Studies on homologous recombination in the green alga <i>Chlamydomonas reinhardtii</i>

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Studies on homologous recombination in the green alga Chlamydomonas reinhardtii

GUMPEL, Nicola J., ROCHAIX, Jean-David, PURTON, Saul

Abstract

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C.

reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C.

reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.

GUMPEL, Nicola J., ROCHAIX, Jean-David, PURTON, Saul. Studies on homologous

recombination in the green alga Chlamydomonas reinhardtii . Current Genetics , 1994, vol.

26, no. 5-6, p. 438-442

DOI : 10.1007/BF00309931

Available at:

http://archive-ouverte.unige.ch/unige:130898

Disclaimer: layout of this document may differ from the published version.

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Curt Genet (1994) 26:438-442

Current Genetics

9 Springer-Verlag 1994

Studies on homologous recombination

in the green alga Chlamydomonas reinhardtii

Nicola J. Gumpel 1, Jean-David Rochaix 2, Saul Purton 1

1 Department of Biology, University College London, Gower Street, London WC1E 6BT, UK

2 Departments of Molecular Biology and Plant Biology, University of Geneva, 30 Quai-Ernest Ansermet, CH-1211-Geneva 4, Switzerland Received: 11 March 1994/23 May 1994

Abstract. The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. re- inhardtii A R G 7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C.

reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of A R G 7 in the nuclear genome. Evidence is presented for homologous recombination between introduced A R G 7 DNA and the resident copy of the gene, and for the de-novo synthesis of the A R G 7 sequence during transformation.

Key words: Chlamydomonas - Homologous recombination - Nuclear transformation - Argininosuccinate lyase

Introduction

The green unicellular alga Chlamydomonas reinhardtii has proven to be an excellent model system for the genetic and biochemical analysis of various cellular processes such as photosynthesis, motility and phototaxis, metabolism, and organelle biogenesis (Harris 1989). More recently, the development of nuclear transformation systems for this organism (Debuchy et al. 1989; Kindle et al. 1989; Kindle 1990) has made possible the detailed molecular study of these cellular processes whereby genes responsible for mutant phenotypes are cloned by complementation (Pur- ton and Rochaix 1994) or by insertion mutagenesis (Tam and Lefebvre 1993). The reintroduction of cloned genes into the C. reinhardtii genome has been used to study ex- pression in vivo (Debuchy et al. 1989; Kindle et al. 1989), to analyse promoter activity through the use of chimeric genes (Blankenship and Kindle 1992; Davies et al. 1992),

Correspondence to: S. Purton

and to study the cellular location of gene products by epi- tope tagging (Kozminski et al. 1993). However, the development of methods for the targeted disruption or modification of nuclear genes (gene targeting) has been hindered by the finding that most transforming DNA integrates at non-homologous sites in the nuclear genome (Debuchy et al. 1989; Kindle et al. 1989; Smart and Selman 1993). Re- cently, Sodeinde and Kindle (1993) have demonstrated the occurrence of homologous recombination during C. rein- hardtii transformation and succeeded in modifying the endogenous nitrate reductase by gene targeting. We present additional evidence for homologous recombination in transformation experiments using the selectable marker ARG7, encoding the enzyme argininosuccinate lyase (ASL), in combination with the non-reverting mutant argT-8. Our data indicate that homologous recombination between co-transforming plasmids occurs readily and that the frequency is related to the amount of A R G 7 DNA shared by each plasmid. We also present evidence for recombination between cloned A R G 7 DNA and the endogenous gene, and for gene conversion in which an intact copy of A R G 7 is generated using a DNA sequence from the arg7 locus. Such studies represent the first step in the development of selection procedures for rare gene-targeting events, similar to those employed in mammalian cell transformation (Capecchi 1989).

Materials and methods

Strains and media. The recA- E. coli strain XLI-Blue (Stratagene) was used in all recombinant DNA work. C.reinhardtii transformation was carried using the cell-wall deficient, arginine-requiring mutant cwdarg7-8 as the recipient. Note that the arg7-8 mutation was originally designated as arg2 (Strijkert et al. 1973). The molecular basis of the mutation is not known. C. reinhardtii was cultured in Tris-acetate-phosphate (TAP) medium (Gorman and Levine 1965) supplemented with 50 mg/1 of arginine where required.

Plasmids. Plasmids pARG7.8, pARG6.5 and pU7.5 have been described previously (Debuchy et al. 1989). Plasmid pARGpvl.3 contains the 1.3-kb PvuII fragment from the 5' end of ARG7 (database accession number X16619) cloned into pBluescript SK-. Plasmids

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439 pARG6.5 and pARG7.8AN were derived from pARG7.8 by delet-

ing the 5' SalI fragment and 3' NdeI fragment, respectively. Similar- ly, p~3AS and p03AN were constructed by deleting the same SalI and NdeI fragments, respectively, from pARG7.8 q~3 (a derivative of pARG7.8 carrying the 394-bp HpaI fragment from q~X174 in the unique HpaI site between exons 7 and 8 of ARG7).

Nuclear transformation of C. reinhardtii. Transformation was car- ried out using a modification of the glass-bead method described by Kindle (1990). Cells were grown in TAP + arginine at 25 ~ under constant illumination of 3 000 lux, to a cell density of 2x106/ml.

Cells were harvested by low-speed centrifugation and resuspended in TAP minus arginine to a final concentration of 2•176 A 0.3-ml volume of cells was transferred to 10-ml Pyrex tubes containing 0.3 g of sterile glass beads (0.5 mm diameter). Plasmid DNA (2 gg) was added and the tubes agitated on a Genie 2 vortex, set at maximum speed, for 15 s. To the tubes 0.3 ml of molten (42 ~ 0.5%

agar in TAP was added and the mixture poured onto the surface of 2% agar TAP plates. Plates were incubated in constant light (2 000 lux) at 25 ~ Colonies appeared after 5-6 days. These were re-streaked to single colonies and grown in liquid TAR

DNA analysis. Total genomic DNA was prepared from C. reinhard- tii using the 'miniprep' method described by Rochaix et al. (1988).

Restriction digests, gel electrophoresis, and transfer to nylon membranes (Hybond-N; Amersham), were according to standard proto- cols (Sambrook et al. 1989). Radiolabelled DNA probes were prepared using the method of Feinberg and Vogelstein (1983). The probes used were (1) the 168 -bp Sau3A - SacI fragment from ARG7 exon 8, (2) uncloned 4~X174 DNA, (3) the 746-bp ScaI fragment of vector pBR329. Hybridisation conditions were 42~ overnight in 50% formamide, 5xSSC, 5xDenhardt's solution, 0.1% SDS, 0.1 mg/ml salmon-sperm DNA. Washing of membranes was at 65~

in 0.1xSSC, 0.1% SDS.

Results

Homologous recombination between overlapping plasmids

B S P S P K H N N (B)

I I I I I I I I I I

I l

1 kb - - ARG7

p A R G 7 . 8 p A R G 6 . 5 A ~ p A R G 7 . 8 A N

pU7.5 p A R G p v 1 . 3 Fig. 1. Restriction map of the ARG7 locus showing the position of the ARG7 gene (open box) and the region contained in each of the plasmids used in the co-transformation experiments. Restriction sites are as follows: B, BamHI; H, HpaI; K, KpnI; N, NdeI; P, PvuII;

S, SalI. (B) is a BamHI site generated during cloning. The vertical broken line marks the position of the SalI site and A indicates the deleted NdeI fragment in pARG7.8AN

Table 1. Transformation rates of plasmids carrying different regions of ARG7. Rates are expressed as a percentage of that obtained with pARG7.8 (approximately 1500 arg+ colonies per transformation). In co-transformation experiments, the amount of overlapping ARG7 sequence shared between plasmids (third column) is expressed in base pairs

Plasmids % Transformation ARG7homology

between plasmids

No DNA 0 -

pARG7.8 100 -

pARG6.5 0.7 -

pARG7.8AN < O. 1 -

pU7.5 0 -

pARGpv1.3 0 -

pARG6.5 + pARG7.8AN 21.9 5 621

pARG6.5 + pU6.5 20.1 1 350

pARG6.5 + pARGpvl.3 6.1 230

Our previous studies ( D e b u c h y et al. 1989) have shown that a complete version o f the A R G 7 gene is required for efficient transformation o f arg7 mutants to arginine pro- totropy. A plasmid ( p A R G 7 . 8 ) carrying the whole gene on a 7.6-kb f r a g m e n t gives transformation rates as high as one transformant per 4 x 104 treated cells (Purton and Rochaix, unpublished) w h e n used in conjunction with the glass- bead m e t h o d (Kindle 1990). However, r e m o v a l o f 5' or 3' A R G 7 sequences abolishes the p l a s m i d ' s ability to trans- f o r m the mutants ( D e b u c h y et al. 1989). We constructed a series o f plasmids containing various portions o f the A R G 7 gene (Fig. 1) and tested their ability to rescue the arg7 mutant, arg7-8 (Eversole 1956). As s h o w n in Table 1, plasmid p A R G 6 . 5 routinely yields a few transformants per plate c o r r e s p o n d i n g to less than 1% o f the numbers obtained with p A R G 7 . 8 . Similarly, p A R G 7 . 8 A N occasion- ally gives rise to arg+ colonies at a f r e q u e n c y o f less than 0.1%. However, in co-transformation experiments using pairs o f plasmids in equal concentration, significant n u m - bers o f transformants are obtained for all pairs. Efficient transformation is o b s e r v e d even when the a m o u n t of A R G 7 sequence shared by the plasmids is as little as 230 bp ( p A R G 6 . 5 and p A R G p v l . 3 ) , indicating that r e c o m b i n a - tion occurs readily even with a minimal region o f h o m o l - ogy. A correlation b e t w e e n the length o f the h o m o l o g o u s

region and the n u m b e r o f transformants is also seen. W h e n the region is increased f r o m 230 bp to 1 350 bp, transformation rates increase by over three-fold. This is further i m p r o v e d by increasing the overlap to 5 621 bp (Table 1).

To c o n f i r m that transformation is the result o f r e c o m - bination between the A R G 7 sequences, thereby giving rise to a complete gene, we designed two overlapping plasmids p03AN and p03AS which lack the NdeI fragment and SalI fragment o f p A R G 7 . 8 , respectively (Fig. 1). Furthermore, each plasmid carries a modification to the A R G 7 D N A in which a 394-bp fragment o f ~X174 D N A (termed a ' f l a g ' ) was inserted into the unique HpaI site between exons 7 and 8. This insertion has no observable effect on the ability of A R G 7 to c o m p l e m e n t arg7 mutations but allows for the discrimination between the e n d o g e n o u s gene and transforming D N A (Gumpel and Purton, unpublished).

The A R G 7 gene contains unique restriction e n z y m e sites for Bsu36I and StuI which are 6.3 kb apart. As depicted in Fig. 2, plasmid p 0 3 A N lacks the StuI site while p03AS lacks the Bsu36I site.

R e c o m b i n a t i o n between the two plasmids would create a complete A R G 7 gene with the two sites 6.7 kb apart (the additional 0.4 kb f r o m the flag), Southern-blot analysis was carried out on five clones obtained by c o - t r a n s f o r m a -

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PROBE

S K ~fS

a r g 7 - - 4 I ~ - - -

, 1 , , 2 , 3

B s u 3 6 I S t u I

[ ~- 6.7 kb " [

~ - t ~ I A ~ P ~3LxN

~1 N I ~ p*3/xS

A R G 7

Fig. 2. Southern-blot analysis of co-transformants generated using plasmids pr and pr Genomic DNA from untransformed cells (wild-type) and five arg+ transformants was digested with Bsu36I and StuI and hybridised with the ARG7 probe. The endogenous 6.3-kb fragment and the 6.7-kb fragment resulting from recombination between the plasmids are indicated. The diagram shows the regions of ARG7 (thick line) in each plasmid. The flag is shown as a shaded box and A represents deleted regions. The location of the probe is indicated by an arrow

A - - - ' ~

B ---t

C ---t

D ---~

E - - q

S t K i S t / S K S

L~ [ ~ / ~ i i ' ' ~ - -

S K S _ _ S K S

' ' t ~ 3 - - / ~ - - B " ' ~ I - - -

S K S S K S

, , , ~ ' ~ ' ] a E l _ _ _

s ~ s

~1 h - -

S K S

Fig. 3A-E. Predicted structures arising from one or two crossovers between the arg7 locus and plasmid p03AN. A single crossover within homologous region 1. B single crossover within region 2. C single crossover within region 3. D gene replacement resulting from crossovers within regions 1 and 2. E gene replacement resulting from crossovers wholely within one of the three regions. The ARG7 sequence is indicated by open boxes; the flag by a shaded box; the pBR329 sequence by a solid line, and genomic DNA by a broken line. SacI and KpnI sites are indicated by S and K, respectively; the deleted ARG7 region by A and the location of the ARG7 probe by an

a r r o w

tion with the two plasmids. Genomic D N A was digested with Bsu36I and StuI, blotted to a nylon membrane and hybridised to the A R G 7 probe. As shown in Fig. 2, the endogenous 6.3-kb band is seen all the transformants together with additional bands representing the integration of one or more copies of the transforming plasmids. In all cases, one of these bands is of the size (6.7 kb) expected for a functional copy of A R G 7 arising through recombination between the two plasmids.

A search f o r gene-targeting events

Plasmid pr lacks exons 10 and 11 of A R G 7 which en- code 65 amino acids of the A S L protein, including a region highly conserved between A S L from different spe- cies. We therefore looked for transformants arising from transformation using p~b3AN, reasoning that arg+ colonies could only arise by restoration of an intact, functional A R G 7 as a result of (1) recombination between pr and the endogenous gene, or (2) reversion of the arg7-8 mutation. Figure 3 shows the possible D N A arrangements at the A R G 7 locus resulting from either one or two recombination events between genomic and plasmid DNA. A single recombination will produce two copies of A R G 7 separated by vector sequence (structures A, B and C). The dis- tance between the SacI sites flanking the vector D N A will be either 6.6 kb (A and B) or 7.4 kb (C) depending which

side of the NdeI deletion the crossover occurs. A gene replacement event, in which the resident A R G 7 D N A is replaced with the introduced D N A by two crossovers (D and E), m a y or m a y not introduce the flag sequence into the genome. In the latter case this is indistinguishable from a reversion of the arg7-8 mutation. Only four arg+ clones were obtained from repeated transformation experiments, representing less than 0.1% of the rate obtained with pARG7.8. These clones were characterised by Southern analysis whereby genomic D N A was restricted with SacI and hybridised with probes specific for the ARG7, ~X174 and pBR329 sequence (Fig. 4). Clone 4 contains no ~X174 or pBR329 D N A and has a single A R G 7 fragment of the same size as the untransformed mutant. This clone is therefore either a revertant or a transformant generated by a gene-replacement event (Fig. 3E). The other three appear to be transformants since they contain an additional copy of A R G 7 carrying the flag and also contain vector DNA.

In the case of clone 3, the vector probe hybridises to a SacI fragment of 6.6 kb. This is the expected size for either structure A or B (Fig. 3). A digestion of D N A from clone 3 with KpnI gives a band of approximately 11 kb which hybridises with probes for both the vector and flag D N A (Fig. 5). This is the expected outcome for structure A where the two KpnI sites are 11.2 kb apart and contain both vector and flag DNA. It therefore appears that clone 3 is a transformant generated by recombination at the A R G 7 locus, although the analysis of clone 3 is complicated by

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Fig. 4. Southern-blot analysis of four arg+ clones obtained by transformation with uncut pO3AN. Genomic DNA from untransformed cells (wt) and the transformants was cut with SacI and hybridised to probes specific for ARG7, q~X 174 and pBR329. Sizes are in kilobase pairs

Fig. 6. Southern-blot analysis of six arg+ clones obtained by transformation with linearised plasmid pq)3AS. Genomic DNA was cut with SacI and hybridised to probes specific for ARG7, q~X174, and pBR329. Sizes are in kilobase pairs. The high-molecular-weight bands present in most of the tracks are artifacts rsulting from incom- plete digestion of the DNA

Fig. 5. Southern-blot analysis of p4)3AN transformant 3. Genomic DNA was cut with KpnI and hybridised to probes specific for pBR329 and 0X174. The 11-kb band is indicated.

Sizes are in kilobase pairs

the presence of additional copies of the p0)3AN D N A in this transformant (Figs. 4 and 5). In the case of clones 1 and 2 the sizes of the fragments hybridising to the vector probe are smaller than expected from the models in Fig. 3 and m a y have resulted from deletion or rearrangement during recombination.

Integration of plasmid D N A at a gene locus as a result of homologous recombination (e.g., Fig. 3 A - C ) can be promoted in m a m m a l i a n (Capecchi 1989) and yeast (Orr- Weaver et al. 1981) cells by linearising the plasmid within the region of homology. We investigated whether homologous recombination in C. reinhardtii could be similarly promoted by transforming the arg7 mutant with linear plasmid pO3AS cut at the unique Asp718 (KpnI) site within the A R G 7 sequence (Fig. 1). Six arg+ clones were isolated and characterised by Southern analysis (Fig. 6). Four of the clones appear indistinguishable from the untransformed strain in that they have the 4.2-kb SacI fragment of A R G 7 and do not possess the flag DNA. However, con-

trol transformation experiments using no D N A never yielded any arg+ colonies. It is most likely, therefore, that these clones are the result of recombination events at the A R G 7 locus in which D N A sequences not including the flag have integrated into the genome. This is supported by the observation that three of the four (clones 2, 3 and 6) have also been transformed with pBR329 D N A (Fig. 6, right panel). An unexpected situation is found in clones 4 and 5. Since the p~3AS plasmid lacks the left-hand SacI site located within the deleted SalI region (Fig. 2), and therefore contains only a single SacI site, any recombination events at the A R G 7 locus would result either in the conversion of the endogenous 4.2-kb SacI fragment to a 4.6-kb fragment (containing the A R G 7 probe D N A and the flag) or leave the fragment unchanged (containing only the A R G 7 probe DNA). However, both fragments are seen in the genomes of the two clones (Fig. 6, left panel) with the larger fragment containing the flag, as expected (Fig. 6, middle panel). We suggest that a process of D N A dupli- cation (gene conversion) has resulted in the synthesis of new A R G 7 D N A corresponding to the 5' untranslated region of the gene upstream of the SaII site. However, we cannot rule out the possibility that these two transformants have arisen as a result of a recombination event at the A R G 7 locus analogous to that seen in the other four transformants, and that the 4.6-kb bands are the result of additional independent integrations of pr elsewhere in the genome fortuitiously placing the SacI site of the plasmid 4.6 kb from an endogenous site.

D i s c u s s i o n

A general and routine system for altering the nuclear gen- otype of C. reinhardtii through gene targeting is an impor-

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442

tant goal in the development of this organism as a model system for molecular-genetic studies of cell function. Pre- vious work has shown that integration of cloned D N A into the nuclear genome occurs principally via non-homologous (illegitimate) recombination even when the introduced D N A contains large regions of D N A homologous to endogenous sequences (e.g., Debuchy et al. 1989). In this paper we show that recombination between two introduced D N A molecules sharing c o m m o n ARG7 sequences occurs readily in C. reinhardtii cells and that the frequency of recombination increases with the size of the c o m m o n sequence. The machinery for homologous recombination therefore exists in vegetative haploid cells and the amount of c o m m o n sequence required for recombination between the two molecules is no more than 230 bp.

Our attempts to demonstrate conclusive evidence of recombination between introduced D N A and the endogenous ARG7 gene have not been successful since a gene replacement event, whereby the endogenous ARG7 sequence is replaced with that harbouring the flag DNA, is not seen in any of the pr or pq~3AN transformants. However, several lines of evidence argue strongly for recombination at the ARG7 locus. Firstly, the expected result for a single crossover event resulting in two copies of ARG7 separated by vector sequence is observed. Secondly, the arg7-8 mutation is extremely stable. Spontaneous reversion of arg7-8 was not detected in the studies of Konvalinkova et al. (1974). Furthermore, we have never obtained any arg+

colonies from arg7-8 in control (no added DNA) transfor- mations experiments. It is most likely, therefore, that the four clones obtained using the linearised p~3AS plasmid (which lacks the 5' region of ARG7) have arisen through crossover events which exclude the incorporation of the flag DNA. This is further supported by the finding that three of the clones have been transformed by vector DNA.

Recently, Sodeinde and Kindle (1993) have published similar evidence on homologous recombination using the nitrate reductase gene NIT1. In these studies, a flag of 84 bp was introduced into a truncated version of the NIT1 marker. O f the 41 transformants they analysed, 30 were indistinguishable from revertants and only three showed a gene-replacement event in which the flag had been introduced at the nitl locus. Our inability to detect a similar gene replacement at the arg7 locus in the few transformants we examined m a y be related to the larger size of the flag (394 bp versus 84 bp). Clearly, further work is required to address this question since the ability to target large fragments of heterologous D N A (e.g., a selectable marker) to a given locus is central to any gene-disruption strategy.

Unlike the NIT1 gene, ARG7 contains repetitive D N A sequences within the introns and the 5' untranslated region (Debuchy et al. 1989). Consequently, recombination events involving ARG7 may be prone to D N A rearrange- ments and this perhaps reduces the frequency of homologous recombination. Of the four pq~3AN transformants (Fig. 4) we examined, Southern analysis revealed that in two of them (1 and 2) the SacI fragment carrying the vec-

tor sequence is smaller than that predicted by the model (Fig. 3) and may have arisen as a result of deletions during recombination.

The development of routine methods for the disruption or modification of C. reinhardtii nuclear genes, similar to the positive-negative techniques developed for m a m m a l - ian genes (Cappechi 1989; Yagi et al. 1993), requires that several criteria be fulfilled. Firstly, the demonstration of homologous recombination in C. reinhardtii and an under- standing of conditions which promote such recombination.

Secondly, the development of dominant markers for the positive selection of transformants, and suicide markers for the negative selection against those transformants arising by non-homologous recombination. The data presented here, together with that from Sodeinde and Kindle (1993), addresses the first criterion. Work on the development of suitable markers will be presented elsewhere.

Acknowledgements. This work was supported by a grant from the Agricultural and Food Research Council and grant 31.26345.89 from the Swiss National Foundation.

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Communicated by C. J. Leaver