Strategy, progress and prospects of transformation in <i>Chlamydomonas reinhardii</i>

Proceedings Chapter

Reference

Strategy, progress and prospects of transformation in Chlamydomonas reinhardii

ROCHAIX, Jean-David, et al.

ROCHAIX, Jean-David, et al. Strategy, progress and prospects of transformation in

Chlamydomonas reinhardii. In: Vloten-Doting, Lous van; Groot, Gert S.P. & Hall, Timothy C.

Molecular Form and Function of the Plant Genome. New York : Plenum Press, 1985. p.

579-592

Available at:

http://archive-ouverte.unige.ch/unige:150726

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

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STPATF.GY, PROGRESS AND PROSPECT'S OF TRANSFO:ffivl..ATION IN 1

!

CHI.AMYIOMJNAS REINHARDII ·· . i

Jean-David Rochab:, Jeanne Erickson, Michel

Goldschmidt-Clenront, Muriel Herz, Robert Spreitzer and Jean-Marie Vallet

Departrrents of Molecular Biology and Plant Biology University of Geneva

30, quai Ernest-Anserrret 1211 Geneva, Switzerland

INI'RODUCTION

Considerable progress has been achieved in recent years in the field of plant transfonnation. Infection of dicotyledonous plants with Agrobacterium tumefaciens containing Ti (turror indu- cing) plasmids provides a natural transformation system which has been exploited efficiently for plant genetic engineering. Consi- derable tirre is hciwever required in this system before the results of an experi.'Te!lt can be assessed. It therefore seerred of interest to develop a transformation system in a photosynthetic euka.I:yotic organism which grows rapidly. Ideally, this organism should have a relatively small genrne , it should be anenable to genetic analysis and it should share major features with higher plants.

The green unicellular heterothallic alga Chlarnvdcrronas reinhardii appears to m::et rrost of these require.rrents. It can be

propagated either phototrcphically. (in t..'1.e light in the abse.rice of a reduced carbon source) or heterotrophically Cin the dark with a reduced carbon source) . Tb.is property has allcwed for the

isolation of m.urerous mutants which are unable to ^gro,.1 phototro- phically because of sorre defective ccrrponent in the photosynthetic aP,?aratus or because of a deficienoJ in chloroplast protein

synthesis. Mutations of this sort have been found both in the nuclear and chloroplast genones of C. reinhaidii (Levine and Gcx::denough, 1970; Gillham, 1978 ). Chloroplast mutations can be recognized easily by their rrode of inheritance. Transmission of these genes is governed by the ^rratin~ type, i.e. in rrost cases only the chloroplast genes of the mt parent are transmitted to the offspring. In rare cases , ha.vever, biparental zygotes occur

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which inherit the chloroplast genorres of both parents. These biparental zygotes allON one to study chloroplast gene recombination.

At least 18 linkage groups have been found in the nuclear gencne of C. reinhardii (Harris, 1982) which contains 99. 7% of the cellular genetic information. Although the chloroplast and

mitochondria contain only 0.3 and 0.03% of the cellulai:: genetic information, their DNAs represent close to 15% and 1% of the rrass of the cell DNA, respectively.

Here we give a progress re:i;:ort on our attempts to develop an efficient nuclear transfornation system in C. reinhardii. Because of recent advances of chloroplast nolecular-genetics in this organism, in particular, the availability of well-characterized chloroplast mutatioas, the possibility is considered of extending this transformation system to the chloroplast canparbrent.

SELECTIVE MAFKERS FOR NUCLEAR TRANSFORMATION

Selective :markers are critical for establishing a transformation system. Arrongst the auxotrophic narkers the arg2/arg7 locus of C reinhardii is the only one that has been characterized both at the genetic and biochemical level. It codes for arginino-succinate lyase, the last enzyme of the arginine biosynthetic pathway which converts arginino-succinate into

arginine and fllm3.rate. This locus has been mapped on linkage group I, but it has not yet been isolated (Levine and Goodenough, 1970).

The corres:i;:onding locus of yeast has been cloned into the plasmid pYearg4 (Clarke and Carbon, 1978), which we have used in our initial transforrration attempts. Colonies growing in the absence of arginine were recovered at a lON frequency and in several cases the yeast DNA was found to be integrated in the nuclear genaoe

('Rochaix and van Dillewijn, 1982). ^H~5ver,

!b7

transforrration efficiency was rather lCM, between 10 to 10 transforrrants per treated cell.

CHLOROPIAST MJLECULAR GENEI'ICS : SELECTIVE MAFKERS FOR CHLOROPIAST TRANSFORMATION

A challenging task for the future is to exter1d the transfor- mation system to the chloroplast compa.rtrrent. At £irst sight, the highly selective chloroplast double envelope appears to be a rrajor obstacle for introducing nucleic acids into the organelle.

Ho.r1ever, microinjection !T'ay be feasible because of the single large-size chlorcplast of C. rei.'1hardii which cccupies 40% of the cell volUrr-e. The availability of well-defined chloroplast markers depends on a detailed knoNledge of chloroplast genare architecture and on the characterization of mutations in chloroplast genes. It

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is therefore appropriate to give a short SUilll\:ilY on the chloroplast genes of _g_. reinhardii.

The chloroplast genorre of this alga consists of 190kb circles

(Behn and Herrmann , 1977 ; Rochaix , 1978l . As in higher plants, it

contains an inverted repeat with the ribosom3.l RNA genes (f ig . 1) . Genes which have been identified on this genane include the r.RNA genes (Rochaix and Malnoe, 1978; Rochaix and Darl ix, 1982\, tRNA

genes (Malnoe and Rochaix , 1978; M. Schneider, unpublished results) and several genes of polypeptides involved in photosyn- thesis and in chloroplast protei n synthesis (cf. fig . 1l . At least two genes , those coding for the 23S rRNA and for a thyla1<oid poly- peptide (psbA in fig . 1, Erickson et al. , 1984al contain introns and both are located ^~thin the inverted repeat.

C. reinhardii is a photosynthetic organism that i s arrenable to an- extensive genetic analysis both at the nuclear and chloro- plast level . We and others have taken advantage of this inportant property by examining two types of chloroplast mutations which map at the rbcL and psbA loci. A list of uniparental mutants which have been characterized at the gene level is given in Table I.

A. rbcL Locus

The rbcL locus codes for the large subunit of

r ibulose-1 ,5-bisphosphate carboxylase/oxygenase (Rubisco). Several light-sensitive, uniparental Rubisto mutants have been

characterized. The first isolated mutant, 10- 6C , was shcwn to produce an inactive Rubisco enzyri'e with a lCMer large- subunit isoelectric point (Spr eitzer and Mets, 1980}. Comparison of the sequences of the rbcL genes of this mutant and wild-type reveals a single nucleotide change which replaces the gly residue 171 near the f irst active site of the large subunit with aspartic acid

(Dron et al. , 1983) . The significance of the base substitution has

been confinred recently when a revertant of this mutant was found to have the original wild type nucleotide (Spreitzer et al., 1982 ; Spreitzer, Rahire and Rochaix, unpublished results) . Since the 10-6C mutation has been shovm to be linked · to other genetic markers in the uniparental linkage group (Mets and C-€ist, 1983 \, this mutation pr ovi des the f irst correlation site betwe⁰_n the genetic and physical chloroplast DNA maps in C. r einhardii. 'I"wo other uniparental Rubisco mutants , 18- SB and l8-7G, were isolated by screening photosynthetic mutants for their inability to

recombine with the original 10-6C rrnltant (Spreitzer and Ogren , 1983l . Both of these mutants l ack Rubi sco enzyrre . ~fuile no l arge subunit can be ^det~5ted when cells of 18-7G are labelled for a short period wi th S- sulfate , a slightly truncat ed product fwhich is imrunoprecipitated with anti-Rubisco antibody\ is seen in the 18-5B mutant. Indeed , sequencing of the rbcL gene of this mutant has revealed the presence of a termination codon near the end of

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Fig. 1 - Chloroplast DNA map of Chlamydorronas reinhardii. The three irmer circles frcrn the outside to the inside represent the F.coRI, BamHI and BglII restriction rnaps (Rocha.ix, 1978). Dark wedges indicate the positions of the 4S RNA genes (Malnoe and Rocha.ix, 1978). The two segrrents of the inverted repeat are drawn on the outside of the map. They contain the rRNA genes and the gene of the 32 kd rrembrane po1 ypeptide , ^psb1~ (Erickson et al. , 1984a). The introns in the 23S rRNA gene and in psbA. are drawn in thirmer lines ^relativE~ to the coding sequences. D2 is the gene for another photosysten II polypeptide. The genes for the large

subunit of ribulose bisphosphate carboxylase , rbcL (Malnoe et al. , 1978) , for the a and f3 subunits of the ATP synthase, atoA and atpB , respectively (Woessner et al., 1984 ; Kovacic andROchai.x , unpublished results) and for the elongation factor EF-Tu , tufA

(Watson and Surzycki , 1982) are also indicated. The other gene locc.tions should be considered as tentative since they are based only on heterol~ous hybridizations with specific probes for the E. coli genes of the ribosarel proteins L22 and/or S19, for S4 and/or Sll and/or Sl3 and for the genes of the Band

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subunits of E. coli RNA polyrrerase (Watson and Surzycki, 1983). The chloro- plast DNA regions whose transcri pts are present in large -. , rredium ^1<2t and lo.v arrounts ^~ are shown . The eight identified

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Table 1. Chloroplast Mutations Characterized at the Gene Level

1-\ltation Polypeptide affected l=is genetic alteratic:n Peferenoe

l0-6C large subunit of Rllbisa> mcr. Point nutation Spreitzer am ^~ts (1980); Cron !! ^~·,

(1983)

lB-SB :rbcL Point rrutaticn Spreitzer and OJ%an (1983)

DCMJ4 J2Kdalttn rreri:lrane polypeptide psbA. Point nutation Ericksai ~ .!!!.· ⁽¹⁹⁸⁴⁾

Dr2 psbA Point rrutatioo. GallOolay and Mets {1982)

/\r7 psbA Point nutatiat GallOolay and Mets {1984)

11-lA S-J6C psbA. psbA Deletion Deletion

3

^Spreitzer and -"lets 11981) cf fig. 4

11-40 psbA. Deleticn

fullll-2 psbA Deleticn

~ llennoJn {unpibli.she:! results) C! fig. 4

full? psbA. Oeleticn

fulllJ psbA Deletion

1'JD-50 S-subunit of ATP synthase atpll Deletion WOessner ~ .!!!_. {1982)

the gene. Pulse-chase experinEnts indicate that the truncated product is unstable. The 18-SB and 18-7G rrutants are not only useful for correlating large-subunit strucbrre to function and enzyrre assembly and for searching for chloroplast suppressors

(Spreitzer and Ogren, 1984), they are also of considerable interest for investigations on the coordination of synthesis of the large and small subunits of Rubisco in the chloroplast and nucleocytoplasmic coropartrrents, respectively. Hybridizations of RNA frcm wild type and from mutant cells with DNA probes specific for the genes of the large and small subunit indicate that RNAs from these two genes accurnulate to nearly the sarre levels in the rrutants and in the wild-type (Goldschmi.dt-Clerrront, 1984) • Irnnunoprecipitation of pulse-labelled cells with antiRubisco antibody shows that the wall subunit is synthesized and processed to its mature size in both mutants, suggesting that it is imp:::>rted into the chloroplast. It is, hooever, rapidly degraded in these mutants suggesting that the stoicbiorretry of the two subunits is achieved at a post-translational level by sorre chloroplast located protease. These results agree with the work of Schmidt and

Mi.sh."<ind {1983) which den:onstrated that under conditions of inhi- bition of chloroplast protein synthesis, the small subunit is

still synthesized but rapidly degraded.

The rrost premising mutant for transformation experim:nts appears to be 18-7G which does not produce detectable levels of Rubisco large subunit even during short pulses . The mutation, which has a low ~ont.aneous reversion frequer1C'J , is therefore

likely to be recessive.

chloroplast ARS sequences are indicated by 01 to 08 (Vallet et al., 1984; Loppes and Denis, 1983). The four chloroplast DNA

sequences prorroting autonorrous replication in Chlar!fy'dorronas are marked bv ARCl, AR::2 and ARC3a,b IRochaix et al. , 1984al . Two

origins of replication, oriA and oriB, are indicated (Waddell et al., 1984).

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B. psbA Locus

The prcxiuct of the psbA locus is usually referred to as the

"32 Kdalton" t=0lypeptide which is associated with photosystern II.

It turns over rapidly in the light (i.e. in cells active in photosynthesisl, but not in the dark (Reisfeld et al., 1982).

Interestingly, this polypeptide appears to be the target site for several herbicides that block electron transport at the reducing side of photosystem II, pres\.mably by interfering with quinone binding {Arntzen et al., 1983). Labelled azido-atrazine has been shCMII'l to bind preferentially to the 32 K dalton polypeptide

(Pfister et al., 1981\. Its amino acid sequence has been highly conserved in higher plants and algae. In contrast to higher plants where the psbA gene has been mapped in the .single oopy region, it

is

located within the inverted repeat and therefore present in two copies per genane in C. reinhardii. As shc:Mn in fig. 2 the gene contains four introns - of 1. 35, 1. 4 , 1.1 and 1. 8 kb and it spans a region of 7 kb (Erickson et al., 19.84al.

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Fig. 2 - Analysis of deletions in the psbA region. The psbA gene is indicated in the upper part of the figure with its 5 e.xons !1!111

and 4 introns rzm . Transcription proceeds fran the left to the right. Restriction endonuclease sites are rrarked by R, EcoRI; K, KnpI; H, HindIII; X, XbaI; B, BamHI. Mutants 11- 1.P., 11-40 and 8-36C were isolated by Spreitzer and Piets (1981 ) , mutants FuDll-2, FuD7 and FuD13 were obtained £rem P. Bennoun. Deletions are

indicated by open bars. The deletion end points were rriapped within the regions marked by double arrows.

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Uniparental mutants resistant to the herbicides diuron, atrazine and branacil have been isolated in C. reinhardii

(Galloway and Mets, 1982; 1984; Tellenbach et al., 1983; Erickson et al., 1984b). Atrazine resistance bas also appeared in several weed species (Arntzen et al., 1983). The first mutant of C.

reinhardii examined, DCMU4, was isolated by P. Bennoun as-a

diuron-resistant mutant. Further studies revealed that this mutant is also highly resistant to atrazine. A comparative sequence analysis of wild type and mutant psbA revealed a single base pair change in the fifth exon at the ser 264 residue which is .replaced by ala. Both copie·s· of psh?\. were found to be rntated (Erickson et al. , 1984bl . Hirschberg and Mcintosh (1983) have previously reported that a similar change occurs in an atrazine-resistant biotype of Amaranthus hybridus where the sarre ser is replaced by gly. Sequence analysis of psbA from two other uniparental

herbicide resistant mutants of C . . reinhardii isolated by Galloway

and Mets (1984) has revealed that other parts of the 32 K dalton polypeptide play a role in herbicide binding. Mutant Dr2 which is 17 fold resistant to diuron and 2 fold resistant to atrazine has the wild-type val 219 changed to ile while mutant Ar7 which is 15 fold resistant to atrazine and rrore sensitive to diuron than wild- type has phe 255 changed to tyr {Erickson, Rahire and Rochaix, unpublished results) . These studies indicate that there is a considerable flexibility in the binding sites for these

herbicides. It is noteworthy that .electron flew is not affected in the Dr2 and Ar7, in contrast to IXMU4 and to the atrazine-

resistant Alraranthus. Mutations of the fo:mer type may therefore be of considerable agronomic value.

The tightly packed structural organization of the

polypeptides in photosystern II may intrcduce sare ccnplications in the interpretation of these results . The fact that amino acid changes occur in these herbicide resistant rrn.itants does not

necessarily imply that the binding site is located entirely on the

psbA product. Allosteric effects on neighborinq polypeptides in the photosystem II unit involved in true herbicide binding cannot be ruled out (Gressel , 1984). It is interesting to note that another PSII associated p::>Lypeptide, D2 ^I displays partial sequence hcrrology with the 32 kdalton p::>lypeptide, eSfeCially in the region where amino acid changes have been found in herbicide resistant mutants (Rochaix et al., 1984b). Whether alterations in this p::ily- peptide can also induce herbicide resistance re:-na.ins on open question. Herbici de resistance would appear to be a useful trans-

formation marker provided that it is a dcminant trait, an issue which has not yet been settled.

Since the psbA genes span 7% of the chloroplast gencrre of ^~­

reinhardii, one might e.xpect that they are particularly sensitive to mutagenesis. Arrongst six mutants examined that are deficient in photosystem II (Spreitzer and Mets, 1981; Ben.noun, unpublished

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results\ all have deleted the entire psbA region <fig. 2; Herz , Erickson and Rochaix, unpublished results). Since the psbA gene is within the inverted repeat, all of these I1U1tants contain double deletions. The preliminary mapping data are consistent with the deletions being entirely within the inverted repeat.

Double deletions at the other end of the inverted repeat have been described previously by Myers et al. (1982). The Irn.ltations affecting psbA are valuable both as genetic and chloroplast trans-

fonration markers. Linkage between the 8-36C mutation (fig. 3) and other na.rkers of the uniparental linkage group has been

denonstrated (Mets and Geist, 1983) and several PSU I1U1tations have been shown to be at a single chloroplast genetic locus

(Spreitzer and Ogren, 1983).

AUIONCMJUSLY :REPLICATING PIASMIDS

The limiting step in transforrnation in C. reinhardii may occur at different stages : delivery of DNA

to

the cells, stabili- zation and/or expression of the foreign DNA. It has been shown that in yeast the transformation yield can be enhanced

considerably through the use of autonan::iusly replicating plasmids (Beggs, 1975). It therefore seemed of interest to use plasmids of this sort in C. reinhardii.

Since no free plasmids exist in this alga except for chloro- plast and mitochondrial DNA, they were constructed in vitro. The 2.7 kb yeast HindIII fragrrent containing the ARG4 locus fran plasmid pYearg4 (Clarke and Carbon, 1978) was inserted into the EcoRI site of pBR322 by blunt-end ligation, thereby producing the plasmid pID2 (fig. 3) • MboI or HindIII fragments from total DNA and purified chloroplast DNA were inserted into the BamHI or Hind.III sites of pJD2. Pools of these recombinant plasmids were prepared and used to transfonn yeast or

c.

reinhardii by selecting for arginine prototrophy. · -

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Fig. 3 - ^~.ap of pID2. The yeast fragrrent containing the ARG4 locus is indicated.

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Because , unlikely replicat two orig by obser these or AAS ele.rr sho.m tl.

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.lS

rray stabili- shown

;ilasmids 5mids of chloro- ro. The

.r-an to the cing the al DNA s were or selecting

RG4 locus

A. Relatibnship Between Chloroplast ARS Sequences and Authentic Replication Origins of Chloroplast DNA

It was shcwn previously that a 2.2 kb mitochondrial restriction fragrrent fran Xenopus laevis containing the mito- chondrial origin of replication prorrotes autonorrous replication in yeast (Zakian, 1980), i.e. it must contain an ARS elerrent

(autononously replicating sequence). Although this observation did not prove the identity bet.veen the ARS element and the

mitochondrial origin of replication, it seemed of interest to examine whether other organellar origins of replication may be related to ARS elerrents •. Pools _of recaroinant plasmids described above were used for transforming yeast. Most of the transfonrants contained autonarously replicating plasmids har'l:ouring distinct chloroplast DNA segrrents. Hybridizing these recanbinant plasmids to EcoRI and BarnHI digested chloroplast DNA allcwed us to map the chloroplast ARS elerrents on the chloroplast DNA rrap {fig. 1, Vallet et al., 1984). To date our studies and those of wpp::s and Denis {1983) have revealed the presence of at least 8 chloroplast ARS elerrents which are interspersed throughout the chloroplast genorre (01, .~. 08 in fig. 1). Plasmids carrying these elerrents are unstable in yeast when the cells are grCMil under non-selective conditions. Sequence analysis of three of these chloroplast ARS fragrrents reveals a high AT content, many short direct and

inverted repeats and the presence of at least one elerrent in each fragment that i s related to the yeast ARS consensus sequence A/T TIT ATPu TIT A/T (Stinchcomb et al., 1981; Broach et al., 1982) • Because of the large nlllt'ber ofchloroplast ARS elerrents it is very unlikely that they all correspond to authentic chloroplast

replication origins. Recently, Waddell et al. (1984) have mapped

two origins of replication of the chloroplast DNA of C. reinhardii by observing replication forks in the electron microscope. One of these origins maps on the EcoRI fragrrent Rl3 which also acts as an

ARS ele.rrent in yeast ^{( fig.~.} Subcloning of the ^fra~t has sho.vn that the ^ARS elerrent is · distinct frcm the authentic origin.

Similarly we have found that the Euglena chloroplast restriction f ragrrent :containing the origin of replication (Koller and Delius, ' 1982 ; Schlunegger et al., 1982) does not prcrrote autonorrous

replication in yeast.""'i'hese results ^indi~te that at least in Euglena -and ^in~· reirlhardii chloroplast origins of replication are not a subclass of chloroplast ARS elerrents.

B. Autonorrously Replicating Plasmids in C. reinhardii

Using the pools of reCGrflbinant plasmids constructed with total cellular DNA for transformation of C. reinhardii, it was possible to r ecover several arginine prototrophs . Hybridization of total DNA frcm these cells with lal:elled pJD2 DNA indicated the presence of free plaSIT'~ds (Rochaix et al ., 1984a)

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Fig. 4 - Restriction map of Rl.3. The localization: of one authentic chloroplast replication origin, oriA, and the ClaI sites were detennined by Wang et al. (1984). Fragrrents able(+) or unable (-) to prarote autonarous replication in yeast are indicated. ARCl

re~ers to a 153 bp fragrrent of Rl3 capable of praroting autonarous replication in C. reinhardii (cf. Table II) . :The restriction sites R, EcoRI; B, BaffiHI and C, ClaI are indicated;

containing ARC sequences (autonorrous replication in

Chlamydaronas) • The arrount of plasmid DNA in these cells is

considerably lower after 60 generations relative to 25 generations after transfonration. Even if the cells are kept under selective pressure the plasmids are eventually lost, presumably because of reversion at th.e "AOC,7 locus. Several of these plasmids could J:e recovered by transfonning E.coli with the DNA isolated frcm the transformants. Four of these plasmids pCAl , pCA2 , J;CA3 and pCA4

were examined in m::>re details. The locations of the corresponding

ARC sequences were determined by hybridizing these plasmids to restriction enzyrre digests of nuclear and chloroplast DNA.

Surprisingly, all four ARC elerrents hybridize to chloroplast DNA.

Their locations on the chloroplast DNA map are indicated in fig. 1 and their properties are surrrnarized in Table II. The inserts in these plasmids are relatively small, ranging between 102 and 414 bp. Only one of the plasmids, pCAl, also has ^ARS activity. Its insert contains a 27 bp inverted repeat which carries a yeast ARS consensus sequence fVallet et al., 19841. It is noteworthy that fCAl traps on the EcoRI fragrrentR13 which contains one of the chloroplast origins of replication (Waddell et al., 1984).

Sequence ccnparison of the four ARC regions hasrevealed two san.i-conserved AT rich sequences of 19 and 12 bp that may play a role in prarotir.g autcnarous reolication in C. rei..nhardii (Rochaix et al. , 1984a) . Fig . 5 displays- the sequence-organization of these regions.

CONCLUSIONS

~- rein."11ardii aP.Jears to be an e..'<cellent rocx:1el system for studyirig the regulation of expression of genes involved in

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ne authentic .es were

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.ls is

; generations : selective

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; could be

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DNA.

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:.nserts in c02 and 414 rity. Its

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Fig. 5 - Sequence organization of four ARC elerrents of C.

reinbardii. I and II refer to two semi conserved elerrents of 19 and 12 bp, respective ly. C is an elerrent identical to the yeast ARS consensus sequence. Arr0"1s in p:Al indicate an 27 bp inverted repeat (from Rochaix et al., 1984a, with permission) •

Plasmid

~

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Table 2. Properties of ARC Plasmids Site Size (bp) U:>cation

of insert

ARC2 414 R18 Ba7

ARCl 153 R13 Ball

ARC3 102

ARC3a b ₂₅₇

R24 Ba4 R24 Ba4

activity ARS

+

R and Ba refer to the chloroplast EcoRI and BarnHI fragrrents shown on fig. 1. ARS activity is defined as the ability to prarote autonorrous replication in yeast.

phot osynthesis and rrore generalLy for examining the integration of organelles within eukaryot ic cells. A major reason is that chloro- plast rrolecular biology, biochemistry and genetics can no.N be coupled fruitfully in at least two chloroplast loci , rbcL and psb.Z\. Mutations at these two l oci have provided new i nsights into

the structure- function relat ionship of two chloroplast

polYf:.eptides and scrre of t.."1ese mutations have wade it possible to anSw"er specific ^qu~stions on chloroplast- nucleoc:ytoplasmic inter- actions . It i s likely that other chloroplast and nuclear loci will

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be uncovered soon that will allCM us to extend this analysis further.

It is possible to introduce foreign DNA into C. reinhardii and it is also feasible to construct plasmids that-replicate autonarcusly in this organism. The transformation efficiency still needs to be improved if this system is to be used for isolating genes by cornplerrentation of defined mutations. Sinci: the problem of expression of our selective markers has been neglected, work is in progress to construct plasmids with chirreric genes under the control of authentic nuclear prorroters of C. reinhardii. An efficient transfomation system in C. reiriFiardii will not only have a great impact on our understanding of regulatory rrechanisms in photosynthesis, but it may also provide a useful test system f?r genes of higher plants.

ACT<NCHLIDGEMENTS

We thank

o.

Jenni for drawings and photography, M. Rahire and J. van Dillewijn for excellent technical assist.ance. This work was supported by grant 3.258-0.82 from the Swiss National Science Foundation.

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. 592

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