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The Plant Journal (1998) 14(4), 441–447

Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron Victoria Lumbreras, David R. Stevens and Saul Purton* Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK

Summary Heterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 59 and 39 sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation-independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga. Introduction The development of the unicellular alga Chlamydomonas reinhardtii as the ‘green yeast’ (Goodenough, 1992; Rochaix, 1995) – a model system for molecular-genetic studies of cellular processes in photosynthetic eukaryotes – has been hindered by the difficulties of foreign gene expression in the nucleus of this organism (Blankenship and Kindle, 1992; Cerutti et al., 1997a; Day et al., 1990). This has prevented, for example, the use of heterologous genes to complement mutants or to manipulate key metabolic pathways, and the application of reporter genes to studies of gene expression and protein localisation. Possible reasons for the failure to express heterologous genes in Chlamydomonas include epigenetic suppression of gene Received 11 September 1997; revised 26 January 1998; accepted 20 February 1998. *For correspondence (fax 144 171 3807096; e-mail [email protected]).

© 1998 Blackwell Science Ltd

expression, inefficient transcription due to the lack of appropriate promoter and/or enhancer elements, the lack of introns required for efficient RNA processing, and poor translation of foreign mRNA lacking the codon bias found in Chlamydomonas genes (see Cerutti et al., 1997a; Kindle and Sodeinde, 1994; Stevens et al., 1996). Recently, some progress has been made in the expression of bacterial genes fused to 59 and 39 regulatory sequences from a Chlamydomonas gene. However, the random integration of these chimeric genes into the nuclear genome frequently results in undetectable or unstable expression (Cerutti et al., 1997a,b; Stevens et al., 1996). Studies of transgenes in higher eukaryotes, including plants, have demonstrated a positive role for introns in gene expression. In some cases, intron splicing appears to be required for efficient nuclear export or stability of transcripts (Huang and Gorman, 1990; Rose and Last, 1997). Alternatively, the rate of transcription can be influenced by transcriptional enhancer or repressor elements located within introns (Brooks et al., 1994; Goto et al., 1996). In those eukaryotic genes for which intronic enhancer elements have been identified, such elements are typically located within the first intron of the gene (Koziel et al., 1996). In plants, most intron effects on transgenes appear to be associated with post-transcriptional events (reviewed by Koziel et al., 1996). However, examples of enhancers in plant introns have been reported recently (Bolle et al., 1996; Gidekel et al., 1996), and Taylor (1997) has noted the importance of such intragenic elements in the regulation of plant gene expression. In this study, we have examined the influence of introns and other factors on the expression of a foreign gene in Chlamydomonas that contains the coding sequence of the bacterial ble gene (conferring resistance to zeomycin) fused to the 59 and 39 non-coding regions of the endogenous RBCS2 gene. We show that the truncation of the 59 region and insertion of Chlamydomonas introns within the ble sequence increases significantly the expression of the gene. A particularly strong effect is observed using the first intron of the RBCS2 gene which results in significant increases in the level of antibiotic resistance and abundance of the recombinant Ble protein. This intron appears to contain a transcriptional enhancer since it is effective when placed upstream of the RBCS2 promoter and in either orientation. Our findings suggest a role for introns in the expression of Chlamydomonas genes, demonstrate that efficient and stable expression of foreign genes is now possible, and provide an efficient dominant marker for transformation of this model organism. 441

442 Victoria Lumbreras et al. Results Analysis of the RBCS2 upstream and downstream regions Our original ble marker (plasmid pSP108, Stevens et al., 1996) comprises the coding region of the bacterial gene fused to 740 bp of the RBCS2 promoter and 231 bp of the RBCS2 39 non-coding region. To investigate whether elements within the RBCS2 sequences influence the expression of ble in Chlamydomonas, we compared the transformation efficiencies of a series of deletion plasmids relative to pSP108 (Figure 1a). Deletion of the 39 region (plasmid pSP107) has a negligible effect on the efficiency, showing that the putative polyadenylation signal (Goldschmidt-Clermont and Rahire, 1986) and any other elements in the 231 bp region are not required for ble expression.

In contrast, removal of both the 59 and 39 sequences (plasmid pSP.ble) reduces the transformation rate by two orders of magnitude, indicating that expression of ble depends on the RBCS2 59 region. We then examined the effect of successive deletions of this region. A deletion of 440 bp of upstream sequence (from –740 to –300, plasmid pSP110) increases the efficiency threefold (Figure 1a). This effect is maintained when the 59 deletion is extended to –180, but is lost when all sequences upstream of –60 are removed. These results suggest the presence of a negative regulatory element between positions –740 and –300 (see Cerutti et al., 1997a and Discussion). However, comparisons of Ble protein levels (see below) or zeomycin resistance (data not shown) amongst transformant populations generated using pSP108 or pSP109 do not reveal significant differences in the level of expression. This suggests that differences in transformation efficiency may be due to structures within the –740 to –300 region affecting the insertion or stability of the marker DNA in the genome, rather than an increase in gene expression. Chlamydomonas introns stimulate ble expression Since many C. reinhardtii nuclear genes contain introns, we investigated whether the introduction of Chlamydomonas introns into ble would increase its transformation efficiency. The first intron of the RBCS2 gene was inserted in the middle of the ble sequence of pSP108, creating plasmid pSP112 (Figure 1b). To generate appropriate 59 and 39 splice sites, two extra codons were added to the ble sequence. These changes map to the variable hinge region of the Ble protein, so they are unlikely to interfere with its activity

Figure 1. Influence of Chlamydomonas elements on the efficiency of the ble marker. (a) Effects of 59 and 39 RBCS2 sequences on ble expression. Various deletion derivatives of pSP108 were assayed for transformation efficiency in Chlamydomonas. Deletion of 39 RBCS2 sequences (pSP107) has little effect on transformation, whereas progressive deletions of the 59 RBCS2 region led to higher (pSP110 and pSP109) or equivalent (pSP111) transformation rates. (b) Influence of intron sequences on ble expression. Introduction of Chlamydomonas introns (RBCS2 intron 1, RBCS2 intron 2 or ARG7 intron 11) into the middle of the ble coding region improves the transformation efficiency, with RBCS2 intron 1 giving the greatest effect. The effect of RBCS2 intron 1 can be further improved by placing it at the start of the coding region (pSP114) and by introducing two copies of the intron (pSP117). (c) A combination of the shortened RBCS2 promoter region and RBCS2 intron 1 results in a dramatic increase in transformation efficiency relative to the original pSP108 plasmid. At the bottom of the figure are the results of co-transformation experiments using the ARG7 and ble markers. Introduction of pSP108 DNA into the Chlamydomonas genome using ARG7 results in ble expression in only 2.4% of ARG1 transformants. This is markedly improved (25%) when pSP124 is the unselected DNA, giving a value similar to that obtained in the reciprocal experiment. Transformation rates are relative to the original pSP108 plasmid and represent the average of at least two independent experiments each comprising 3–6 replicas. Black boxes represent the ble coding sequence and open boxes represent the various introns. ‘TS’ marks the transcription start.

© Blackwell Science Ltd, The Plant Journal, (1998), 14, 441–447

Foreign gene expression in Chlamydomonas 443 (Dumas et al., 1994). Two further plasmids were made using a similar strategy: pSP116, containing the second intron of RBCS2, and pSP113, containing the 11th intron of the Chlamydomonas ARG7 gene (Purton and Rochaix, 1995; Figure 1b). As shown in Figure 1(b), the presence of RBCS2 intron 1 increases the transformation efficiency approximately sixfold. The other two introns are less effective but still improve the efficiency several-fold. These results show that the introduced introns are accurately spliced from the ble transcript, resulting in a functional gene product, and that introns do improve the transformation efficiency of the marker, although the magnitude of the effect depends on the choice of intron. To examine further the specific effect of RBCS2 intron 1, we relocated it to a site immediately downstream of the translation start (pSP114, Figure 1b). In addition, a second construct was made containing a copy of the intron at both sites (pSP117). We found that the insertion of the intron near the translation start gives rise to a 10-fold increase as compared to the intron-less control, and that the presence of two introns leads to a 19-fold stimulation (Figure 1b). These findings suggest that the position of the intron influences the expression of ble (see below), and that insertion of two copies of the intron has an additive effect. We also placed the intron in the RBCS2 leader sequence 8 bp upstream of the ATG. However, this construct yielded very few transformants possibly because of the disruption of important sequences within the leader region (data not shown). Development of an efficient dominant marker The above results demonstrate that the efficiency of the ble marker is improved by using a shorter RBCS2 promoter and by the insertion of endogenous introns. To see whether these effects can be combined, we made plasmid pSP115, in which the –180 RBCS2 promoter region drives expression of a ble construct containing the RBCS2 intron 1 (Figure 1c). This plasmid has a transformation rate 28-fold higher than the original pSP108 construct. However, the addition of a second copy of the intron (pSP124) gives only a marginal further improvement, suggesting that a maximal transformation efficiency has been reached (approximately one transformant/105 treated cells). This efficiency is comparable to that of homologous markers such as NIT1 and ARG7 (Kindle, 1990; Purton and Rochaix, 1995). To compare further the functioning of the ble marker, we performed co-transformation experiments using the ARG7 marker (Debuchy et al., 1989). Introduction of pSP108 DNA into the arginine-requiring strain 363 using ARG7 as the selectable marker resulted in arg1 transformants in which 2.4% were resistant to zeomycin (Figure 1c; see also Stevens et al., 1996). In contrast, introduction of pSP124 by ARG7 selection resulted in 25% of the transformants © Blackwell Science Ltd, The Plant Journal, (1998), 14, 441–447

Figure 2. Northern analysis of ble expression. Four random clones were picked from transformants generated using pSP108 (no intron), pSP115 (one intron) and pSP124 (two introns). RNA was size-fractionated on an agarose gel, blotted to a membrane and hybridised with a radiolabelled fragment containing the ble coding sequence (upper panel). The filter was then stripped and re-probed with a DNA fragment containing the 5.8S rRNA gene as a loading control (lower panel). In transformants two and eight, which have particularly high levels of ble RNA, additional larger transcripts are seen that may represent unspliced, or partially spliced, species. The larger transcript in lane 9 may be a consequence of genomic rearrangement at the integration site. Each transformant has 1–2 copies of ble as judged by Southern analysis.

displaying zeomycin resistance. We then performed the reciprocal experiment in which pSP124 was used to introduce the ARG7 marker into strain 363 and zeomycinresistant transformants scored for arginine-independent growth. In this case, a similar percentage of transformants (29%) expressed the non-selected marker. This demonstrates that the improved version of ble now contains the regulatory elements required for efficient expression and behaves essentially as a homologous transgene when introduced into the Chlamydomonas genome. Finally, we examined the stability of the zeomycinresistance phenotype, since Cerutti et al. (1997a) observed that transformants generated using the bacterial aadA gene often exhibit unstable expression in the absence of selective pressure. We never observed a loss of zeomycin resistance in ble transformants maintained on non-selective medium for many months, suggesting that expression of the ble gene is stable in Chlamydomonas. Increased expression of ble To confirm that the increase in transformation efficiency in the new marker corresponds to an increase in gene expression, we compared the steady-state levels of ble RNA and protein in four randomly picked transformants generated using the original marker (pSP108) and the modified versions carrying one copy (pSP115) or two copies of RBCS2 intron 1 (pSP124). Northern analysis of total RNA from these transformants was performed using probes specific for the ble sequence (Figure 2). Although some variability is observed between colonies of the same class, the results suggest that the intron does increase the steady state level of ble RNA.

444 Victoria Lumbreras et al.

Figure 3. Western analysis for ble expression in Chlamydomonas cells. Total soluble protein was fractionated on a 15% denaturing polyacrylamide gel, transferred to nitrocellulose and probed using a polyclonal antibody to Ble. (a) Equal amounts of protein from four randomly selected individual colonies of each transformation class pSP108 (no intron), pSP115 (one intron) and pSP124 (two introns) was run per lane. WT corresponds to untransformed host cells. (b) Protein from 100 randomly selected individual transformants from constructs pSP108 (none), pSP114 (one*), pSP115 (one) and pSP124 (two) was pooled and an equal amount of protein was run per lane. Note that the recombinant protein in each transformant has an identical size (14 kDa), confirming the correct splicing of introns from the ble transcripts.

An improvement in the expression of ble is clearly seen when Ble protein levels are compared by Western blot analysis. A three- to fourfold increase in Ble protein is observed in all eight transformants as compared to the four carrying the intron-less marker (Figure 3a). The level of variation seen at the RNA level is not apparent at the protein level, suggesting that other factors such as protein turnover may limit the accumulation of the recombinant protein in these transformants. To confirm the effect of the intron on protein level, we also examined protein samples obtained from pools of 100 transformants of the three classes. As shown in Figure 3(b), the presence of one or two copies of the intron does increase the average amount of protein within each pool of transformants. To test that it is the intron rather than the truncated RBCS2 promoter in the pSP115 and pSP124 constructs that is responsible for the increase in protein levels, we also analysed a pool of 100 transformants (labelled ‘one*’ in Figure 3b) generated using pSP112, which differs only from pSP108 by the presence of the intron (see Figure 1b). Again we found an increase in the protein level relative to the pSP108. Finally, we examined the expression of ble in colonies transformed with pSP108, pSP115 and pSP124 by measuring their range of zeomycin resistance. Since the Ble protein binds zeomycin in a 1:1 stoichiometry (Dumas et al., 1994), the level of zeomycin resistance is directly related to the concentration of Ble protein in the cell. We find that all three sets of transformants show a wide range of resistance, from 20 mg ml–1 to .1000 mg ml–1 (Figure 4). This illustrates the influence of genomic context (‘position effects’) on the expression of the transgene. Consistent with the Western

Figure 4. The RBCS2 intron 1 increases the level of zeomycin resistance. Colonies transformed with pSP108 (no intron), pSP115 (one intron 1) and pSP124 (two introns) were tested for growth at different concentrations of zeomycin. N represents the number of transformants analysed for each construct. The percentage displaying zeomycin resistance at the different drug concentrations was calculated by scoring each transformant as 3 (resistant), 1 (partial resistance) or 0 (sensitive).

analysis, the presence of the intron increases the percentage of transformants displaying resistance at higher antibiotic concentrations. The presence of two introns increases the percentage still further with approximately 30% of the transformants resistant to the highest concentration tested. The RBCS2–1 intron contains an enhancer element The specific increase in transformation efficiency conferred by RBCS2 intron 1 (Figure 1b) led us to investigate whether cis-acting elements within the intron are responsible for this effect. First, we tested the activity of RBCS2 intron 1 lacking an internal fragment of 91 bp (construct pSP118; Figure 5a). This construct does not produce transformant colonies, suggesting that this ‘mini-intron’ cannot be spliced from the primary transcript. This may be due to the intron’s small size (56 bp), or may be due to the loss of elements required for splicing. The former seems more likely since Chlamydomonas introns appear to lack any conserved motifs, apart from a pyrimidine-rich tract towards the 39 end (Liss et al., 1997). To distinguish between these alternatives, we replaced the missing 91 bp with an unrelated fragment (from pBluescript) of similar size (Figure 5a; plasmids pSP120a,b). Insertion of this fragment in either orientation produced functional constructs, but their transformation efficiency was significantly lower than that obtained with the original intron. These observations indicate that increasing the size of the mini-intron is sufficient to allow efficient splicing, but that specific elements within RBCS2 intron 1 (that are not present in the chimeric introns) contribute to the marked effect on ble expression. © Blackwell Science Ltd, The Plant Journal, (1998), 14, 441–447

Foreign gene expression in Chlamydomonas 445 RBCS2 gene contains sequences that modulate the rate of transcription in vivo. Discussion

Figure 5. Functional analysis of RBCS2 intron 1. (a) The sequence requirements for the activity of the intron were tested by examining several mutant constructs. Plasmid pSP118 contains a 91 bp deletion of internal RBCS2 intron 1 sequences. In pSP120a,b a pBluescript fragment of 90 bp was inserted into pSP118 in either orientation. (b) To test for enhancer activity of the RBCS2 intron 1, the DNA was placed upstream of the 60, 180 and 740 bp RBCS2 promoter fragments (pSP122a,b, pSP125 and pSP121, respectively). A significant increase in the efficiency of transformation is observed when the DNA is located in either orientation close to the transcription start site (pSP122a,b), suggesting that it acts by stimulating the level of transcription. A transformation efficiency of 1 was arbitrarily assigned to each control construct without the intronic element.

The requirement for specific sequences within the RBCS2 intron 1 could reflect their role in splicing or transcript stability. Alternatively, they could act at the DNA level by stimulating the rate of transcription. We examined the latter possibility by placing the intron outside of the transcriptional unit, 60 bp upstream of the transcriptional start, and in either orientation (Figure 5b). We observed a 7– 8-fold improvement in the transformation efficiency that is independent of the intron orientation. This effect, outside of the transcriptional unit, indicates that the intron contains a transcriptional enhancer. A similar enhancement is also seen when the intron is placed at positions –740 and –180, relative to the transcription start, although the magnitude of the improvement is progressively reduced as the element is moved further from the basal promoter. This enhancer activity is specific for RBCS2 intron 1 since the insertion of RBCS2 intron 2 at the –60 position has no effect on the transformation efficiency of ble (data not shown). Taken together, these findings suggest that the first intron of © Blackwell Science Ltd, The Plant Journal, (1998), 14, 441–447

Our results show that endogenous introns introduced into the bacterial ble gene are correctly spliced in the Chlamydomonas nucleus and that their presence improves significantly the efficiency of the marker. Such intron effects on transgene expression have been described in other organisms including plants (Koziel et al., 1996), animals (Huang and Gorman, 1990), fungi (Dequard-Chablat and Ro¨tig, 1997) and the green alga Volvox cateri (Gruber et al., 1996), demonstrating that introns play an important role in the efficient expression of eukaryotic genes. Introns may exert a general effect at the post-transcriptional level whereby pre-mRNA splicing contributes to the overall process of mRNA formation, stabilisation and export from the nucleus (Huang and Gorman, 1990). Alternatively, specific introns may contain transcriptional enhancer or repressor elements that regulate promoter activity (e.g. Brooks et al., 1994). Our findings indicate both transcriptional and post-transcriptional effects on ble expression. An improvement of two- to threefold is obtained when RBCS2 intron 2, ARG7 intron 11 or the artificial intron are introduced into ble. However, no improvement is seen when RBCS2 intron 2 is placed upstream of the transcription start, suggesting that this intron acts at the posttranscriptional level. In contrast, the marked improvement observed with RBCS2 intron 1 is due primarily to an enhancer element within the intron. This element is functional when placed upstream of the RBCS2 promoter and in either orientation. The efficacy of the enhancer appears to correlate with its proximity to the promoter and with its copy number. Recently, we have mapped the enhancer element to the 39 end of the intron (V. Lumbreras and S. Purton, unpublished data). At present we do not know if the enhancer serves a regulatory role in RBCS2 expression since this gene does not show any light-induced or circadian changes in expression and is generally considered to be constitutively transcribed (Nelson et al., 1994). The transformation efficiency of the ble marker is also improved several-fold by eliminating sequences upstream of the RBCS2 promoter. This has been reported independently by Cerutti et al. (1997a) who suggested that an acetate-repressive element is contained within the –740 to –300 region. However, we see no difference in the range of resistance levels amongst transformants generated using constructs with or without this region, and no change in zeomycin resistance when transformants containing this element are grown in the presence or absence of acetate (V. Lumbreras and S. Purton, unpublished data). The increase in transformation efficiency in the absence of this region may therefore reflect an improved stability or rate

446 Victoria Lumbreras et al. of integration of the transforming DNA, rather than a direct effect on gene expression. In either case, the combination of a truncated promoter element and RBCS2 intron 1 provides a dominant marker that gives rates of transformation comparable with that of auxotrophic markers. These cis elements therefore appear to be necessary and sufficient for efficient expression of ble in Chlamydomonas and it should be possible to achieve similar success with other foreign genes (or Chlamydomonas cDNAs). Importantly, selection is not required for the establishment of stable expression since the introduction of the improved ble marker by co-transformation leads to a functional gene product in 25% of transformants. Typically, co-transformation in Chlamydomonas is very efficient with up to 80% of transformants harbouring the unselected gene (Stevens et al., 1996). The difference between these two values may reflect the epigenetic silencing of transgenes in some transformants as described by Cerutti et al. (1997b). Nonetheless, with such a high percentage showing stable expression, the recovery of functional transformants should be straightforward. Finally, we find that the range of expression levels in ble1 transformants is very broad with a .50-fold range of zeomycin resistance. Such variation in transgene expression has been well documented in plant and animal systems (Wilson et al., 1990). It may therefore be possible to obtain Chlamydomonas cell lines over-expressing a foreign gene by screening populations of transformants.

Experimental procedures

internal 91 bp fragment of the RBCS2 intron by a BglII site in a two-step PCR reaction (Higuchi et al., 1988). pSP120a and pSP120b were constructed from pSP118 by re-inserting a 90 bp fragment from pBluescript (Stratagene), in either orientation, at the BglII site. pSP115 and pSP124 are similar to pSP112 and pSP117, respectively, except that they contain the same RBCS2 promoter fragment as pSP109. All constructs containing Chlamydomonas introns have appropriate donor and acceptor sites to ensure efficient splicing of introns. pSP121 and pSP125 were derived from pSP108 and pSP109, respectively, by inserting the first RBCS2 intron upstream of the promoter region using a unique HindIII site. pSP122a and pSP122b were made from pSP111 in a similar manner by cloning the intron in both orientations upstream of the promoter (see Figure 5b). All constructs were confirmed by restriction or PCR analysis, and by nucleotide sequencing. A detailed description of the strategy used to create each plasmid can be found on our Web site at http://www.ucl.ac.uk/biology/prg.htm.

Nuclear transformation of Chlamydomonas reinhardtii All transformations were carried out using the glass-bead method of Kindle (1990). Co-transformation of the arginine-requiring strain 363 using plasmid pARG7.8 (Debuchy et al., 1989) together with pSP108, pSP115 or pSP124, was carried out as described in Gumpel and Purton (1994). For direct selection of zeomycin resistant transformants, cells were agitated with glass beads and DNA, diluted in 20 ml TAP liquid medium and left to express the ble gene by incubating at 25°C in the light (80 µE m–2 s–1) for 15–18 h with gentle shaking. Cells were then pelleted by centrifugation, resuspended in 5 ml of TAP containing 0.5% molten agar, and poured onto the surface of a TAP-2% agar plate containing zeomycin at 20 mg ml–1 (Cayla-France). For spot tests, aliquots of diluted cells were pipetted onto TAP plates containing zeomycin at concentrations between 0 and 1 mM. In all cases, plates were incubated at 25°C in the light (45 µE m–2 s–1) and transformants appeared after 7–10 days.

Strains and media Chlamydomonas cells used were the cell wall deficient strain CC-849 (cw10, mt–) obtained from the Chlamydomonas Culture Collection at Duke University and the cell wall deficient, arginine requiring strain 363 (arg7.8cwd, mt–) from R. Matagne (University of Liege). Chlamydomonas cells were grown in Tris-acetatephosphate (TAP) medium (Gorman and Levine, 1965), and supplemented with 50 mg l–1 arginine where required. Cultures were incubated in the light (45 µE m–2 s–1) at 25°C.

Construction of ble fusions Plasmids pSP109, pSP110 and pSP111 were prepared from pSP108 (Stevens et al., 1996) by deleting distal promoter sequences using SacI, SmaI and SphI sites, respectively, present in the RBCS2 promoter. pSP107 was generated during the construction of pSP108 and differs from the latter in that it lacks a 231 bp Eco47III fragment corresponding to the RBCS2 39 region. pSP112, pSP113 and pSP116 were made from pSP108 by inserting PCR fragments corresponding to the RBCS2 intron 1, ARG7 intron 11 and RBCS2 intron 2, respectively, in the unique SexAI site within the ble coding region. pSP114 contains the first RBCS2 intron inserted at the unique MscI site that overlaps the ble ATG initiator codon. pSP117 contains two copies of RBCS2 intron 1 cloned at the MscI and SexAI sites. pSP118 was made from pSP116 by replacing an

RNA analysis For Northern blots of ble transformants, total RNA was extracted from 10 ml of Chlamydomonas cultures as described by Goldschmidt-Clermont et al. (1990). 10 µg of RNA from each sample was electrophoresed on 2% denaturing agarose-formaldehyde gels and transferred to nitrocellulose membranes (Sambrook et al., 1989). Transcripts for ble were detected using a 395 bp fragment from the ble coding sequence as a probe and were normalised using a DNA probe for 5S rRNA. Hybridisations were performed at 42°C overnight in 50% formamide, 1 M NaCl, 50 mM sodium phosphate (pH 6.5), 7.53 Denhardt’s solution, 1.0% SDS and 0.1 mg ml–1 salmon sperm DNA.

Western analysis Total soluble protein was isolated from 10 ml cultures of Chlamydomonas grown to 5 3 106 cells ml–1. Cells were washed in 5 mM HEPES pH 7.5, 10 mM EDTA, 2 mM benzamidine, 2 mM DTT, and resuspended in 250 µl of the same buffer. Cells were lysed by freeze-thawing twice and insoluble cellular material removed by centrifugation. Protein concentration was determined by the method of Bradford (1976). Samples were boiled for 1 min and separated by SDS-PAGE using a 17.5% Tris-tricine gel as described by Scha¨gger and von Jagow (1987). Proteins were transferred to © Blackwell Science Ltd, The Plant Journal, (1998), 14, 441–447

Foreign gene expression in Chlamydomonas 447 nitrocellulose ECL membrane (Amersham) and probed using rabbit anti-Ble antibodies (Cayla, France). Signals were visualised using the ECL assay (Amersham) according to the manufacturer’s instructions.

Acknowledgements We thank Laura Winskill for valuable technical assistance. This work was supported by grants from The Leverhulme Trust and the Biotechnology and Biological Sciences Research Council.

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