Chloroplasts Richard B. Hallick University of Arizona, Tucson, Arizona, USA New features of chloroplast gene expression are continually being discovered, particularly in the area of post-transcriptional RNA processing. RNA editing of chloroplast pre-mRNAs occurs in both monocotyledons and dicotyledons, and involves both initiator and internal codons. The view of introns as mobile genetic elements expands both with the identification of additional twintrons in Euglenachloroplast genes and with studies on the homing group I introns of Chlamydomonas. Current Opinion in Genetics and Development 1992, 2:926-930
Introduction The complete DNA sequences of several chloroplast genomes, including those of tobacco [1], liverwort [2] and rice [3], have now been reported. A nearly complete sequence is also available for Euglena gracilis. The transcription units, promoters and terminators are well characterized, and group II introns abound. What is left to discover in the chloroplast molecular biology field? A remarkable amount. New and unexpected results seem to be the order of the day in this ever exciting field. In this review, I will focus on the new types of plastid post-transcriptional RNA processing reactions, introns-within-introns, and the importance of chloroplast transformation in unraveling the biology of the plastid.
Editing of mRNA RNA-processing events are central to chloroplast gene expression. Various modes of post-transcriptional precursor-mRNA processing in chloroplasts have previously been described, including endonuclease cleavage of polycistronic pre-mRNAs, cZs-splicing of group I and group II introns, and tran~splicing of group II introns. We can now add mRNA editing to the known chloroplast RNA-processing reactions. RNA editing is the term applied to the post-transcriptional modification of pre-mRNA to alter its nucleotide sequence. RNA editing in various systems includes the insertion and deletion of nucleotides, or specific nucleotide modifications. K6ssel and colleagues have demonstrated C to U editing of initiator and internal codons in several chloroplast transcripts. Most chloroplast genes begin with the normal AUG initiator codon. One exception is the rpl2 gene, encoding chloroplast ribosomal protein L2, in the polycistronic rpoA operon of maize and rice, which begins with an
ACG codon. Using PCR-amplification of cDNA, a single edited AUG-initiator codon in the first exon of processed rpl2 mRNA of maize was identified [4°°]. RNA editing is not confined to the chloroplasts of monocots. The initiator codon of the spinach and tobacco psbL gene for a hydrophobic 3.2 kD polypeptide of photosystem (PS) II is ACG. Following C to U editing, the canonical AUG initiator codon is found in the mature mRNA [5]. RNA editing is neither limited to initiator codons nor to the editing of ACG codons. The chloroplast ndhA gene encodes a homolog of a mitochondrial NADH dehydrogenase subunit. Four internal serine codons in the maize chloroplast nd/~, either UCG, UCA or UCC, are edited to the corresponding leucine codons UUG, UUA and UUC, respectively [6"]. The edited codons are. at sites where leucine residues are nearly universally conserved among both chloroplast and mitochondrial gene products. One of the sites edited in maize chloroplasts corresponds exactly to an editing site in mitochondria of Oenothera, wheat and petunia. All edited codons to date are believed to be functionally significant. They restore either the translateability of the mRNA or the presumed critical residues for protein function, and no neutral editing has been observed. What is the mechanism of editing? As noted by Hodges and Scott [7], plant mitochondrial and chloroplast editing may be most similar to "substitutional editing" in mammalian nuclear-RNA editing. The most likely mechanism is a cytidine deamination reaction. Thus, mRNA editing may be most similar to post-transcriptional base-modification reactions well known in pre-tRNA maturation pathways, including those of plant chloroplasts [8]. A more significant question is the mechanism for recognition of the pre-edited codon. It seems evident that the structure of the pre-mRNA itself may be central to proper editing. An in vitro RNA-editing system would be very useful in answering these questions.
Abbreviations ORF~open reading frame; PS---photosystem. 926
(~) Current Biology Ltd ISSN 0959-437X
Chloroplasts Hallick 927 Introns and twintrons The chloroplast genome of E gracilis is approximately 150kb in size. The genes are arranged primarily in polycistronic transcription units. Euglena genes contain at least 64 group 1I intror/s. All of the Euglena group II introns possess both the highly conserved catalytic domain V and the nucleophile-containing domain VI. These introns are shorter than other group II introns, and have abbreviated versions of domains I-IV [9°o,10]. However, many Euglena chloroplast group U introns retain the two- and three-dimensional base-pairing interactions of known or suspected functional significance, exon-binding sites, 'guide pair', a-e' and 7-7' (see [10] for a description of the key structural and functional domains of a group 11intron). The chloroplast genes of Euglena also contain more than 62 introns of a unique class that is designated group 11I (see [11]). Group HI introns appear to be highly degenerate versions of group II introns. They retain conserved group II-like 5' boundary sequences with a U at the second position and the G at the fifth position, and a domain VI like structure at the 3' end of the intron 9 [11]. These introns are small and uniform in size at approximately 100 bp. Group Ill introns have also been found in the plastid genes of the heterotrophic flagellate Astasia longa [12], a non-photosynthetic protist with a 73 kb circular plastid genome lacking photosynthetic operons. Group II and group III introns are probably evolutionarily related, and may have some splicing mechanisms in common.
Euglena chloroplast genes also contain new genetic elements called twintrons, or introns-within-introns. One class of twintron, a group II twintron, is located within the psbF gene, which encodes the [3-subunit of cytochrome b-559 of the PS U reaction center [13°]. The psbF twintron is composed of a 618 bp group II intron inserted within domain V of a 424 bp external group II intron. The mechanism for twintron formation is unknown, although a three-step process of reverse splicing, reverse transcription and homologous recombination has been suggested [9°o,13o]. Recently, two new types of twintrons in the Euglena chloroplast rpl23 ribosomal protein operon, both predicted from the DNA sequence data [ 11], have been characterized. The rpl23 operon is composed of 11 chloroplast ribosomal protein genes, an internal tRNA cistron, and an unidentified open reading frame (ORF). The gene order is: rp123-rp12-rpslg--rp122-rps3-or~16/514rpl16-rpl14-rp15-rps8-rp136-trnI-rps14 [11,14] (see EMBL, Accession No z11874). This gene arrangement is very similar to the related operons in higher plant chloroplast genomes. All 13 cistrons are transcribed as a primary transcript of length 11.8 kb [15]. The pre-mRNA undergoes a very complex RNA-maturation pathway [15]. There are at least 24 introns in the operon, including 14 group His, 6 group Fls, and two intercistronic group III introns [16]. The two new types of twintrons are a mixed twintron, which incorporates group II and group 1II introns in rps3 [9 °°] and a group III twintron in rpll6 [17].
The rps3 gene is interrupted by a 409 bp 'mixed' twintron [9"°]. This intron was found to be composed of a
311 bp group II intron internal to a 98 bp group rn intron. In this mixed twintron, excision also occurred through a sequential splicing pathway, with removal of the group 11 intron preceding group HI intron splicing. How was the rps3 twintron formed during the evolution of the rps3 gene? This ribosomal protein cistron is an ancient gene in an ancient operon, and was probably present in the common ancestor of eubacteria, archaebacteria, plastids, and eukaryotes. The most parsimonious explanation for mixed twintron formation is the addition of a group II intron into a group HI intron after the descent of this gene from a common, intronless ancestral gene [9°°]. The hypothesis that introns are vestiges of an 'exon shuffling' mechanism reflecting the modular assembly of ancient proteins from a limited universe of exons 18 [18,19], is not supported by the existence of twintrons. The rpH6 gene for protein L16 of the large ribosomal subunit contains a 208bp group III twintron, formed from the insertion of one group HI intron into another. The rpll6 twintron is excised by a two-step sequential splicing pathway [17]. Remarkably, the internal group III intron is excised using multiple 5' and 3' splice sites. Three additional group III twintrons are found in the RNA polymerase subunit gene rpoC1 of the rpoB-rpoCl-rpoC2 operon [17]. The internal group III introns of two of the rpoC1 twintrons are also spliced from multiple splice sites. The utilization of multiple 5' and/or 3' splice sites during the excision of the internal introns of the group III twintrons may have biological significance in terms of gene evolution. Alternative splice-site selection of these pre-mRNAs containing multiple 5' and 3' splice sites could further evolve to yield two or more different gene products from a progenitor cistron. If twintrons form as a result of a mobile intron inserting into another intron, could higher order twintrons also be formed from insertion of an intron into a twintron? The 434 bp rpsl8 intron within the rps2 operon is neither a group II intron, nor a simple twintron [20], but a complex twintron that is excised by four sequential splicing reactions. The external intron is interrupted by one internal intron, which in turn is split by two additional introns. Two of the internal introns are excised using multiple 5' and/or 3' splice sites (RG Drager and RB Hallick, unpublished data). The existence of twintrons and complex twintrons has two important implications concerning the origin, evolution and function of introns. First, the concept of group I1 and group III introns as mobile genetic elements that insert into genes is strengthened. Second, intron insertion into existing introns is a possible evolutionary mechanism for the assembly of large introns with multiple 5' and 3' splice sites.
Analysis of structure-function relationships using transgenic plastids Major advances in biology often follow advances in technology. The most significant development in the chloroplast technology field in the past five years has been the
928
Genomes and evolution advent of biolistic plastid-transformation procedures for both Chlamydomonas [21] and tobacco [22]. Transformation of plastid genomes of other species is sure to follow. Some recent results with transgenic Chlam3~ domonas chloroplast are particularly noteworthy. One important result from chloroplast transformations is the characterization of chloroplast promoters in t,ivo, complementing earlier in vitro studies on promoter ac* tivity. Klein et al. [23"] have coupled both the aq~B and 16S rRNA promoter regions to a reporter gene, the E. coli 13-glucuronidase u/d.A locus. Deletion analysis was used to compare structural features of the two promoters. As Chlamydomonas will grow non-photosynthetically on acetate-containing medium, disruptions of non-essential genes, including those involved in biogenesis of the photosynthetic apparatus, are candidates for targeted gene disruptions. Two different approaches have been used to obtain targeted disruptions of Chlamydomonas chloroplast-encoded photosynthetic genes. Newman et aL [24.] have bombarded cells with microprojectiles coated with a mixture of two different plasmid DNAs. In this cotransformation, one DNA encodes selectable markers for antibiotic resistance in the rRNA operon, and the other a cassette-inactivated, non-essential gene, such as aq~B or rbcL Some of the resulting antibioticresistant transfonnants became homoplastic for the disrupted gene. An alternative approach developed by Goldschmidt-Clermont [25"] is direct gene disruption with a selectable marker in a single plasmid transformation vector. The bacterial aadA gene, encoding aminoglycoside adenine transferase, when combined with Chlamydomonas transcription and translation signals, confers both spectinomycin and streptomycin resistance as dominant selectable markers on transformed cells. This approach was used for the site-directed disruption of two genes, psaC and tacA, which are required for photosynthesis. The aadA-dismpted psaClocus has been characterized in detail [26"]. The psaCgene product was believed to be involved in PSI assembly. In the mutants, neither PSI reaction-center subunits nor the seven small subunits of PSI accumulate. This has led to the conclusion that psaC is required both for assembly and PS I activity in chloroplasts [26"]. The gene-disruption strategy is of obvious importance for further characterization of components of the photosynthetic apparatus. The tacA gene was previously identified as a locus required for trans-splicing o f p s a A pre-mRNA [27..]. The tacA gene product is not a protein, because the 0.7 kb region required for in vivo splicing lacks significant reading frames, and insertional inactivation of the locus does not completely block splicing [27.°]. It was concluded that tacA encodes a small RNA required for splicing, perhaps the central domain of a group II intron [27"]. With the aadA-disrupted tar_A, mutants completely deficient is psaA trans-splicing could be obtained [25"]. This result paves the way for more detailed structure-function analysis of this novel chloroplast trans-splicing reaction.
Mobile group I intron Man), group I introns contain an internal ORF that encodes a site-specific DNA endonuclease. The endonuclease activity confers genetic mobility on the intron in crosses between intron-containing and intronless strains [28]. The single 888 bp group I intron of the Chlam3~ domonas reinhardtii 23S rRNA encodes a polypeptide of 163 residues [29]. This is the first example of a serf-splicing chloroplast group I intron [30], which is serf-splicing in vitro [30,31"]. There are also several group I introns in the 23S rRNA gene of Chlamydomonas eugametos. Intron5 is mobile during interspecific crosses with Chlamydomona.~ moewuaii [32]. The C eugametos intron encodes a 218 amino-acid double-strand DNA endonuclease, designated I-Ceul, with in vitro activity specific for the 'homing' site of this mobile intron [33]. The C reinhardtii intron-encoded ORF was also shown to encode a double-strand DNA endonuclease [31"], designated I-CreI. What are the activities of these intron-encoded ORFs in vivo? Are they required for splicing in vivo? Are they necessary and sufficient for intron mobility? Thompson and Herrin [34"] have studied in detail the in vitro selfsplicing of the C. reinhardtii group I intron, and in addition have characterized in vivo splicing of mutants deleted internally within the intron-encoded I-O*ellocus. Biolistic gene replacement was used to create homoplastic deletion mutants. The mutants spliced normally in vivo, indicating that I-O'el does not encode a maturase required for splicing. The mobility in vivo of the group I intron was elegantly demonstrated by Durrenberger and Rochaix [31"]. When an artificial intron homing site corresponding to a region of the intronless form of the C reinhardtii 23S rRNA was introduced into the C reinhardtii chloroplast genome by biolistic gene transfer, many of the resulting transformants had the 888 bp 23S rRNA group I intron integrated precisely at the e x o n - e x o n boundary in the artificial homing site. This demonstrates that the intron behaves as a site-specific mobile genetic element in vivo if a target sequence is present. The implication is that the I-CreI endonuclease activity encoded by the intron is required for homing actMty. This can be tested by directed mutagenesis within the I-O'el gene.
Conclusions Many important questions about chloroplast gene expression remain unanswered. Although we now have DNA sequences for numerous chloroplast-encoded polypeptides, a very large number of essential genes have not been identified. With the advent of biolistic transformation, the time is now ripe for rapid progress in gene identification by a reverse genetic approach, employing sitedirected gene disruption. Because of the ease of transformation, most progress to date has involved chloroplasts
Chloroplasts Hallick of Chlamydomonas.This work is no doubt a harbinger of advances to come in other systems, including higher plants, as the technology advances. Among the interesting aspects of gene expression awaiting detailed study are various post-transcriptional RNA-processing events. What is the mechanism of RNA editing. How are pre-edited codons selected? What is the splicing machinery for the ubiquitous group II introns? Are maturases required for group I intron splicing? We can eagerly await answers to these and other important questions.
existence of twintrons strongly support the introns late model for the origin of introns. 10.
MICHELF, UMESONOK, OZEKI H: Comparative and Functional Anatomy of Group II Catalytic Introns - - a Review. Gene 1989, 82:5-30.
11.
CHRISTOPHERDA, HAIa.ICK RB: Euglena gracilis Chloroplast Ribosomal Protein Operon: a New Chloroplast Gene for Ribosomal Protein L5 and Description of a Novel Organelle Intron Category Designated Group Ill. Nucleic Acids Res 1989, 17:7591-7608.
12.
SIEMEISTERG, BUCHHOLZC, HACHTELW: Genes for the Plasrid Elongation Factor Tu and Ribosomal Protein $7 and 6 Transfer RNA Genes on the 73-kb DNA from Astasia longa that Resembles the Chloroplast DNA of Euglena. Mol Gen Genet 1990, 220:425--432.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •. of outstanding interest 1.
2.
3.
SHINOZAKIK, OHME M, TANAKA M, WAKASUGI T, HAYASHIDA N, MATSUBAYASHIT, mAlTA N, CHUNWONGSE J, OBOKATA J, YAMAGUCHI-SHINOZAKI K, ET AL: The Complete Nucleotide Sequence of the Tobacco Chloroplast Genome: Its Gene Organization and Expression. EIVlBO J 1986, 5:2043-2049. OHYAMAK, FUKUZAWAH, KOHCHI T, SHIRAI H, SANO T, SANO S, UMESONOK, SHIKIY, TAKEUCHIM, CHANGZ, ETAL: Chloroplast Gene Sequence Deduced from the Complete Sequence of Liverwort Marchantia Polymorpha Chloroplast DNA. Nature 1986, 322:572-574. HIRATSUKA j, SH1MADAH, WHITI'IER R, ISHIBASHIT, SAKAMOTOM, MORI M, KONOO C, HONJI Y, SON CR, MENG B-Y, Er m.: The Complete Sequence of the Rice (Oryza sativa) Chloroplast Genome: Intermolecular Recombination b e t w e e n Distinct tRNA Genes Accounts for a Major Plastid DNA Inversion during the Evolution of the Cereals. Mol Gen Genet 1989, 217:185-194.
4. •.
HOCH B, MAmR RM, APPEL K, IGLOI GL, KOSSELH: Editing of a Chloroplast mRNA by Creation of an Initiation Codon. Nature 1991, 353:178--180. The initial report of mRNA editing in chloroplasts. PCR was used to amplify cDNA copies of edited mRNA. The possibility of a mitochondrial origin for the cDNA was ruled out. 5.
KUDLAJ, IGt.OI GL, METZLAFFM, I-L,'~GEMANNR, KOSSELH: RNA Editing in Tobacco Chloroplasts Leads to the Formation of a Translatable p~bL mRNA by a C to U Substitution within the Initiation Codon. ~14B0 J 1992, 11:1099-1103.
MAIER RIM, HOCH B, ZELTZ P, KOSSEL H: Internal Editing of the Maize Chloroplast ndhA Transcript Restores Codons for Conserved Amino Acids. Plant Cell 1992, 4:609-616. Using PCR, evidence is given for several internal editing sites in the nd-hA transcript. The comparison of mRNA editing between mitochondrial and chloroplast-encoded pre-mRNAs is of particular interest.
13. •
COPERT1NODW, HAmCK RB: Group ll Twintron: an Intron within an Intron in a Chloroplast Cytochrome b-559 Gene. EMBO J 1991, 10:433-442. Original experimental evidence for the existence of twintrons (introns within introns). 14.
CHRISTOPHERDA, CUSHMANJC, PRICE CA, HALUCKRB: Organization of Ribosomal Protein Genes rpi23, rpl2, rpsl9, rp122 and rps3 on the Euglena gracilis Chloroplast Genome. Cure" Genet 1988, 14:275-285.
15.
CHRISTOPHERDA, HALUCK RB: Complex RNA Maturation Pathway for a Chloroplast Ribosomal Protein Operon with an Internal tRNA Cistron. Plant Cell 1990, 2:659-671.
16.
STEVENSONJK, DRAGER RG, COPERT1NO DW, ET AL: Intercistronic Group III lntrons in Polycistronic Ribosomal Protein Operons of Chloroplasts. Mol Gen Genet 1991, 228:183-192.
17.
COPERTINODW, SHIGEOKAS, HALL1CKRB: Chloroplast Group HI Twintron Excision Utilizing Multiple 5'- and 3'-Splice Sites. EMBO J 1992, in press.
18.
DORITRL, SCHOENBACHL, GILBERTW: How Big is the Universe of Exons? Science 1990, 250:1377-1382.
19.
DORITRL, GILBERTW: The Limited Universe of Exons. Curr Opin Genet Dev 1991, 1:464-469.
20.
DRAGERRG, HALIACKRB: A Novel E u g l e n a gracilis Chloroplast Operon Encoding four ATP Synthase Subunits and Two Ribosomal Proteins Contains Seventeen Introns. Curr Genet 1992, in press.
21.
BOYNTONJE, GILLHm~I NW, HARRIS EFI, HOSLER JP, JOHNSON AM, JONES AR, RANDOLPH-ANDERSONBL, ROBERTSON D, KLEIN TM, SHARK K13, SANFORDJC: Chloroplast Transformation in Chlamydomonas with High Velocity Microprojectiles. Sci. ence 1988, 240:1534-1538.
22.
SVAB Z, HAJDUKIEW1CZP, MAHGA P: Stable Transformation o f Plastids in Higher Plants. Proc Natl Acad Sci USA 1990, 87:852643530.
6.
•.
7.
HODGESP, Sco'rr J: Apolipoprotein B mRNA Editing: a New Tier for the Control of Gene Expression. Trends Biochem Sci 1992, 17:77-81.
8.
GREENBERGBM, GRUISSEMW, HALUCK RB: Accurate Processing and Pseudouridylation of Chloroplast Transfer RNA in a Chloroplast Transcription System. Plant Mol Biol 1984, 3:97-109.
COPERTINODW, CHPdSTOPHERDA, HALLICKRB: A Mixed Group H/Group III Twintron in the E u g l e n a gracilis Chloroplast Ribosomal Protein $3 Gene: Evidence for Intron Insertion during Gene Evolution. Nucleic Acids Res 1991, 19:6491-6497. This is the initial report of a 'mixed' twintron, comprised of one group II intron internal to a group Ill intron. An extensive phylogenetic analysis of the known rps3 genes is presented. It is also argued that the 9.
•.
23. •
KLEINU, DE CJ, BOGORAD L: Two Types of Chloroplast Gene Promoters in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 1992, 89:3453--3457. Analysis of promoters for Chlamydomonas chloroplast a~OB or 16S rRNA-encoding genes coupled to the E. coli [~-glucuronidase (GUS) reporter gene in transgenic chloroplasts. 24.
NEWMANSM, GIUMAM NW, HARRISEH, JOHNSON AM, BOYNTON JE: Targeted Disruption of Chloroplast Genes in Chlamydomonas reinhardtii. 291ol Gen Genet 1991, 230:65-74. Describes an elegant cotransformation approach to modifying two plastid genes in a single particle bombardment. •
25. .,
GOLDSCHMIDT-CLERMONTM: Transgenic Expression of Aminoglycoside Adenine Transferase in the Chloroplast: a Selectable Marker of Site-directed Transformation o f Chlamydomonas. Nucleic Acids Res 1991, 19:4083-4089. Describes the targeted gene disruption with a dominant selectable marker based on a bacterial antibiotic-resistance gene. One important
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Genomes and evolution experiment is the disruption of art ORF of unknown function. Transformants were persistently heteroplasmic, perhaps indicating that the disrupted locus is essential for cell growth. 26. •
TAKAHASHIY, GOLDSCHMIDT-CLERMONTM, SOEN SY, FRANZEN LG, ROCHAIXJD: Directed Chloroplast Transformation in Chlamydomonas reinhardtii: Insertional Inactivation of the psaC Gene Encoding the Iron Sulfur Protein Destabilizes Photos}stem I. EMBO J 1991, 10:2033-2040. An excellent example of the use of transgenic chloroplasts to detemline the function of a chloroplast gene product in photos3~tem membrane assembly. 27.
GOLDSCHMIDT-CLERMONTM, CHOQUETY, GIRARDBJ, MICHELF, SCHIRMERRM, ROCHAIXJD: A Small Chloroplast RNA May be Required for Trans-splicing in Chlamydomonas reinhardtii. Cell 1991, 65:135-143. First demonstration of a tram-acting RNA required for chloroplast splicing. Often cited as a potential model for the origin of transacting RNAs in nuclear splicing.
31. **
DURRENBERGERF, ROCHA~JD: Chloroplast Ribosomal Intron of Chlamydomonas reinhardtii: in Vitro Self-splicing, DNA Endonuclease Activity and in Vivo Mobility. EMBO J 1991, 10:3495-3501. A extremely elegant experimental approach to demonstrating intron mobility in viva This system may be amenable to the assay of mobility of foreign introns. 32.
MARSHALL P, LEMIEUXC: Cleavage Pattern of the Homing Endonuclease Encoded by the Fifth Intron in the Chloroplast Large Subunit rRNA-encoding Gene of Chlamydomonas eugametos. Gene 1991, 104:241-245.
33.
GAUTHIERA, TURMEL M, LEMIEUXC: A Group I Intron in the Chloroplast Large Subunit rRNA Gene of Chlamydomonas eugametos Encodes a Double-strand Endonuclease that Cleaves the Homing Site of this Intron. Curt Genet 1991, 19:43-47.
do
28.
DUJONB, BELFORTM, BUTOW RA, JACQ C, LEMIEUXC, PERLMAN PS, VOGTVM: Mobile Introns: Definition of Terms and Recommended Nomenclature. Gene 1989, 82:115-118.
29.
ROCHAIXJD, RAHIREM, MICHELF: The Chloroplast Ribosomal lntron of Chlamydomonas reinhardii Codes for a Polypeptide Related to Mitochondrial Maturases. Nucleic Acids Res 1985, 13:975-984.
30.
HERRINDL, CHEN Y'F, SCHMIDTGW: RNA Splicing in Chlamydomonas Chloroplasts. Self-splicing of 23 S preRNA. J Biol Cbem 1990, 265:21134-21140.
THOMPSONAJ, HERRIN DL: In Vitro Serf-splicing Reactions of the Chloroplast Group I Intron Cr.LSU from Chlamydomonas reinhardtii and in Vivo Manipulation via GeneReplacement. Nucleic Acids Res 1991, 19:6611-6618. Using gene disruption, the authors anab'ze the role of the I-Crel endonuclease in splicing. Includes an in t,itro characterization of the self-splicing reaction. 34. •
RB Hallick, Department of Biochemistry, University of A_rizona, Tucson, Arizona 85721, USA.
Introduction The complete DNA sequences of several chloroplast genomes, including those of tobacco [1], liverwort [2] and rice [3], have now been reported. A nearly complete sequence is also available for Euglena gracilis. The transcription units, promoters and terminators are well characterized, and group II introns abound. What is left to discover in the chloroplast molecular biology field? A remarkable amount. New and unexpected results seem to be the order of the day in this ever exciting field. In this review, I will focus on the new types of plastid post-transcriptional RNA processing reactions, introns-within-introns, and the importance of chloroplast transformation in unraveling the biology of the plastid.
Editing of mRNA RNA-processing events are central to chloroplast gene expression. Various modes of post-transcriptional precursor-mRNA processing in chloroplasts have previously been described, including endonuclease cleavage of polycistronic pre-mRNAs, cZs-splicing of group I and group II introns, and tran~splicing of group II introns. We can now add mRNA editing to the known chloroplast RNA-processing reactions. RNA editing is the term applied to the post-transcriptional modification of pre-mRNA to alter its nucleotide sequence. RNA editing in various systems includes the insertion and deletion of nucleotides, or specific nucleotide modifications. K6ssel and colleagues have demonstrated C to U editing of initiator and internal codons in several chloroplast transcripts. Most chloroplast genes begin with the normal AUG initiator codon. One exception is the rpl2 gene, encoding chloroplast ribosomal protein L2, in the polycistronic rpoA operon of maize and rice, which begins with an
ACG codon. Using PCR-amplification of cDNA, a single edited AUG-initiator codon in the first exon of processed rpl2 mRNA of maize was identified [4°°]. RNA editing is not confined to the chloroplasts of monocots. The initiator codon of the spinach and tobacco psbL gene for a hydrophobic 3.2 kD polypeptide of photosystem (PS) II is ACG. Following C to U editing, the canonical AUG initiator codon is found in the mature mRNA [5]. RNA editing is neither limited to initiator codons nor to the editing of ACG codons. The chloroplast ndhA gene encodes a homolog of a mitochondrial NADH dehydrogenase subunit. Four internal serine codons in the maize chloroplast nd/~, either UCG, UCA or UCC, are edited to the corresponding leucine codons UUG, UUA and UUC, respectively [6"]. The edited codons are. at sites where leucine residues are nearly universally conserved among both chloroplast and mitochondrial gene products. One of the sites edited in maize chloroplasts corresponds exactly to an editing site in mitochondria of Oenothera, wheat and petunia. All edited codons to date are believed to be functionally significant. They restore either the translateability of the mRNA or the presumed critical residues for protein function, and no neutral editing has been observed. What is the mechanism of editing? As noted by Hodges and Scott [7], plant mitochondrial and chloroplast editing may be most similar to "substitutional editing" in mammalian nuclear-RNA editing. The most likely mechanism is a cytidine deamination reaction. Thus, mRNA editing may be most similar to post-transcriptional base-modification reactions well known in pre-tRNA maturation pathways, including those of plant chloroplasts [8]. A more significant question is the mechanism for recognition of the pre-edited codon. It seems evident that the structure of the pre-mRNA itself may be central to proper editing. An in vitro RNA-editing system would be very useful in answering these questions.
Abbreviations ORF~open reading frame; PS---photosystem. 926
(~) Current Biology Ltd ISSN 0959-437X
Chloroplasts Hallick 927 Introns and twintrons The chloroplast genome of E gracilis is approximately 150kb in size. The genes are arranged primarily in polycistronic transcription units. Euglena genes contain at least 64 group 1I intror/s. All of the Euglena group II introns possess both the highly conserved catalytic domain V and the nucleophile-containing domain VI. These introns are shorter than other group II introns, and have abbreviated versions of domains I-IV [9°o,10]. However, many Euglena chloroplast group U introns retain the two- and three-dimensional base-pairing interactions of known or suspected functional significance, exon-binding sites, 'guide pair', a-e' and 7-7' (see [10] for a description of the key structural and functional domains of a group 11intron). The chloroplast genes of Euglena also contain more than 62 introns of a unique class that is designated group 11I (see [11]). Group HI introns appear to be highly degenerate versions of group II introns. They retain conserved group II-like 5' boundary sequences with a U at the second position and the G at the fifth position, and a domain VI like structure at the 3' end of the intron 9 [11]. These introns are small and uniform in size at approximately 100 bp. Group Ill introns have also been found in the plastid genes of the heterotrophic flagellate Astasia longa [12], a non-photosynthetic protist with a 73 kb circular plastid genome lacking photosynthetic operons. Group II and group III introns are probably evolutionarily related, and may have some splicing mechanisms in common.
Euglena chloroplast genes also contain new genetic elements called twintrons, or introns-within-introns. One class of twintron, a group II twintron, is located within the psbF gene, which encodes the [3-subunit of cytochrome b-559 of the PS U reaction center [13°]. The psbF twintron is composed of a 618 bp group II intron inserted within domain V of a 424 bp external group II intron. The mechanism for twintron formation is unknown, although a three-step process of reverse splicing, reverse transcription and homologous recombination has been suggested [9°o,13o]. Recently, two new types of twintrons in the Euglena chloroplast rpl23 ribosomal protein operon, both predicted from the DNA sequence data [ 11], have been characterized. The rpl23 operon is composed of 11 chloroplast ribosomal protein genes, an internal tRNA cistron, and an unidentified open reading frame (ORF). The gene order is: rp123-rp12-rpslg--rp122-rps3-or~16/514rpl16-rpl14-rp15-rps8-rp136-trnI-rps14 [11,14] (see EMBL, Accession No z11874). This gene arrangement is very similar to the related operons in higher plant chloroplast genomes. All 13 cistrons are transcribed as a primary transcript of length 11.8 kb [15]. The pre-mRNA undergoes a very complex RNA-maturation pathway [15]. There are at least 24 introns in the operon, including 14 group His, 6 group Fls, and two intercistronic group III introns [16]. The two new types of twintrons are a mixed twintron, which incorporates group II and group 1II introns in rps3 [9 °°] and a group III twintron in rpll6 [17].
The rps3 gene is interrupted by a 409 bp 'mixed' twintron [9"°]. This intron was found to be composed of a
311 bp group II intron internal to a 98 bp group rn intron. In this mixed twintron, excision also occurred through a sequential splicing pathway, with removal of the group 11 intron preceding group HI intron splicing. How was the rps3 twintron formed during the evolution of the rps3 gene? This ribosomal protein cistron is an ancient gene in an ancient operon, and was probably present in the common ancestor of eubacteria, archaebacteria, plastids, and eukaryotes. The most parsimonious explanation for mixed twintron formation is the addition of a group II intron into a group HI intron after the descent of this gene from a common, intronless ancestral gene [9°°]. The hypothesis that introns are vestiges of an 'exon shuffling' mechanism reflecting the modular assembly of ancient proteins from a limited universe of exons 18 [18,19], is not supported by the existence of twintrons. The rpH6 gene for protein L16 of the large ribosomal subunit contains a 208bp group III twintron, formed from the insertion of one group HI intron into another. The rpll6 twintron is excised by a two-step sequential splicing pathway [17]. Remarkably, the internal group III intron is excised using multiple 5' and 3' splice sites. Three additional group III twintrons are found in the RNA polymerase subunit gene rpoC1 of the rpoB-rpoCl-rpoC2 operon [17]. The internal group III introns of two of the rpoC1 twintrons are also spliced from multiple splice sites. The utilization of multiple 5' and/or 3' splice sites during the excision of the internal introns of the group III twintrons may have biological significance in terms of gene evolution. Alternative splice-site selection of these pre-mRNAs containing multiple 5' and 3' splice sites could further evolve to yield two or more different gene products from a progenitor cistron. If twintrons form as a result of a mobile intron inserting into another intron, could higher order twintrons also be formed from insertion of an intron into a twintron? The 434 bp rpsl8 intron within the rps2 operon is neither a group II intron, nor a simple twintron [20], but a complex twintron that is excised by four sequential splicing reactions. The external intron is interrupted by one internal intron, which in turn is split by two additional introns. Two of the internal introns are excised using multiple 5' and/or 3' splice sites (RG Drager and RB Hallick, unpublished data). The existence of twintrons and complex twintrons has two important implications concerning the origin, evolution and function of introns. First, the concept of group I1 and group III introns as mobile genetic elements that insert into genes is strengthened. Second, intron insertion into existing introns is a possible evolutionary mechanism for the assembly of large introns with multiple 5' and 3' splice sites.
Analysis of structure-function relationships using transgenic plastids Major advances in biology often follow advances in technology. The most significant development in the chloroplast technology field in the past five years has been the
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Genomes and evolution advent of biolistic plastid-transformation procedures for both Chlamydomonas [21] and tobacco [22]. Transformation of plastid genomes of other species is sure to follow. Some recent results with transgenic Chlam3~ domonas chloroplast are particularly noteworthy. One important result from chloroplast transformations is the characterization of chloroplast promoters in t,ivo, complementing earlier in vitro studies on promoter ac* tivity. Klein et al. [23"] have coupled both the aq~B and 16S rRNA promoter regions to a reporter gene, the E. coli 13-glucuronidase u/d.A locus. Deletion analysis was used to compare structural features of the two promoters. As Chlamydomonas will grow non-photosynthetically on acetate-containing medium, disruptions of non-essential genes, including those involved in biogenesis of the photosynthetic apparatus, are candidates for targeted gene disruptions. Two different approaches have been used to obtain targeted disruptions of Chlamydomonas chloroplast-encoded photosynthetic genes. Newman et aL [24.] have bombarded cells with microprojectiles coated with a mixture of two different plasmid DNAs. In this cotransformation, one DNA encodes selectable markers for antibiotic resistance in the rRNA operon, and the other a cassette-inactivated, non-essential gene, such as aq~B or rbcL Some of the resulting antibioticresistant transfonnants became homoplastic for the disrupted gene. An alternative approach developed by Goldschmidt-Clermont [25"] is direct gene disruption with a selectable marker in a single plasmid transformation vector. The bacterial aadA gene, encoding aminoglycoside adenine transferase, when combined with Chlamydomonas transcription and translation signals, confers both spectinomycin and streptomycin resistance as dominant selectable markers on transformed cells. This approach was used for the site-directed disruption of two genes, psaC and tacA, which are required for photosynthesis. The aadA-dismpted psaClocus has been characterized in detail [26"]. The psaCgene product was believed to be involved in PSI assembly. In the mutants, neither PSI reaction-center subunits nor the seven small subunits of PSI accumulate. This has led to the conclusion that psaC is required both for assembly and PS I activity in chloroplasts [26"]. The gene-disruption strategy is of obvious importance for further characterization of components of the photosynthetic apparatus. The tacA gene was previously identified as a locus required for trans-splicing o f p s a A pre-mRNA [27..]. The tacA gene product is not a protein, because the 0.7 kb region required for in vivo splicing lacks significant reading frames, and insertional inactivation of the locus does not completely block splicing [27.°]. It was concluded that tacA encodes a small RNA required for splicing, perhaps the central domain of a group II intron [27"]. With the aadA-disrupted tar_A, mutants completely deficient is psaA trans-splicing could be obtained [25"]. This result paves the way for more detailed structure-function analysis of this novel chloroplast trans-splicing reaction.
Mobile group I intron Man), group I introns contain an internal ORF that encodes a site-specific DNA endonuclease. The endonuclease activity confers genetic mobility on the intron in crosses between intron-containing and intronless strains [28]. The single 888 bp group I intron of the Chlam3~ domonas reinhardtii 23S rRNA encodes a polypeptide of 163 residues [29]. This is the first example of a serf-splicing chloroplast group I intron [30], which is serf-splicing in vitro [30,31"]. There are also several group I introns in the 23S rRNA gene of Chlamydomonas eugametos. Intron5 is mobile during interspecific crosses with Chlamydomona.~ moewuaii [32]. The C eugametos intron encodes a 218 amino-acid double-strand DNA endonuclease, designated I-Ceul, with in vitro activity specific for the 'homing' site of this mobile intron [33]. The C reinhardtii intron-encoded ORF was also shown to encode a double-strand DNA endonuclease [31"], designated I-CreI. What are the activities of these intron-encoded ORFs in vivo? Are they required for splicing in vivo? Are they necessary and sufficient for intron mobility? Thompson and Herrin [34"] have studied in detail the in vitro selfsplicing of the C. reinhardtii group I intron, and in addition have characterized in vivo splicing of mutants deleted internally within the intron-encoded I-O*ellocus. Biolistic gene replacement was used to create homoplastic deletion mutants. The mutants spliced normally in vivo, indicating that I-O'el does not encode a maturase required for splicing. The mobility in vivo of the group I intron was elegantly demonstrated by Durrenberger and Rochaix [31"]. When an artificial intron homing site corresponding to a region of the intronless form of the C reinhardtii 23S rRNA was introduced into the C reinhardtii chloroplast genome by biolistic gene transfer, many of the resulting transformants had the 888 bp 23S rRNA group I intron integrated precisely at the e x o n - e x o n boundary in the artificial homing site. This demonstrates that the intron behaves as a site-specific mobile genetic element in vivo if a target sequence is present. The implication is that the I-CreI endonuclease activity encoded by the intron is required for homing actMty. This can be tested by directed mutagenesis within the I-O'el gene.
Conclusions Many important questions about chloroplast gene expression remain unanswered. Although we now have DNA sequences for numerous chloroplast-encoded polypeptides, a very large number of essential genes have not been identified. With the advent of biolistic transformation, the time is now ripe for rapid progress in gene identification by a reverse genetic approach, employing sitedirected gene disruption. Because of the ease of transformation, most progress to date has involved chloroplasts
Chloroplasts Hallick of Chlamydomonas.This work is no doubt a harbinger of advances to come in other systems, including higher plants, as the technology advances. Among the interesting aspects of gene expression awaiting detailed study are various post-transcriptional RNA-processing events. What is the mechanism of RNA editing. How are pre-edited codons selected? What is the splicing machinery for the ubiquitous group II introns? Are maturases required for group I intron splicing? We can eagerly await answers to these and other important questions.
existence of twintrons strongly support the introns late model for the origin of introns. 10.
MICHELF, UMESONOK, OZEKI H: Comparative and Functional Anatomy of Group II Catalytic Introns - - a Review. Gene 1989, 82:5-30.
11.
CHRISTOPHERDA, HAIa.ICK RB: Euglena gracilis Chloroplast Ribosomal Protein Operon: a New Chloroplast Gene for Ribosomal Protein L5 and Description of a Novel Organelle Intron Category Designated Group Ill. Nucleic Acids Res 1989, 17:7591-7608.
12.
SIEMEISTERG, BUCHHOLZC, HACHTELW: Genes for the Plasrid Elongation Factor Tu and Ribosomal Protein $7 and 6 Transfer RNA Genes on the 73-kb DNA from Astasia longa that Resembles the Chloroplast DNA of Euglena. Mol Gen Genet 1990, 220:425--432.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •. of outstanding interest 1.
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SHINOZAKIK, OHME M, TANAKA M, WAKASUGI T, HAYASHIDA N, MATSUBAYASHIT, mAlTA N, CHUNWONGSE J, OBOKATA J, YAMAGUCHI-SHINOZAKI K, ET AL: The Complete Nucleotide Sequence of the Tobacco Chloroplast Genome: Its Gene Organization and Expression. EIVlBO J 1986, 5:2043-2049. OHYAMAK, FUKUZAWAH, KOHCHI T, SHIRAI H, SANO T, SANO S, UMESONOK, SHIKIY, TAKEUCHIM, CHANGZ, ETAL: Chloroplast Gene Sequence Deduced from the Complete Sequence of Liverwort Marchantia Polymorpha Chloroplast DNA. Nature 1986, 322:572-574. HIRATSUKA j, SH1MADAH, WHITI'IER R, ISHIBASHIT, SAKAMOTOM, MORI M, KONOO C, HONJI Y, SON CR, MENG B-Y, Er m.: The Complete Sequence of the Rice (Oryza sativa) Chloroplast Genome: Intermolecular Recombination b e t w e e n Distinct tRNA Genes Accounts for a Major Plastid DNA Inversion during the Evolution of the Cereals. Mol Gen Genet 1989, 217:185-194.
4. •.
HOCH B, MAmR RM, APPEL K, IGLOI GL, KOSSELH: Editing of a Chloroplast mRNA by Creation of an Initiation Codon. Nature 1991, 353:178--180. The initial report of mRNA editing in chloroplasts. PCR was used to amplify cDNA copies of edited mRNA. The possibility of a mitochondrial origin for the cDNA was ruled out. 5.
KUDLAJ, IGt.OI GL, METZLAFFM, I-L,'~GEMANNR, KOSSELH: RNA Editing in Tobacco Chloroplasts Leads to the Formation of a Translatable p~bL mRNA by a C to U Substitution within the Initiation Codon. ~14B0 J 1992, 11:1099-1103.
MAIER RIM, HOCH B, ZELTZ P, KOSSEL H: Internal Editing of the Maize Chloroplast ndhA Transcript Restores Codons for Conserved Amino Acids. Plant Cell 1992, 4:609-616. Using PCR, evidence is given for several internal editing sites in the nd-hA transcript. The comparison of mRNA editing between mitochondrial and chloroplast-encoded pre-mRNAs is of particular interest.
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COPERT1NODW, HAmCK RB: Group ll Twintron: an Intron within an Intron in a Chloroplast Cytochrome b-559 Gene. EMBO J 1991, 10:433-442. Original experimental evidence for the existence of twintrons (introns within introns). 14.
CHRISTOPHERDA, CUSHMANJC, PRICE CA, HALUCKRB: Organization of Ribosomal Protein Genes rpi23, rpl2, rpsl9, rp122 and rps3 on the Euglena gracilis Chloroplast Genome. Cure" Genet 1988, 14:275-285.
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CHRISTOPHERDA, HALUCK RB: Complex RNA Maturation Pathway for a Chloroplast Ribosomal Protein Operon with an Internal tRNA Cistron. Plant Cell 1990, 2:659-671.
16.
STEVENSONJK, DRAGER RG, COPERT1NO DW, ET AL: Intercistronic Group III lntrons in Polycistronic Ribosomal Protein Operons of Chloroplasts. Mol Gen Genet 1991, 228:183-192.
17.
COPERTINODW, SHIGEOKAS, HALL1CKRB: Chloroplast Group HI Twintron Excision Utilizing Multiple 5'- and 3'-Splice Sites. EMBO J 1992, in press.
18.
DORITRL, SCHOENBACHL, GILBERTW: How Big is the Universe of Exons? Science 1990, 250:1377-1382.
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DORITRL, GILBERTW: The Limited Universe of Exons. Curr Opin Genet Dev 1991, 1:464-469.
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DRAGERRG, HALIACKRB: A Novel E u g l e n a gracilis Chloroplast Operon Encoding four ATP Synthase Subunits and Two Ribosomal Proteins Contains Seventeen Introns. Curr Genet 1992, in press.
21.
BOYNTONJE, GILLHm~I NW, HARRIS EFI, HOSLER JP, JOHNSON AM, JONES AR, RANDOLPH-ANDERSONBL, ROBERTSON D, KLEIN TM, SHARK K13, SANFORDJC: Chloroplast Transformation in Chlamydomonas with High Velocity Microprojectiles. Sci. ence 1988, 240:1534-1538.
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SVAB Z, HAJDUKIEW1CZP, MAHGA P: Stable Transformation o f Plastids in Higher Plants. Proc Natl Acad Sci USA 1990, 87:852643530.
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HODGESP, Sco'rr J: Apolipoprotein B mRNA Editing: a New Tier for the Control of Gene Expression. Trends Biochem Sci 1992, 17:77-81.
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GREENBERGBM, GRUISSEMW, HALUCK RB: Accurate Processing and Pseudouridylation of Chloroplast Transfer RNA in a Chloroplast Transcription System. Plant Mol Biol 1984, 3:97-109.
COPERTINODW, CHPdSTOPHERDA, HALLICKRB: A Mixed Group H/Group III Twintron in the E u g l e n a gracilis Chloroplast Ribosomal Protein $3 Gene: Evidence for Intron Insertion during Gene Evolution. Nucleic Acids Res 1991, 19:6491-6497. This is the initial report of a 'mixed' twintron, comprised of one group II intron internal to a group Ill intron. An extensive phylogenetic analysis of the known rps3 genes is presented. It is also argued that the 9.
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23. •
KLEINU, DE CJ, BOGORAD L: Two Types of Chloroplast Gene Promoters in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 1992, 89:3453--3457. Analysis of promoters for Chlamydomonas chloroplast a~OB or 16S rRNA-encoding genes coupled to the E. coli [~-glucuronidase (GUS) reporter gene in transgenic chloroplasts. 24.
NEWMANSM, GIUMAM NW, HARRISEH, JOHNSON AM, BOYNTON JE: Targeted Disruption of Chloroplast Genes in Chlamydomonas reinhardtii. 291ol Gen Genet 1991, 230:65-74. Describes an elegant cotransformation approach to modifying two plastid genes in a single particle bombardment. •
25. .,
GOLDSCHMIDT-CLERMONTM: Transgenic Expression of Aminoglycoside Adenine Transferase in the Chloroplast: a Selectable Marker of Site-directed Transformation o f Chlamydomonas. Nucleic Acids Res 1991, 19:4083-4089. Describes the targeted gene disruption with a dominant selectable marker based on a bacterial antibiotic-resistance gene. One important
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Genomes and evolution experiment is the disruption of art ORF of unknown function. Transformants were persistently heteroplasmic, perhaps indicating that the disrupted locus is essential for cell growth. 26. •
TAKAHASHIY, GOLDSCHMIDT-CLERMONTM, SOEN SY, FRANZEN LG, ROCHAIXJD: Directed Chloroplast Transformation in Chlamydomonas reinhardtii: Insertional Inactivation of the psaC Gene Encoding the Iron Sulfur Protein Destabilizes Photos}stem I. EMBO J 1991, 10:2033-2040. An excellent example of the use of transgenic chloroplasts to detemline the function of a chloroplast gene product in photos3~tem membrane assembly. 27.
GOLDSCHMIDT-CLERMONTM, CHOQUETY, GIRARDBJ, MICHELF, SCHIRMERRM, ROCHAIXJD: A Small Chloroplast RNA May be Required for Trans-splicing in Chlamydomonas reinhardtii. Cell 1991, 65:135-143. First demonstration of a tram-acting RNA required for chloroplast splicing. Often cited as a potential model for the origin of transacting RNAs in nuclear splicing.
31. **
DURRENBERGERF, ROCHA~JD: Chloroplast Ribosomal Intron of Chlamydomonas reinhardtii: in Vitro Self-splicing, DNA Endonuclease Activity and in Vivo Mobility. EMBO J 1991, 10:3495-3501. A extremely elegant experimental approach to demonstrating intron mobility in viva This system may be amenable to the assay of mobility of foreign introns. 32.
MARSHALL P, LEMIEUXC: Cleavage Pattern of the Homing Endonuclease Encoded by the Fifth Intron in the Chloroplast Large Subunit rRNA-encoding Gene of Chlamydomonas eugametos. Gene 1991, 104:241-245.
33.
GAUTHIERA, TURMEL M, LEMIEUXC: A Group I Intron in the Chloroplast Large Subunit rRNA Gene of Chlamydomonas eugametos Encodes a Double-strand Endonuclease that Cleaves the Homing Site of this Intron. Curt Genet 1991, 19:43-47.
do
28.
DUJONB, BELFORTM, BUTOW RA, JACQ C, LEMIEUXC, PERLMAN PS, VOGTVM: Mobile Introns: Definition of Terms and Recommended Nomenclature. Gene 1989, 82:115-118.
29.
ROCHAIXJD, RAHIREM, MICHELF: The Chloroplast Ribosomal lntron of Chlamydomonas reinhardii Codes for a Polypeptide Related to Mitochondrial Maturases. Nucleic Acids Res 1985, 13:975-984.
30.
HERRINDL, CHEN Y'F, SCHMIDTGW: RNA Splicing in Chlamydomonas Chloroplasts. Self-splicing of 23 S preRNA. J Biol Cbem 1990, 265:21134-21140.
THOMPSONAJ, HERRIN DL: In Vitro Serf-splicing Reactions of the Chloroplast Group I Intron Cr.LSU from Chlamydomonas reinhardtii and in Vivo Manipulation via GeneReplacement. Nucleic Acids Res 1991, 19:6611-6618. Using gene disruption, the authors anab'ze the role of the I-Crel endonuclease in splicing. Includes an in t,itro characterization of the self-splicing reaction. 34. •
RB Hallick, Department of Biochemistry, University of A_rizona, Tucson, Arizona 85721, USA.
