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Molecular and Cellular Biology, May 2001, p. 3472-3481, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3472-3481.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Obed W. Odom, Stephen P. Holloway,dagger Nita N. Deshpande,Dagger Jaesung Lee, and David L. Herrin*

Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 78712

Received 25 August 2000/Returned for modification 10 November 2000/Accepted 27 February 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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/µg 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.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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 mechanisms (references 24 and references therein).

The chloroplast genome of the unicellular chlorophytes Chlamydomonas spp. is a rich source of group I introns (23, 39-42). They are known mostly from the rRNA genes, a frequent home for group I introns in general, but also occur in photosynthesis genes. 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 (~6 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). The target DNA was efficiently invaded by the endogenous intron, and this process was subsequently shown to be dependent on the intron's ORF (12). Thus, the chloroplast transformation system in C. reinhardtii makes it an excellent system for studying intron homing in a eucaryote.

The C. reinhardtii psbA gene, which codes for the D1 protein of photosystem II (13), contains four self-splicing group I introns, Cr.psbA1 to Cr.psbA4 (7, 14, 20, 23). Cr.psbA2 and Cr.psbA4 contain free-standing ORFs with motifs representative of the GIY-YIG and H-N-H homing endonuclease families, respectively (23). It was also suggested that the Cr.psbA4 ORF contains the unusual arrangement of having both motifs in the same polypeptide (23). The Cr.psbA4 intron is clearly related to the first intron of the C. moewusii psbA gene (Cm.psbA1), and it was recently reported that the ORF in the Cm.psbA1 intron encodes an endonuclease that cuts intronless psbA DNA near the intron insertion site (9). Here we report the results of experiments on the mobility of introns Cr.psbA2 and Cr.psbA4 in vivo.

Initially, we tried the previously used approach of introducing the target (i.e., fragments and/or an intact copy of an intronless psbA gene) into a strain with a wild-type psbA gene; however, analysis of the target DNAs in the transformants gave complicated products that were difficult to interpret. Thus, we tested whether exogenous intron DNA, cloned into an expression vector, could home into an intronless psbA gene on the chloroplast chromosome (26). The results demonstrate that homing of exogenous intron DNA is quite efficient and gives easily interpretable results. Using this approach, data indicating that both Cr.psbA2 and Cr.psbA4 are mobile introns were obtained. Cr.psbA4 homing was also used to demonstrate exon coconversion and the trans functioning of the intron ORF. Surprisingly, homing of exogenous Cr.psbA4 does not require an external promoter, and expression of the ORF can be driven from a promoter within the intron itself, a phenomenon which heretofore has been reported only for bacteriophage T4 introns (17). The highly efficient homing of exogenous Cr.psbA4 using a DNA fragment that contains little more than the intron may have implications for lateral intron movement and for improving organelle transformation.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Strains and culture conditions. The C. reinhardtii wild-type 2137 and the intronless-psbA IL (26) strains were used; the latter was obtained from Udo Johanningmeier (Martin Luther University, Halle-Wittenberg, Germany). The cells were maintained on 2% agar plates containing Tris-acetate-phosphate (TAP) medium (19) plus 100 µg of ampicillin per ml.

Construction of plasmids. The construction of plasmid pb4c110, which contains a 7-kb BamHI fragment of C. reinhardtii chloroplast DNA that includes the spectinomycin resistance marker spr-u-1-6-2 in the 16S rrn gene, has been described (37). The plasmids used to study homing of Cr.psbA2 were created by subcloning the XbaI fragment of the psbA gene (Fig. 1), obtained from plasmid pGEM.R14.2 (20), into the XbaI-digested atpX plasmid (16) in both orientations. These plasmids were called atpX-i2S (sense) and atpX-i2A (antisense), respectively.


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  		FIG. 1.   Schematic map of the C. reinhardtii psbA gene and of cloned fragments used to study homing of introns 2 and 4. (A) Map of the psbA gene. The DCMU resistance marker in exon 5 is indicated. The arrow indicates the direction of transcription. (B to F) Inserts from plasmid clones, the names of which are indicated to the right. Those designated with a Delta  have the portion of the Cr.psbA4 ORF between the EcoNI and MluI sites deleted. S and A refer to sense and antisense, respectively. Restriction sites are as follows: B, BstEII; E, EcoNI; K, KpnI; M, MluI; R, EcoRI; X, XbaI.

For Cr.psbA4, the starting plasmids were pKS4, which contains the XbaI-KpnI fragment of psbA, and pBX4, which contains the BstEII-XbaI fragment (Fig. 1 and reference 23). These DNAs also contained the DCMU4 mutation in exon 5, which provides resistance to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (13). The pBX4 insert was obtained in the atpX vector as follows. pKS4 was cleaved with BstEII, end filled with Klenow polymerase, and then digested with XbaI. The gel-purified 2.1-kb fragment was ligated into atpX, which had been digested with either BamHI or PstI, end filled with Klenow fragment polymerase, and then cleaved with XbaI; cloning into BamHI-cut atpX yielded plasmid atpX-i4S (sense), and cloning into PstI-cut atpX yielded plasmid atpX-i4A (antisense). A 544-bp fragment of the intron ORF was removed from both pKS4 and pBX4 by digestion with EcoNI and MluI, end filling with Klenow polymerase, purification of the larger fragment, and religation. The resulting plasmids, containing a severely truncated ORF, were called pKS4.Delta and pBX4.Delta , respectively (Fig. 1).

Plasmids with Cr.psbA4 progressively deleted from the 5' end were constructed as follows. Plasmid pBX4 was digested with EcoRI and PstI, and the ca. 1,430-bp gel-purified fragment was ligated into EcoRI and PstI-digested pBluescript SK to give pRP4. Plasmid pRP4 was digested with HincII and PstI, and the ca. 1,230-bp gel-purified fragment was cloned into HincII and PstI-digested pBluescript SK to give pHP4. pRP4 was also digested with SspI and PstI, and the ca. 1,120-bp gel-purified fragment was cloned into HincII- and PstI-digested pBluescript SK to give pSP4. PCR was used for further paring of the intron. To obtain PCR products beginning at bp 564 or 626 of the intron, oligonucleotides 206 and 207, respectively, were used together with oligonucleotide 100 (see Table 1) and pBX4 as the template. Pfu DNA polymerase (Stratagene) was used to maximize accuracy of the amplification according to the supplier's recommendations. The respective PCR products were gel purified and digested with PstI, and the larger fragments were cloned into HincII- and PstI-cut pBluescript SK to give p564P4 and p626P4, respectively; at least two clones of each plasmid were used in the homing experiments. The inserts from pHP4, pSP4, p564P4(2), and p626P4(2) were also cloned into both pUC18 and pUC19 by digestion with PstI and KpnI, gel purification, and ligation into PstI-and KpnI-digested vector DNA. The resultant plasmids were called pHP4-pUC18, pSP4-pUC18, p564P4-pUC18(2), p626P4-pUC18(2), pHP4-pUC19, pSP4-pUC19, p564P4-pUC19(2), and p626P4-pUC19(2).

Chloroplast transformation. DNA was introduced into C. reinhardtii chloroplasts by particle bombardment (2, 27) using a helium-driven apparatus (Bio-Rad He 1000). The conditions were 28.5 in. of vacuum, use of a 1,100-lb/in2 rupture disk, the plate on shelf 4, and the macrocarrier in position 2. The recipient cells were grown in liquid TAP to 2 × 106 to 4 × 106 cells/ml, collected by centrifugation, and resuspended to 1 × 108 cells/ml in TAP. Then, 0.5 ml of cells (~5 × 107 cells) were mixed with 0.5 ml of molten (42°C) 0.25% agar in TAP-minimal medium, pipetted onto the center of a TAP plate (2% agar and 100 µg of ampicillin per ml), and spread over roughly three-fourths of the surface (110-mm-diameter plates). Typically, 7 µg of DNA was precipitated onto 3 mg of tungsten particles (M17; Bio-Rad), and one-eighth of this slurry (~0.4 mg of tungsten and 875 ng of DNA) was shot at each plate. After the shooting, the plates were kept overnight in dim light. The next day, the cell layer was scraped off and respread onto two selective plates (100 µg of spectinomycin per ml in TAP or 3 µM DCMU in minimal medium), which were incubated under moderately bright light (~2,000 lx) at 23°C. Spectinomycin-resistant transformants usually began appearing after about 1 week and continued to appear for up to 3 weeks thereafter. DCMU-resistant transformants required about 2 weeks to begin appearing and continued to appear for 3 to 4 weeks thereafter.

PCR analysis of transformants. Cells for PCR analysis were regrown on selective plates, and DNA was prepared by the method of Dürrenberger et al. (12). Standard protocols were used for PCR with Taq polymerase. The temperature regimen was as follows: 4 min at 94°C and 24 cycles of 60°C (1 min), 70°C (5 min), and 94°C (30 s), which was followed by 1 min at 60°C and 8 min at 70°C. The primers are given in Table 1. The PCR products were analyzed on 1% agarose gels containing 40 mM Tris-acetate (pH 8.0), 1 mM EDTA, and 0.2 µg of ethidium bromide per ml. In some experiments, the PCR products were eluted from the gel and subjected to automated sequencing using the same primers.

                              
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  		TABLE 1.   Oligonucleotides used as primers within the psbA gene

Primer extension analysis of transcript 5' ends. Primer extension was performed using oligonucleotides 161 and 276, which are complementary to portions of the Cr.psbA4 ORF (Table 1). For the annealing step, 50 µg of total RNA (extracted from log-phase cultures) was mixed with 1 ng of an oligonucleotide primer whose 5' end had been labeled with 32P, in 10 mM Tris-HCl (pH 7.5)-300 mM NaCl-2 mM EDTA (10 µl), heated for 3 min at 85°C, and cooled to 42°C for 30 min. To this was added 40 µl of prewarmed (42°C) reverse transcription mix, which contained 1 mM (each) deoxynucleoside triphosphate, 75 µg of actinomycin D per ml, 125 µg of acetylated bovine serum albumin per ml, 20 U of placental RNase inhibitor (Roche), and 12.5 U of avian myeloblastosis virus reverse transcriptase MP (Life Sciences, Inc.) in 10 mM Tris-HCl (pH 8.4)-7.5 mM MgCl2-10 mM dithiothreitol. The reaction mixtures were incubated for 30 min at either 42 or 50°C, and then the reactions were stopped by adding EDTA to 10 mM followed by RNase A to 20 ng/µl. After digestion of the RNA for 30 min at 37°C, the samples were phenol-chloroform extracted and precipitated with ammonium acetate and ethanol. The cDNAs were taken up in 2.3 µl of 1× Tris-borate-EDTA, and then a 1.2 volume of gel-loading solution (90% formamide, 50 mM EDTA [pH 8.0], 0.01% xylene cyanole, 0.01% bromophenol blue) was added, and the cDNAs were heated at 80°C for 3 min. The cDNAs were separated by electrophoresis on a 6% polyacrylamide sequencing gel, alongside sequence ladders that were prepared using the same end-labeled primer and plasmid pBX4 (23) as the template. The ladders were made by using the fmol DNA cycle sequencing system (Promega). For precise alignment of the cDNAs with those of the sequence ladder, it was necessary that all samples be in the same loading buffer and that equal volumes be loaded on the gel. The gels were run at 60 W (~50°C) until the xylene cyanole migrated ~90% of the length of the gel.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Integration of introns Cr.psbA2 and -A4 from atpX constructs. To test for Cr.psbA2 homing, the intron, plus flanking exon sequences, was cloned into the chloroplast expression vector atpX (16) in both orientations (Fig. 2A). The short exon sequences (82 and 141 bp) were not expected to promote integration via simple (i.e., nonhoming) homologous recombination with any efficiency. Each construct was then introduced into the IL strain along with plasmid pb4c110, which contains a spectinomycin resistance marker in the 16S rrn gene (35). Transformants were selected on spectinomycin, and then integration of the intron was assessed by PCR with primers in exons 2 and 3; Figure 2B and C show the analysis of several representative transformants. Intron integration was observed in >95% (33 of 34) of the transformants obtained with pb4c110 and the sense plasmid (atpX-i2S) but in 0% (0 out of 23) of the transformants obtained with pb4c110 and the antisense plasmid (atpX-i2A). Southern blot analysis of several of the transformants agreed with the PCR analysis (data not shown). This result is consistent with integration of Cr.psbA2 by intron homing. Also, all of the intron-containing transformants grew normally on minimal medium in bright light, indicating that the intron integrated correctly.


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  		FIG. 2.   PCR analysis of Cr.psbA2 integration in spectinomycin-resistant transformants. The IL strain was bombarded with pb4c110, which confers spectinomycin resistance, and with either atpX-i2S or atpX-i2A. DNAs were extracted from several spectinomycin-resistant transformants and analyzed by PCR using primers that flank the intron. (A) Diagram of the chimeric genes created by insertion of Cr.psbA2 and flanking sequences into the atpX vector in both orientations. The diagram as written represents the sense orientation (atpX-i2S). The shaded portion of the intron is its ORF; E2, E3, and I3 are, respectively, part of exon 2, the complete exon 3, and a portion of intron 3. The promoter and 5' untranslated region from the atpA gene are indicated (5'atpA), as is the 3' region of the rbcL gene (3'rbcL). (B) Diagram of the locations of primers used for PCR and the sizes of the products expected with and without intron integration. Numbered boxes represent the psbA exons, which are not drawn to scale. The primers were 118 and 136, respectively (Table 1). (C) Agarose gel analysis of the PCR products obtained from the atpX-i2S and atpX-i2A transformants (six clones of each type). For reference, PCR products obtained from the untransformed IL strain (far left lane) and from the atpX-i2S plasmid (far right lane) are also shown. Numbers to the left refer to the positions and sizes (in kilobases) of selected markers from the 1-kb Plus DNA marker (Life Technologies).

To test for homing of Cr.psbA4, the BstEII-XbaI fragment from pBX4 (Fig. 1) was subcloned into atpX in both orientations, and the DNAs were separately cotransformed into IL along with the spectinomycin resistance marker. The results of PCR analysis of several randomly selected spectinomycin-resistant transformants are shown in Fig. 3. In this analysis, the PCR was performed with one primer in exon 2 and one in the intron, so that a product would be obtained only if Cr.psbA4 was present. All of the cotransformants that were examined from the sense (atpX-i4S) transformation (10 of 10) contained the integrated intron; however, 80% (i.e., 8 of 10) of those examined from the antisense (atpX-i4A) transformation also contained the intron (the two intron-negative transformants gave a larger than expected PCR product that, upon sequencing, proved to be unrelated to intron homing). As with the Cr.psbA2 intron, all of the Cr.psbA4-containing transformants grew similarly to the wild type on minimal medium in bright light, indicating normal psbA expression.


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  		FIG. 3.   PCR analysis of Cr.psbA4 integration in spectinomycin-resistant transformants. The IL strain was bombarded with the spectinomycin resistance plasmid and either atpX-i4S or atpX-i4A, and DNAs from spectinomycin-resistant transformants were analyzed by PCR. (A) Diagram of the insert in the atpX-i4S (sense orientation) and atpX-i4A (antisense) expression plasmids. The insert as drawn is in the sense orientation. E4 and E5 refer to parts of exons 4 (50 bp) and 5 (269 bp), and the intron ORF is shaded. (B) Diagram showing the locations of the primers used for PCR. Numbered boxes are the psbA exons, which are not to scale. The primers were oligonucleotides 118 and 116 (Table 1). A 0.74-kb PCR product was expected if Cr.psbA4 had integrated; no product was expected from intronless DNA. (C) Agarose gel analysis of the PCR products from spectinomycin-resistant transformants (five clones of each type). The position of the 0.5-kb HindIII-lambda DNA marker is indicated.

The efficient cointegration of Cr.psbA4 from the expression vector in both orientations was unexpected and was investigated further by cotransforming the IL strain with the pKS4 and pBX4 plasmids (Fig. 1). These plasmids use a pBluescript vector (Stratagene), which lacks any chloroplast DNA sequences. The same result was obtained (data not shown). In addition, cotransformation of IL with just the inserts from pKS4 and pBX4 resulted in cointegration of the intron in spectinomycin-resistant transformants (data not shown). These results seemed to indicate that either Cr.psbA4 integration was not dependent on expression of the intron ORF or a promoter for transcription of the ORF was internal to the intron.

Determining if the ORF is required for efficient integration of Cr.psbA4. To determine if efficient Cr.psbA4 integration required the intact ORF, plasmids pKS4.Delta and pBX4.Delta , which have a truncated ORF, were created (Fig. 1). The ORF deletion (544 bp) also caused a frameshift, which placed a new stop codon (TAA) 6 bp downstream of the deletion. Hence, the new ORF codes for a 33-amino-acid peptide that lacks the conserved motifs (23). These plasmids were cotransformed into IL along with pb4c110, and DNAs from spectinomycin-resistant transformants were analyzed by PCR using primers that flank the intron; representative analyses are shown in Fig. 4. None (0 of 14) of the pBX4.Delta cotransformants and only one (of 14) of the pKS4.Delta cotransformants analyzed contained the intron. In contrast, all of the cotransformants examined from the pKS4 or pBX4 bombardments (15 of each), which were performed in parallel, were homoplasmic for the intact intron (Fig. 4). This result indicates that efficient Cr.psbA4 integration requires the ORF and is consistent with homing.


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  		FIG. 4.   Effect of severely truncating the ORF on Cr.psbA4 integration in spectinomycin-resistant transformants. Plasmids pKS4 and pBX4 and their truncated derivatives pKS4.Delta and pBX4.Delta were separately introduced into the IL strain, along with the spectinomycin resistance plasmid (pb4c110). Transformants were selected on spectinomycin, and the presence of Cr.psbA4 was examined by PCR. (A) Diagrams showing the locations of primers used for PCR and the expected sizes of the products if the intact intron (1.97 kb), truncated-ORF intron (1.42 kb), or no intron (0.14 kb) had integrated. Numbered boxes are the psbA exons, which are not drawn to scale. The primers used were 99 and 100 (Table 1). (B) Agarose gel analysis of the PCR products from transformants selected on spectinomycin plates. The + sign before a plasmid indicates that the analysis is of a transformant bombarded with that plasmid; all the transformations also included plasmid pb4c110. PCR products from the pKS4 plasmid itself (pKS4) or from the untransformed IL strain were included as markers. Numbers at the left refer to positions and sizes (in kilobases) of DNA markers.

All of the constructs used to test for homing of Cr.psbA4 also contained a DCMU resistance marker in exon 5 (13), 34 bp downstream of the intron (Fig. 1). This marker provided a convenient test for exon coconversion. When the spectinomycin-resistant cotransformants that had acquired Cr.psbA4 were tested for growth on minimal medium plus DCMU (3 µM), all of them grew well, whereas the spectinomycin-resistant transformants that lacked the intron did not (not shown), indicating that coconversion of the downstream exon accompanied intron integration. Also, sequencing of the PCR products from three of the pBX4 transformants confirmed the coconversion of exon 5 and the accuracy of intron insertion (data not shown).

We also determined if DCMU resistance could be used as a primary selection for Cr.psbA4 integration. Thus, the IL strain was transformed with the spectinomycin resistance plasmid and either pKS4 or pBX4; then half the transformants were plated on spectinomycin and half were plated on DCMU. The results of this experiment are summarized in Table 2. The number of transformants obtained with DCMU selection was 100- to 200-fold greater than that obtained with spectinomycin, and moreover, all of the cotransformants selected on spectinomycin also contained the DCMU resistance marker. Two repeats of these transformations gave similar results. Importantly, all of the DCMU-resistant transformants that were examined by PCR contained the Cr.psbA4 intron. These results indicate that the integration of Cr.psbA4 (and its flanking marker) is much more efficient than the integration of the ~7-kb chloroplast DNA fragment containing the spectinomycin-resistant 16S rRNA gene. The table also shows that none of the DCMU-selected transformants that were examined further contained the spectinomycin resistance marker. This presumably reflects the great difference in the integration efficiencies of these two markers. A large number, perhaps hundreds, of DCMU-resistant transformants would probably have had to be analyzed to find ones with the spectinomycin marker.

                              
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  		TABLE 2.   Comparison of results of spectinomycin selection (i.e., cotransformation) with those of DCMU selection for assessing Cr.psbA4 integration

The Cr.psbA4 ORF can promote intron integration in trans. The foregoing results suggested that an intact ORF was needed for intron 4 integration. However, at least one alternative explanation, that the truncated-ORF intron is incompatible with cell growth, was also possible. To address this, a wild-type strain was transformed with the pKS4.Delta plasmid (Fig. 1) and transformants were selected for DCMU resistance. In this experiment, the DNA should integrate by simple homologous recombination. The number of transformants was 10- to 20-fold lower than with the IL strain, but ~50% of those analyzed by PCR were homoplasmic for the ORF-truncated intron (data not shown). Thus, the ORF-truncated version of Cr.psbA4 is not incompatible with cell growth.

With this encouraging result, we decided to see if the Cr.psbA4 ORF could promote integration of the truncated-ORF intron in trans (1). The IL strain was cotransformed with three plasmid DNAs: pb4c110, pKS4.Delta , and pRP4, which donates the ORF but should be incapable of integrating itself, since it lacks exon sequences (Fig. 5A). Transformants were selected on spectinomycin, and the PCR analysis of several is shown in Fig. 5B and C. In this series, the downstream primer annealed to exon 5 as shown in Fig. 4, but the upstream primer annealed to exon 3 instead of exon 4. All 10 of the transformants examined by PCR contained the intron from pKS4.Delta . They also contained the DCMU marker, based on their ability to grow on DCMU plates (not shown). Sequencing of the PCR products from two of the transformants confirmed the presence of the nucleotide substitution that confers DCMU resistance (13) and that the correct exon-intron junctions were present (data not shown). When a similar transformation was plated on DCMU, a large number of transformants was obtained (similar to that with pKS4) (Table 2) and all 10 that were examined by PCR contained the truncated-ORF intron (data not shown). Finally, transformation with pb4c110, and either pRP4 or pKS4.Delta , did not give any colonies on DCMU plates or any intron integration among spectinomycin-resistant transformants, as expected. These results confirm that Cr.psbA4 undergoes homing and, moreover, indicate that the ORF can act in trans to promote integration of the disabled intron.


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  		FIG. 5.   The Cr.psbA4 ORF can act in trans to promote integration of the truncated-ORF intron. The IL strain was cotransformed with three plasmids, pb4c110, pRP4, and pKS4.Delta ; pb4c110 provided spectinomycin resistance, pRP4 provided the intact intron ORF, and pKS4.Delta provided the truncated-ORF intron with flanking exonic sequences (and DCMU resistance). The transformants were selected on spectinomycin, and DNAs from several transformants were analyzed by PCR for intron integration. (A) Diagram of the inserts of plasmids pRP4 and pKS4.Delta . The shading (dark for exons and light for intron ORFs) and restriction sites are as in Fig. 1, except for the addition of P (PstI). The ORF in pKS4.Delta is reduced to 33 amino acids. (B) Diagram showing the locations of the primers used for PCR and the expected sizes of the PCR products if the transformants contained the truncated intron or no intron. Numbered boxes are the psbA exons, which are not to scale. The primers were oligonucleotides 176 and 100 (Table 1). (C) Agarose gel analysis of the PCR products obtained from five spectinomycin-resistant transformants (+pRP4+pKS4.Delta ). The PCR product from the recipient strain IL was also analyzed as a control. The positions and sizes (in kilobases) of selected DNA size markers are indicated to the left.

Identification of a promoter upstream of the Cr.psbA4 ORF. The fact that plasmid pRP4 promoted homing of the ORF-disabled Cr.psbA4 in trans suggested that if a promoter was present in Cr.psbA4, it should reside in this fragment, presumably upstream of the ORF. Thus, the RP fragment was progressively deleted from its 5' end and the effect on trans homing was assessed. Transformation efficiency with DCMU selection was used as the indicator for homing efficiency; this greatly increased the range and sensitivity compared to cotransformation with the spectinomycin resistance marker (Table 2). Figure 6A shows the intron subfragments tested as ORF donors, and Fig. 6B shows the relative transformation efficiency (which was found to be less variable than the absolute numbers of transformants) with each. The data show no decrease and even show an increase in homing efficiency as the ORF donor DNA was deleted downstream of the ORF and upstream of it beyond the HincII site. However, deleting the DNA between the HincII and SspI sites caused a marked decrease in homing efficiency. Additional downstream deletions further reduced the efficiency to a very low level, but somewhat surprisingly, homing was still detectable even when the first 30 bp (i.e., 10 amino acids) of the ORF were missing (626P fragment). This result indicates that the intronic sequence between the HincII and SspI sites is required for efficient homing in trans.


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  		FIG. 6.   Effect of deletions in the ORF donor DNA on trans homing of Cr.psbA4. The trans homing assay was performed as described in the legend to Fig. 5, except for varying the plasmid that donates the ORF and selecting the transformants on DCMU. With fragment BX, however, it was not necessary to add plasmid pKS4.Delta , since sufficient exon sequences were present for homing of BX. (A) Series of Cr.psbA4 fragments used as ORF donors. Restriction site abbreviations are as described in the legend to Fig. 1, with the following additions: H, HincII; P, PstI; and S, SspI. (B) Relative transformation efficiency as a function of the ORF donor construct. The entry "NONE" indicates that no ORF donor plasmid was added. The data were normalized relative to the BX transformation, which was set to 100%. The values are averages of two to three transformations that varied <25%. (C) Sequence of Cr.psbA4 from the HincII site 5' of the ORF to just past the EcoNI site within the ORF and possible -10 and -35 promoter elements. Also shown are the initiator ATG (double underlined), the 5' restriction sites used to generate fragments HP (HincII) and SP (SspI), the positions of the 5' ends of fragments 564P (564) and 626P (626), a possible Shine-Dalgarno sequence (SD, boxed), and a possible alternative translation initiator, ATT (dashed underline). The positions of the oligonucleotides (oligo) used for primer extension (161 and 276) (Fig. 7) are indicated by horizontal arrows over the sequence. The 5' ends of the transcripts mapped by primer extension (Fig. 7) are indicated by the vertical arrows. Below the Cr.psbA4 sequence are the consensus E. coli -10 and -35 promoter elements.

To assess any potential effects of the vector (pBluescript) in the ORF donor plasmids, the HP, SP, 564P, and 626P intron fragments were also subcloned into pUC18 and pUC19. These vectors lack phage promoters, and with pUC19, the inserts are in the antisense orientation with respect to the lac promoter and thus have no identifiable outside promoter. The results of the trans homing assay with the pUC vectors were similar to those obtained with the pBluescript derivatives (data not shown).

Figure 6C shows the sequence of the Cr.psbA4 intron from the HincII site to 105 bp downstream of the putative translation start site. Between the HincII and SspI sites is a sequence that exactly matches the Escherichia coli consensus -10 promoter element, and 17 bp upstream is a putative -35 sequence. The latter diverges more from the consensus -35 hexamer, but overall, these sequences constitute the strongest putative promoter upstream of the ORF. The data in Fig. 6B suggest that this might not be the only promoter in this region, however, since some trans homing occurred with constructs lacking this motif. We therefore show other possible -10 motifs located further downstream, including one that is 30 bp into the ORF. Interestingly, there is also a possible alternative start codon, ATT (i.e., AUU), 26 bp downstream of the putative -10 motif in the ORF. The -10 motif and alternative start codon located downstream of nt 626 might account for the residual homing seen with the 626P fragment. AUU has been found to function, albeit poorly, as a start codon in the C. reinhardtii petA and petD genes (4). Initiation at the indicated ATT would result in a protein missing the first 21 amino acids of the N terminus, which are not conserved between this protein and the Cm-psbA1 ORF (23).

Primer extension analysis was performed to look for additional evidence of a promoter upstream of the Cr.psbA4 ORF. Figure 7 shows the results obtained with a primer that annealed to the 5' end of the ORF beginning with the G of the initiation codon (oligonucleotide 276) (Fig. 6C). We obtained a major doublet of cDNAs with RNA from the wild type, but not from IL, that mapped 5 and 6 bp downstream of the consensus -10 element between the HincII and SspI sites (Fig. 6C). It is common for chloroplast transcripts to map a little closer to the -10 element than in E. coli (5 to 7 bp versus 7 bp), and these are certainly in the normal range (8, 18). Increasing the temperature of the reaction from the standard 42 to 50°C had no affect on the major doublet (Fig. 7), consistent with these bands corresponding to transcript ends. Primer extension analysis of wild-type and IL RNA preparations was also performed with an oligonucleotide that annealed 40 nucleotides (nt) into the ORF (oligonucleotide 161) (Fig. 6C), and the same results were obtained (data not shown). There was no evidence for a major transcript starting at the possible -10, -35 promoter that is closer to the ORF, between the SspI site and the Shine-Dalgarno sequence (Fig. 6C). Perhaps this is not surprising since these putative -10 and -35 elements are further apart (24 bp) than is typical (18). Also, although we have identified a possible Shine-Dalgarno sequence 22 nt upstream of the ORF, it should be noted that the function of this sequence is RNA specific (15, 33). Finally, the relatively long film exposure time (~48 h) required for the autoradiographic image in Fig. 7 suggests that the doublet of transcripts at -91 (relative to the ORF) are of fairly low abundance.


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  		FIG. 7.   Primer extension analysis with an end-labeled oligonucleotide that anneals 3 nt into the Cr.psbA4 ORF. Total RNAs from the wild type (WT lanes) and the IL strain (IL lanes) were used for primer extension with end-labeled oligonucleotide 276 (Fig. 6C) at 42°C (standard temperature) and at 50°C. The sequence ladder (lanes A, T, C, and G) was generated with the same primer, and pBX4 was the template. The reaction mixtures were separated on a 6% polyacrylamide sequencing gel, which was dried and exposed to BioMax MS X-Ray film (Kodak) at -70°C with a Dupont Cronex intensifying screen for ~48 h. The locations of the major bands and the sequences of this region and its complement (i.e., coding strand) are indicated to the left.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

These results demonstrate that Cr.psbA4 and probably Cr.psbA2 are mobile introns capable of efficiently and correctly inserting into an intronless psbA gene. 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. The ability of Cr.psbA4 to home may explain why it has been present in the Chlamydomonas lineage for a long time (23), despite being unessential (26).

The presence of the DCMU resistance marker in exon 5 of the Cr.psbA4 constructs allowed us to demonstrate that exon coconversion accompanies Cr.psbA4 homing (at least for the 3' exon). Exon coconversion, which was not addressed in previous studies of Cr.LSU homing (11, 12), is consistent with the double-strand break repair model for intron homing (29, 36). We also showed that the Cr.psbA4 ORF could mediate homing of the ORF-disabled intron in trans, thus providing genetic evidence that the ORF encodes a protein required for homing. This ORF has been expressed in E. coli, and the protein has been found to have site-specific endonuclease activity against an exon 4-exon 5 fusion fragment (H.-H. Kim and D. L. Herrin, unpublished data).

Perhaps the most surprising finding is that a promoter external to intron Cr.psbA4 was not required for homing of this intron. Deletion analysis indicated that an intronic region between bp -88 and -194 (relative to the ORF) stimulates Cr.psbA4 homing in trans, and sequence analysis revealed a consensus -10 and possible -35 promoter elements in this region. It should be noted that Klein et al. (28) showed that a -35 element was not necessary for transcription of the atpB gene in C. reinhardtii chloroplasts; thus, the consensus -10 element may be sufficient to drive ORF expression. Consistent with this being a functional promoter, primer extension analysis with two different oligonucleotides revealed a major doublet of RNAs that mapped 5 and 6 nt downstream of the consensus -10 sequence. Northern blot hybridization of wild-type RNA with a large internal fragment of Cr.psbA4 produced a fairly complex pattern of transcripts (7), including one just smaller than the free intron (~1.7 kb), which might correspond to the transcript that we have mapped (assuming that its 3' end is the same as that of mature psbA mRNA [23]). The doublet of bands obtained with the primer extensions may result from adjacent transcription start sites, from the reverse transcriptase adding a nontemplated nucleotide to the shorter cDNA (6), or from 5' processing in vivo. The primer extension data are also suggestive of a low level of these transcripts, which is consistent with previous studies indicating that organellar intron-encoded endonucleases typically occur at very low to undetectable levels in vivo (11, 25). Indeed, the trans-homing experiments with the 5' deletion derivatives (Fig. 6) suggest that the level of intron-encoded endonuclease needed for homing of Cr.psbA4 must be very low.

To our knowledge, intron-internal promoters for homing endonuclease ORFs have been reported for only one other system, the group I introns of T4 phage (17). The T4 intron ORFs are expressed from these promoters at fairly high levels late in infection and, interestingly, are not expressed from the early pre-mRNAs. It was proposed that, in the pre-mRNAs, the Shine-Dalgarno sequence (for ribosome binding) was occluded by a secondary structure but was not in the intron-internal transcripts. In Cr.psbA4, we do not know if the intron-internal promoter is absolutely necessary for expression of the ORF from the wild-type psbA gene. In other words, our data indicate that the internal promoter is sufficient for intron homing but do not address if it is necessary. A definitive answer to this question would require experimentation that is beyond the scope of this paper. However, given that the DNA constructs lacking all of psbA upstream of the Cr.psbA4 ORF still promoted a low level of homing in the trans assay (Fig. 6), it seems unlikely that this promoter is essential. On the other hand, it might contribute to the homing efficiency of Cr.psbA4 from the wild-type gene. The levels of free intron RNA and of unspliced precursors are very low in the light, although the latter accumulate in the dark (7). Moreover, the translation of the ORF from these transcripts may be inhibited by a secondary structure. The large size (>500 nt) and nonconserved nature of the region of Cr.psbA4 upstream of the ORF makes it difficult to predict the secondary structures in these RNAs. However, thermodynamically based predictions of the secondary structure of the first 200 nt of the RNA derived from the internal promoter indicate that the initiator AUG is in a favorable structure for translation (unpublished results). Finally, we note that the corresponding region of the homologous C. moewusii intron (Cm.psbA1) is not very similar to the Cr.psbA4 sequence and does not contain the same -10, -35 promoter.

In contrast to what occurred with Cr.psbA4, homing of exogenous Cr.psbA2 required expression from the external atpA promoter. Presumably, this is because it lacks an internal promoter, homing of Cr.psbA2 requires higher expression than does Cr.psbA4, or both. The Cr.psbA2 ORF has also been expressed in E. coli, but no endonuclease activity against intronless psbA DNA has yet been detected (H.-H. Kim and D. L. Herrin, unpublished results). This may be a technical problem, or alternatively, a subunit of the enzyme is encoded outside of the intron, possibly in the nucleus. This appears to be the case for the mitochondrial intron-encoded endonuclease I-SceIV from yeast (43).

The present work differs from previous studies of organellar intron homing because the intron donor is exogenous rather than endogenous. In a somewhat analogous experiment, a nuclear rRNA intron from the slime mold Physarum polycephalum was shown to home into yeast nuclear ribosomal DNA from an exogenous plasmid (34). The efficiency of homing of the exogenously applied psbA introns is high, based on the nearly 100% cointegration of the introns in cotransformation experiments and on the very high frequency of transformants (>10,000/µg of DNA) obtained with the DCMU resistance marker. This transformation frequency, which is 100 to 200 times that obtained with the spectinomycin resistance plasmid (50 to 100 transformants/µg of DNA with the IL strain), is, we believe, due in part to the fact that the DNA is integrating via homing instead of simple recombination and also because of the recombination "hot spot" located at the 3' end of exon 5 (35). This conclusion is supported by the fact that transformation of the pBX4 and pKS4 constructs (Fig. 1) into the wild type, which would integrate via simple homologous recombination, gave transformation frequencies of ~1,000 transformants/µg of DNA with DCMU selection (O. W. Odom and D. L. Herrin, unpublished results). The spectinomycin resistance plasmid gave 100 to 300 transformants/µg of DNA with this same strain, indicating that the wild type is a somewhat better recipient for chloroplast transformation than IL. The inference from this data is that the increase in transformation frequency due to homing appears to be at least 10-fold. This may be an underestimate, however, since we may have reached the limit of transformation frequency that can be achieved by biolistics, at least under these conditions.

Homing also increased the efficiency of chloroplast transformation by decreasing the amount of homology between the donor and target required for DNA integration. This increased efficiency is most apparent from the facts that neither pKS4.Delta nor pBX4.Delta gave a significant number of DCMU-resistant transformants with the IL strain and that the ORF-containing versions (pBX4 and pKS4, respectively) gave ~10,000 transformants/µg of DNA. The ORF-deleted derivatives have the same amount of homology with the target DNA (i.e., intronless psbA) as the parental DNAs; thus, it is homing that makes the difference. The results with pBX4 also indicate that as little as 50 bp of flanking homology is sufficient for intron homing. However, the absolute lower limit of homology required for group I intron homing in chloroplasts remains to be determined.

The fact that an exogenous self-splicing intron, without an external promoter and with minimal flanking sequences, efficiently integrates into the chloroplast genome may have implications for the horizontal transfer of group I introns. Comparative evidence for horizontal transfer of several group I introns, including Cr.LSU and Cr.psbA3, has been published (e.g., references 5, 23, 32, and 40). DNA- and RNA-level mechanisms have been proposed to explain the transposition of group I introns, but which mechanism is mainly responsible is not clear (29, 44). The relative instability of RNA in most biological systems, coupled with the high efficiency of DNA homing argues for DNA-level transfer. In addition, an intron that is self-splicing, carries its own promoter for ORF expression, and integrates efficiently from an exogenous source, as with Cr.psbA4, would certainly be more likely to move to new locations than one lacking these features. It will be interesting to see if future studies turn up evidence for horizontal transfer of Cr.psbA4.

These results may also have practical implications for improving organelle transformation in other systems, since they suggest that the principal factor limiting chloroplast transformation (at least with biolistic DNA delivery) is recombination of the introduced DNA with the chromosome. Thus, it may be possible to improve organelle transformation by inducing a double-strand break in the target. This may require transforming in the target site for a rare-cutting endonuclease as a first step; then subsequent integration events would be at the same locus.


    ACKNOWLEDGMENTS

We thank Udo Johanningmeier for the IL strain, Michel Goldschmidt-Clermont for the atpX vector, and Lib Harris and the Chlamydomonas Genetics Center for plasmid DNAs.

This research was supported by grants from the USDA (NRICGP 96-35301-3420 and 99-35301-7847) and the Robert A. Welch Foundation (F-1164).


    FOOTNOTES

* Corresponding author. Mailing address: MCDB A6700, Bio 311, University of Texas at Austin, Austin, TX 78712. Phone: (512) 471-3843. Fax: (512) 471-3843. E-mail: DLHerrin{at}utxvms.cc.utexas.edu.

dagger Present address: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7760.

Dagger Present address: San Diego Supercomputer Center, University of California at San Diego, La Jolla, CA 92093-0537.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Molecular and Cellular Biology, May 2001, p. 3472-3481, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3472-3481.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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