain of 300–400 amino acids with several putative transmembrane do- mains, and a highly basic hydrophilic C-terminus of variable length (Wolfe et al. 1992). The degree of se-
Fig. 1 Physical map of theC. reinhardtiicpDNA region containing theorf1995and strategy used to inactivate this gene. The map shows theEcoRI fragments 23, 28, 33 and part ofEcoRI fragment 11 (from the left). Restriction sites for the following enzymes are indicated: B, BamHI; E, EcoRI; EV,EcoRV; H, HindIII; P,PstI. For the in- activation experiments, theaadAcassette was inserted into the unique NcoI site (N) of plasmid pBSE23 and into the uniqueHpaI (Hp) site of plasmid pBSH13
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quence identity between theC. reinhardtii orf1995 pro- tein and the tobaccoycf1product is only 20%, and the blocks conserved between theycf1protein sequences of dicots and Marchantia polymorpha (Wolfe et al. 1992) were not found in the C. reinhardtii orf1995 sequence.
The Chlorella orf819 sequence, however, shares 24.7%
identity with a stretch of 219 amino acids ofMarchantia
polymorpha ycf1 (orf1068), and may thus represent an evolutionary link between orf1995 and ycf1. The land plant ycf1 is under few evolutionary constraints as it varies extensively in sequence and size (Wolfe et al.
1992). It is therefore possible that the most recent common ancestor of land plants and green algae pos- sessed a chloroplast gene functionally similar toorf1995
Fig. 2 Alignment of the C. reinhardtii (Cr) orf1995, the Chlorella vulgaris (Cv) orf819 and the Nephroselmis olivacea (No) orf956 deduced amino acid sequences. Asterisks below the sequences designate the residues conserved in all three sequences whereas the crosses indicate residues conserved in two of the three sequences.
Dashes indicate gaps inserted in the sequences. Solid lines in the alignment indicate long sequence insertions in the C. reinhardtii protein, the sizes of these insertions are indicated inbrackets. The putative transmembrane domains near the N-termini of the three sequences areunderlined
and that the sequence of this gene evolved differently in the green algal and land plant lineages, but retained its principal structural features.
Interestingly, no ORF similar to either orf1995 or ycf1 has been described in the complete cpDNA se- quences of the rhodophyte Porphyra purpurea, the chromophyte Odontella sinensisor the glaucophyte Cy- anophora paradoxa (reviewed by Reardon and Price 1995).
Disruption oforf1995
In order to determine whetherorf1995encodes a protein important for cell function, this gene was disrupted near its 5′ and 3′ ends by means of chloroplast transforma- tion. The aadA cassette, which confers spectinomycin resistance (Goldschmidt-Clermont 1991), was inserted into the NcoI site of the plasmid pBSE23 and into the HpaI site of pBSH13 (Fig. 1). These sites are located at residues 517 and 4818, respectively, relative to the orf1995initiation codon.
Sixteen pBSE23-NcoI:aadA transformants (eight transformants with the aadA cassette in each orienta- tion) and nine pBSH13-HpaI:aadA transformants were subjected to three or four rounds of single colony pur- ification under selective conditions and their total cel- lular DNAs were analyzed by Southern blot hybridization (Fig. 3A, B). All of the transformants from both disruption experiments were found to be heteroplasmic; i.e. to contain both the disrupted and the wild-type alleles at the orf1995 locus (see Fig. 3A, B).
EcoRI digests of the pBSE23-NcoI:aadA(+/)) trans- formant DNAs, hybridized with EcoRI fragment 23, showed 3.2 and 5.1 kb fragments corresponding to the wild-type and disrupted alleles, respectively. Only the 3.2 kb fragment was observed in the DNA of the wild-type strain (Fig. 3A), whereas only the 5.1 kb fragment was observed in the DNAs of the transformants when the blot was rehybridized with a fragment containing the 2.0 kbaadAcassette (data not shown).BamHI digests of the pBSH13-HpaI:aadA transformant DNAs, hybridized with a 0.9 kbEcoRV-PstI fragment (Fig. 1), revealed 7.6 and 9.5 kb fragments. Only the 7.6 kbBamHI fragment was detected in the wild-type strain (Fig. 3B), while only the 9.5 kb fragment was observed when the blot was rehybridized with theaadA coding sequence. These re- sults indicate that orf1995 has a vital function, since disruptions of non-essential chloroplast genes in C. re- inhardtii have been shown to segregate to produce homoplasmic lines within three or fewer cycles of re- cloning (Goldschmidt-Clermont 1991).
Concluding remarks
TheC. reinhardtii orf1995encodes an exceptionally long transmembrane protein that is essential for cell survival.
Homologues of this gene have been clearly identified in
the cpDNAs of other green algae. It is possible that the Chlamydomonas orf1995 and the land plant ycf1 genes have a common origin. The fact that ycf1is one of the few remaining genes found in the cpDNA of the non- photosynthetic flowering plantEpifagus virgiana(Wolfe et al. 1992) suggests that this gene is also essential in land plants. The strongly back character of the C-terminus of theorf1995- andycf1-encoded proteins suggests that this domain is able to bind nucleic acids. We have shown that an overexpressed domain of the orf1995-encoded protein is indeed able to bind DNA and RNA; however, competition experiments with different DNA and RNA samples did not reveal significant specificity of this binding (unpublished results of S. L.). We speculate that the protein may be involved in binding cpDNA to either the chloroplast envelope or the thylakoid membrane.
Attachment of the chloroplast genome to these mem- branes might be required for genome replication, seg- regation and for the transcription of several genes (Possingham and Lawrence 1983; Sato et al. 1993). Al- ternatively, the orf1995-encoded protein may be in- volved in docking mRNAs to the thylakoid membranes for translation; many chloroplast mRNAs are associated to thylakoid-bound chloroplast polysomes when they are being translated (Breidenbach et al. 1988, Brei- denbach et al. 1990).
Acknowledgements We thank E. Harris from theChlamydomonas Genetics Center at Duke University for the clones P-581, P-582 and P-17; M. Sugiura for the unpublishedChlorella orf819 sequence;
and D. Therrien for his technical assistance. This research was supported in part by grants from the Natural Sciences and En- gineering Research Council of Canada and Le Fonds pour la Formation de Chercheurs et l’Aide a` la Recherche (93-ER-0350), by a grant from the German-Israeli Foundation for Scientific Re- search and Development (1-251-129.03/92), by the Bertie and Doris Fig. 3A Examples of Southern blot analysis of the pBSE23- NcoI:aadA transformants. EcoRI digests of total cellular DNAs from wild-typeC. reinhardtii(lane 1), from six pBSE23-NcoI:aadA+
(lanes 2–7) and from five pBSE23-NcoI:aadA-. Transformants were hybridized with the 3.2 kbEcoRI fragment 23 (see Fig. 1).BExamples of Southern blot analysis of the pBSH13-HpaI:aadA+ transformants.
DNA digests from wild-type C. reinhardtii(lane 1) and from four pBSH13-HpaI:aadA+ transformants were hybridized with a 0.9 kb EcoRV-PstI fragment (see Fig. 1)
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Black Center for Bioenergetics in Life Sciences (Beer Sheva) and by grant 31.34014.92 from the Swiss National Science Foundation.
M.T. is a scholar in the Evolutionary Biology Program of the Canadian Institute for Advanced Research.
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