Nonreciprocal recombination between alleles of the chloroplast 23S rRNA gene in interspecific
            <i>Chlamydomonas</i>
            crosses

Lemieux, Claude; Lee, William R.

doi:10.1073/pnas.84.12.4166

Cited by 58 publications

(21 citation statements)

References 31 publications

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“…5) in the chloroplast large subunit rRNA gene (rrnL) of the green alga Chlamydomonas eugametos introduces a double-strand break near the insertion site of the intron in cognate intronless allele (Gauthier et al, 1991 ;Marshall and Lemieux, 1991). In interspecific crosses between C. eugametos and Chlamydomonas moewusii, this double-strand break initiates a very efficient unidirectional gene conversion event (Lemieux and Lee, 1987) during which the intron is most probably inserted by the double-strand break repair mechanism proposed by Szostak et al (1983). This genetic phenomenon, known as 'intron homing', was first observed at the omega locus in the mitochondrial rrnL gene of Saccharomyces cerevisiae (Jacquier and Dujon, 1985;Zinn and Butow, 1985) and was later reported for a number of introns present in various organisms (see Lambowitz and Belfort, 1993 (Colleaux et al, 1988;Marshall and Lemieux, 1992;Sargueuil et al, 1990;Wernette et al, 1992), the insertion of group I introns into target genomes is highly specific.…”

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The I‐Ceu I endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns

Marshall

Davis

Lemieux

1994

European Journal of Biochemistry

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During genetic crosses between the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii, the I‐Ceul endonuclease encoded by the fifth group I intron (CeLSU · 5) in the C. eugametos chloroplast large subunit rRNA gene mediates the mobility of this intron by introducing a double‐strand break near the insertion site of the intron in the corresponding C. moewusii intronless allele. To characterize the biochemical properties of this endonuclease, we have purified I‐CeuI and determined the optimal reaction conditions for cleavage. I‐CeuI activity is maximal at 50°C, pH 10.0, 2.5 mM MgCl2 and in the absence of NaCl. Unlike the well‐characterized I‐SceI endonuclease, I‐CeuI remains stable following preincubation in the absence of substrate. We discuss the role that homing endonucleases may have played in the evolution of Chlamydomonas chloroplast group I introns.

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The I‐Ceu I endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns

Marshall

Davis

Lemieux

1994

European Journal of Biochemistry

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“…More recently, endonucleases have been reported for mobile introns in the nuclear rDNA of P. polycephalum (I-Ppo; 28), in the coxl gene of yeast mitochondria 11,37), and in the td and sunY genes of T-even bacteriophage (I-TevI and I-TevII; 34). It seems likely that the mobility of other group I introns, for example in the chloroplast rDNA of Chlamydomonas eugametos, will be explained by similar endonucleases (24,25). Some group I introns, as well as group II introns, also encode maturases (proteins involved in the splicing of the intron RNA in vivo), and at least in the case of I-Scell, an intron-encoded endonuclease can function as a maturase under special circumstances (14, 20).…”

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Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

Muscarella¹,

Ellison²,

Ruoff³

et al. 1990

Mol. Cell. Biol.

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A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.Group I introns are defined by the presence of conserved sequences and structural elements (reviewed in reference 2). Many group I introns can undergo autocatalytic splicing (self-splicing) in vitro, as first described for the prototypic intron of Tetrahymena cells (5). Of the more than 60 group I introns that have been identified to date (4), most are present in the DNA of mitochondria or chloroplasts or in the DNA of T-even bacteriophage. In only three organisms have group I introns been observed in nuclear genes, and in each of these they are located in the DNA coding for ribosomal RNA (rDNA). Pneumocystis carinii contains an intron in the rDNA encoding the small-subunit rRNA (18), but neither it nor the rDNA of this organism has been extensively characterized. Several but not all Tetrahymena species and strains contain the well-known self-splicing intron in extrachromosomal rDNA coding for the large-subunit rRNA (22,23,38). Depending on the strain, Physarum polycephalum contains two or three group I introns in the extrachromosomal rDNA coding for the large-subunit rRNA (28,30,31). Some group I introns have been found to be mobile elements. They rapidly and efficiently spread in vivo from a locus that contains the intron (I+) to the same locus in a homologous gene that lacks the intron (I). This process, which has been termed intron homing (15,16), is initiated by a double-strand break in the I-locus that is introduced by a site-specific endonuclease encoded by the intron itself. The actual homing of the ...

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“…These are the first mRNA introns from chloroplasts shown to be mobile. Previously, the only known mobile chloroplast introns, Cr.LSU and Ce.LSU5, were from rRNA genes (11,31). Thus, including Cr.LSU, three of the five known group I introns in C. reinhardtii are mobile.…”

Section: Discussionmentioning

confidence: 99%

“…It is not known what fraction of the Chlamydomonas introns are mobile, but at least two seem to be mobile, and several others have been shown to encode endonucleases (reviewed in reference 21). The fifth intron of the large rRNA from Chlamydomonas eugametos (Ce.LSU5) was shown to be unidirectionally inherited (characteristic of homing) in crosses with Chlamydomonas moewusii (31), as was a region (ϳ6 kb) of the chloroplast genome from the latter species that contained group I introns in the psbA gene (3). In Chlamydomonas reinhardtii, the single intron of the large rRNA subunit (Cr.LSU) was shown to be mobile by using chloroplast transformation to introduce the target sequence (i.e., fused exons) into the genome (11).…”

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Mobile Self-Splicing Group I Introns from the psbA Gene of Chlamydomonas reinhardtii: Highly Efficient Homing of an Exogenous Intron Containing Its Own Promoter

Odom

Holloway

Deshpande

et al. 2001

Molecular and Cellular Biology

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Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 and Cr.psbA4, respectively) contain large free-standing open reading frames (ORFs). We used transformation of an intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into a chloroplast expression vector in both orientations and then cotransformed into IL along with a spectinomycin resistance marker (16S rrn). For Cr.psbA2, the sense construct gave nearly 100% cointegration of the intron whereas the antisense construct gave 0%, consistent with homing. For Cr.psbA4, however, both orientations produced highly efficient cointegration of the intron. Efficient cointegration of Cr.psbA4 also occurred when the intron was introduced as a restriction fragment lacking any known promoter. Deletion of most of the ORF, however, abolished cointegration of the intron, consistent with homing. The Cr.psbA4 constructs also contained a 3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating exon coconversion. Remarkably, primary selection for this marker gave >100-fold more transformants (>10,000/g of DNA) than did the spectinomycin resistance marker. A trans homing assay was developed for Cr.psbA4; the ORF-minus intron integrated when the ORF was cotransformed on a separate plasmid. This assay was used to identify an intronic region between bp ؊88 and ؊194 (relative to the ORF) that stimulated homing and contained a possible bacterial (؊10, ؊35)-type promoter. Primer extension analysis detected a transcript that could originate from this promoter. Thus, this mobile, self-splicing intron also contains its own promoter for ORF expression. The implications of these results for horizontal intron transfer and organelle transformation are discussed.Some group I introns are characterized by the ability to self-splice in vitro and to promote their insertion into intronless copies of the host gene in vivo (reviewed in references 10 and 29). The latter process is a type of homologous recombination known as intron homing. Such mobile introns typically contain a large open reading frame (ORF) that codes for a site-specific endonuclease. The endonuclease promotes homing by producing a double-strand break in the intronless allele near the site of intron insertion. A popular model for the subsequent events is based on the double-strand break repair model of DNA recombination in Saccharomyces cerevisiae (36), a postulate of which is that coconversion of flanking exon sequences accompanies intron acquisition.Mobile group I introns have been found in phylogenetically diverse eucaryotes and in certain bacteriophages (29). In eucaryotes, they occur in mitochondrial, chloroplast, and nuclear genes but are typically more common in the organelles. Group I intron homing has been studied intensively, however, only in bacteriophage T4, which has provided an elegant viral-procaryotic system for studying homing mechanism...

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Nonreciprocal recombination between alleles of the chloroplast 23S rRNA gene in interspecific Chlamydomonas crosses

Cited by 58 publications

References 31 publications

The I‐Ceu I endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns

The I‐Ceu I endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns

Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

Mobile Self-Splicing Group I Introns from the psbA Gene of Chlamydomonas reinhardtii: Highly Efficient Homing of an Exogenous Intron Containing Its Own Promoter

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