Copyright 0 1990 by the Society of America

Transformation of Ribosomal RNA Genesin Chlamydomonas: Molecular and Genetic Characterizationof Integration Events

Scott M. Newman,* John E. Boynton,* Nicholas W. Gillham,?Barbara L. Randolph-Anderson,* Anita M. Johnson* and Elizabeth H. Harris*

*Department of Botany and tDepartment of Zoology, Duke University, Durham, North Carolina27706 Manuscript received May 25, 1990 Accepted for publication August10, 1990

ABSTRACT Transformation of chloroplast ribosomalRNA (rRNA) genesin Chlamydomonas has been achieved by the biolisticprocess using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance.The sites of exchange employed during the integration of the donor DNA into the recipient have been localized using a combination of antibiotic resistance mutations in the 16s and 23s rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacementof a region of the chloroplast genomein the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertionof the donor rRNA genes and flanking sequences into one of the recipient genome was followed by intramolecular copy correction so that both copiesof the inverted repeat acquired identical sequences. Increased frequencies ofrRNA gene transformants were achievedby reducing the copy number of the chloroplast genomein the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bonajde chloroplast rRNA transformants, the biolistic processinduced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.

ECOMBINATION occursbetween introduced genes from opposite parents occurs following organ- R donor fragments of chloroplast DNA and the elle fusion (LEMIEUX andLEE 1987; s. M. NEWMAN, recipient chloroplast genomeduring biolistic transfor- E. H. HARRIS,N. W. GILLHAMand J. E. BOYNTON,in mation of the Chlamydomonas chloroplast (BLOWERS preparation). Theseevents have been particularlywell et al. 1989; BOYNTONet al. 1988, 1990a). To study characterized genetically in Chlamydomonas reinhardtii the mechanism and site specificity of intermolecular (HARRISet al. 1977) where a physical map of ca. 7 kb chloroplast gene recombination in Chlamydomonas, of the chloroplast genome can be correlated with the we have developed methodsfor transformation of the detailed genetic map obtained by recombination of 16s and 23s ribosomal RNA (rRNA) genes located antibiotic resistance mutations (HARRISet al. 1989). in the inverted repeat region of the chloroplast ge- Localization of individual exchange events during nome (BOYNTONet al. 1990a). Mutant alleles exist at recombination to specific sites on the chloroplast ge- five different sites within the 16s rRNA gene that nome is made possible through the use of restriction confer resistance to streptomycin, spectinomycin, and fragment length polymorphisms (RFLPs) that distin- neaminelkanamycin and at two loci in the 23s gene guish the chloroplast of C. reinhardtii and that confer resistance to erythromycin, and the base the interfertile strain Chlamydomonas smithii (HARRIS, pairchanges that cause eachmutation have been BOYNTONand GILLHAM1987; PALMERet al. 1985). determined (HARRISet al. 1989). The entire chloro- RFLPs surrounding the rRNAgenes have been char- plast rRNA gene operon has been sequenced, includ- acterized by fine structure mapping and in some cases ing the intergenic spacer and flanking regions, and by sequence analysis. Differences in the number and the operon has been shown to be transcribed in the spacing of small (0.1-0.5 kb) dispersed repeat (SDR) 16S-23s gene order (DRON, RAHIRE andROCHAIX sequences located in the intergenicregions (BOYNTON 1982;LEMIEUX et al. 1989; ROCHAIXand DARLIX et al. 1990b; SCHNEIDERet al. 1985; SCHNEIDERand 1982; ROCHAIXand MALNOE 1978; SCHNEIDERet al. ROCHAIX1986) and marked by KpnI and AatII re- 1985; SCHNEIDERand ROCHAIX1986). In progenyof striction sites generate most of the RFLPs that distin- meiotic zygotes exhibiting biparental inheritance of guish the chloroplast genomes of these two strains chloroplast antibiotic resistance mutations, recombi- (PALMERet al. 1985). nation within and between 16s and 23s chloroplast In this paper we more fully document our prelimi-

Genetics 126 875-888 (December, 1990) 876 S. M. Newman et al. nary report of chloroplast rRNAgene transformation 3-5 X 10" cells/ml) at 200 pE/m2 per second and 25" in (BOYNTONet al. 1990a) and demonstrate the utility of HSHA medium (HS medium supplemented with 29.4 mM sodium acetate; HARRIS1989) and bubbled with air. Afilter- this system for studying chloroplast gene recombina- sterilized solution of FdUrd was added to some cultures tion. Transformation frequencies were maximal when (final concentration 0.5 mM) at a cell density of 5 X 104/ml the recipient cells were grown in 5-fluorodeoxyuri- and thecultures grown for ca. 6 cell generations (cf: HOSLER dine(FdUrd) before bombardment to reduce the et al. 1989). chloroplast genome copy number(WURTZ, BOYNTON Cloning of donor DNA fragments: Chloroplast DNA was isolated by sodium iodide gradientcentrifugation as and GILLHAM1977) and when sequence heterology previously described (GRANT,GILLHAM and BOYNTON betweenthe donor and recipient was mini- 1980). Restriction fragments containing16s and 23srRNA mized. The most common integration event resulted genes from C. reinhardtii (CC-227, CC-110) or C. smithii in complete or nearly complete replacement of the (CC-2477) were cloned into pUC vectors (see Figure 1) residentchloroplast DNA sequence by thecorre- using standard methods(MANIATIS, FRITSCH and SAMBROOK 1982) anddesignated P-183, P-228 and P-229, respectively. sponding sequence in the donor plasmid without in- Plasmid DNA used in the transformation experiments was tegration of vector sequences. Positions of exchange isolated as the covalently closed circular band from cesium events between the homologous recipient and donor chloridegradients (MANIATIS, FRITSCH and SAMBROOK sequences were biased and tended to occur near the 1982). insert:vector junctions. Furthermore the integrated Transformation procedures: Cells grown to midlog phase were concentrated by centrifugation at 20"to a donor sequences were presentin both inverted repeat density of 1.14 X 1Os per ml in HSHA medium, diluted 1: 1 elements of all copies of the chloroplast genome in with 0.2% Difco agar inHS medium at 42" and 0.7-ml thetransformed recipient cells, consistent with the aliquots containing 4X lo7cells were rapidly dispersed onto presence of an active copy correction mechanism for the surfaces of 10 cm petri dishes containing 1.5% agar in maintaining the homogeneity of the inverted repeat HSHA medium. Cells will not withstand 42" for more than a few minutes. Sixty milligrams of tungsten M10 micropro- (MYERSet al. 1982; PALMERet al. 1985).We also jectiles (Du Pont) were resuspended in 1 ml of 95% EtOH, foundthat the process of bombardingwild-type vortexed for 2 min to deglommerate, and spun 2 min in the Chlamydomonas cells can induce mutations, especially microfuge the morning of each experiment. Particles were those conferring resistance to low levels of strepto- washedtwo times by carefully removing the supernatant mycin, which are typical of nuclearsr-1 mutants (HAR- (except for residual 80-100 PI), and resuspending them in 1 ml of sterile distilled H2O by vigorous vortexing. Twenty- RIS 1989). five microliters of resuspended tungsten particles were added to a microfuge tube, immediately followed by 2.5 PI MATERIALS ANDMETHODS of the donor plasmid DNA at 1 pg/ml, 25111 of 2.5 M CaC& and 10pl of 0.1 M spermidine (free base). Tubes were finger Strains: All Chlamydomonas strains were obtained from flicked 8-10 times to mix, allowed to sit at room tempera- the Chlamydomonas Genetics Center, Department of Bo- ture 8-12 min, spun 30 sec in the microfuge and 50 rl of tany, Duke University. CC-124 and CC-125 are wild-type supernatant discarded, leaving enough particle suspension mt- and mt+ strains, respectively (HARRIS1989). CC-1852 for three bombardments. The DNA-coated particles were is a mt- wild-type F1 hybrid between C. reinhardtii and C. resuspended by vortexing vigorously and 2 PI immediately smithii that contains the organelle genomes of C. smithii loaded per macroprojectile. Blank charges (0.22 caliber (GILLHAM,BOYNTON and HARRIS 1987). CC-227 is a C. gray, power level #1) were used as accelerators for bom- reinhardtii sr-u-2-60 spr-u-1-6-2 er-u-37 mt+ strain that car- bardment in a Biolistic PDS-1000 Particle Delivery System ries streptomycin (sr) and spectinomycin (spr) resistance (Du Pont). mutations in the 16s rRNA gene (nucleotide numbers 474 Bombarded plates were respread within 2 hr orincubated and 1 123, respectively; HARRISet al. 1989) anda mutation in darkness overnight and respread the next morning onto that confers resistance to erythromycin (er) located in the selective HSHA media containing 100 pg/ml streptomycin, 23s rRNA gene at a position equivalent to nucleotide num- 100 pg/ml spectinomycin or 250 pg/ml erythromycin singly ber 2057 of Escherichia coli, now shown to be position 2067 or in various combinations. To reduce the possibilityof in the C. reinhardtii gene (LEMIEUXet al. 1989). CC-130 is bacterial contamination, all media contained 50 pg/ml am- a C. reinhardtii sr-u-2-60 nr-u-2-1 mt+ strain that possesses a picillin which has no effect on the growth of Chlamydomo- kanamycin/neamine resistance (kr) mutation at nucleotide nas (HARRIS1989). Colonies, usuallyvisible after 1 to 2 1341 in the 16s rRNA gene (HARRISet al. 1989), as well as weeks, were retested for growth onselective media. Putative the sr-u-2-60 mutation, while CC-1 10 is a C. reinhardtii strain transformants were scored for their ability to grow on the carrying the spectinomycin resistance mutation spr-u-1-6-2. nonselected antibiotics to which the donor DNA conferred CC-2477 is a spectinomycin resistant isolate from CC-1852, resistance or sensitivity. In addition, certain transformants containing a mutation in the C. smithii chloroplast genome were tested for growth on 500 pg/ml streptomycin or 100 that alters the same AatII restriction site in the 16s rRNA pg/ml kanamycin. The frequency of transformants was cal- gene as do the other spectinomycin resistant mutants that culated by dividing the number of colonies appearing on have been sequenced in C. reinhardtii (HARRISet al. 1989). selective plates by the number of cells in the spray pattern Culture conditions: For DNA isolation cells were grown of the gun (14% of the 4 X 10' cells on a 10-cm shot plate) phototrophically as described by HARRISet al. (1989) in and by the fraction of cells (25%) recovered after biolistic shaking cultures of 300 ml HS medium (SUEOKA1960) bombardment and dilution replating on nonselective me- bubbled with 5% CO, under continuous cool white fluores- dium. Both values were determined empirically for condi- cent light (200 pE/m2 per second) at 25". For transforma- tions used in our laboratory and represent constants applied tion, cells were grown mixotrophically to mid log phase (ca. to all experiments. Ch loroplast rRNA Gene Transformation 877Transformation Gene rRNAChloroplast

TABLE 1 Recovery of antibiotic-resistant isolates after biolistic bombardment

Frequency of resistant colonies (X10-6)" Bombarded, Unbombarded Bombarded, Bombarded, P-183Bombarded, Bombarded, Unbombarded Selection controlSelection no DNA P-17 (atpB) (sr spr er) Phenotype 19.5-srb Streptomycin (1Streptomycin 00 rg/ml) 0.0 13 17.0 19.8 42.7 <19.6-sr spr 3.6-sr spr er

' In each treatment, 4.2 X 10' cells were bombarded and 3.9 X 10" cells were plated on 100 rg/ml streptomycin for unbombarded controls. * Ninety percent were resistant only to 100 Pg/ml streptomycin, suggestive of nuclear mutations rather than chloroplast transformants, which are resistant to 500 rg/ml.

DNA isolation and characterization:A rapid procedure half of the latter streptomycin resistant isolates were for isolation of total cell DNA from Chlamydomonas colo- resistant to one or both of the unselected antibiotic nies growing on solid media was developed by modifying the methodsof GRANT,GILLHAM and BOYNTON(1 980) and resistant markers (spr or spr er) carried by the donor GILLHAM,BOYNTON and HARRIS(1 987). Cells were spread plasmid. Furthermore, all of the sr spr and ST spr er by toothpick into1-2-cm2 patches on HSor HSHA-YA agar isolates wereresistant to500 pg/mlstreptomycin, platesand grown 3-7 days in thelight (80 pE/m2 per characteristic of the sr resistance allele in the donor second). Each patch was scraped up with a sterile toothpick plasmid, whereas only 10% of the isolates resistant to and resuspended in a 1.5 ml microfuge tube containing1 .O ml cold TEN buffer (150 mM NaCI, 10 mM Na'EDTA, 10 streptomycin alone grew on the higher concentration mM Tris-HCI, pH 8.0), pelleted at room temperature and of this antibiotic. Our data suggest that the vast ma- resuspended in 0.4 ml TEN, to which 40 PI 20% SDS, 40 jority of the isolates resistant to only 100 pg/ml strep- ~120%Sarkosyl and35 pl of 50 mg/ml heat-treated Pronase tomycin were in fact new mutations equivalent to the (Boehringer Mannheim) were added. Samples were gently vortexed, and rotated for 5 min at 4" and then extracted sr isolates obtained following bombardment with with 0.65 ml TEN-saturated phenol:chloroform:isoamyl al- tungsten particles carrying no DNA or atpB DNA. cohol 25:24:1 (v:v) for 10 min at room temperature with These low levelstreptomycin resistantisolates are very gentle intermittent vortexing.The top aqueous phase(<0.5 likely new mutations in the single nuclear sr-1 gene ml) was recovered after a 10 min centrifugation at room (HARRIS1989), whereas the ST spr and ST spr er isolates temperature and the nucleic acids precipitated with 1.0 ml ice-cold 95% ethanol at -20" for 30 min. The precipitate resistant to 500 pg/ml streptomycin from the P-183 was collected by centrifugation for 10 min at room temper- bombardment are almost certainly bona fide trans- ature, washed with cold 70% ethanol, dried, resuspended in formants. Genetic analyses of representative low level 0.05 ml TE (10 mM Tris, pH 8.0, 1 mM EDTA) and 5-10- streptomycin resistant isolates from a second experi- PI aliquots digested.All single and double restriction digests were carried out according to the manufacturer's specifica- ment involving P-183 bombardment of wild type tions. Fragments were separated by electrophoresis in 0.8, showed thatthe vast majority exhibitedbiparental 1.0, or 1.2% aqarose gels andblotted to nitrocellulose, transmission of that phenotype typical of nuclear mu- hybridized with 'P nlck-translated probes and washed un- tations (data not shown). der stringent conditionsas described by GILLHAM,BOYNTON and HARRIS(1987). Transformation strategies: Our experimental de- signs utilized donor fragments and recipient chloro- plast genomes that differed by both genetic and phys- RESULTS ical markers. This allowed us to rule out thepossibility Induction of mutations by microprojectile bom- that antibioticresistant colonies appearing on the bardment: Experimentsindicate that the biolistic bombarded plates arose from antibioticresistance mu- transformation process using tungsten particles with- tations induced by the transformation process itself out DNA is mutagenic to Chlamydomonas.When (Table 1) or by growth of therecipient cells in FdUrd wild-type C. reinhardtii cells were bombarded with (Table 2; WURTZet al. 1979). either uncoated tungsten microprojectiles or micro- First, wild-type cells carryingthe chloroplast ge- projectiles coated with P-17 DNA encoding the chlo- nomes of C. reinhardtii (CC-124) and C. smithii (CC- roplast atpB gene (BOYNTONet al. 19SS), thefre- 1852), respectively, weregrown with or without quency of colonies capable of growth on 100 pg/ml FdUrd for 5-7 cell generations and bombarded with streptomycin increased over 1000-fold compared to the plasmid P- 183 carrying theer spr and sr mutations the unbombarded control (Table 1). When the P-183 (Figure1). Putative transformants selected on me- plasmid containing the er spr sr markers was used as dium containing both spectinomycin and streptomy- the donorDNA, the frequency of streptomycinresist- cin were subsequently scored for resistance to eryth- ant colonies increased an additional twofold. About romycin. Doublesr spr resistant CC-124 and CC-1852 878 S. M. Newman et al.

TABLE 2 Transformation frequency for Chlamydomonas chloroplast rRNA genes

~~ Pre- Transformants growth Experiment Donor Recipient Donor Experiment in Total cells FrequencyPercent withunselected No. FdUrdSelection strainplasmid (X 107 No. (X 10-6) markerdonor 1 P-183 1 CC-124 sr6.8 spr No 297 0.41 1 P-183 CC-124 sr5.5 spr Yes94 6.8 27 1 CC-1852 P-183 srspr No 6.8 0

1.I 0.6 1.5 0.7 "", " ". ,I -"" I I Recipient CC-1852 B BH HH KK K E E K KB 5s 235 16s II 3' -spr 5' t Plasmid P-229 t I

H 1.0 kb FIGURE1.-Physical and genetic maps of the rRNA region of the inverted repeat of the chloroplast genomes of C. reinhardtii strain CC- 130 andhybrid strain CC-1852 containing thechloroplast genome of C. srnithii that were used as transformation recipients. Chloroplast DNA fragments were cloned into pUC vectors for use as donor plasmids as indicated by the solid bars above or below the restriction maps of strains CC-130 and CC-1852. Positions of the antibiotic resistance alleles carried by the chloroplast inserts in P-183 (er, spr, sr) and P-228 and P-229 (spr)plasmids and the kr and sr alleles of the CC-130 recipient strain areshown. Locations of the tRNA genes forisoleucine (trnl) and alanine (trnA) on the chloroplast genome of C. reinhardtii are indicated by 0. The position and size of the RFLPs scored in this study are marked by dashed lines on the restriction map. The unique 0.5-kb KpnI fragment present between the 16s and 23s rRNA genes in C. smilhii but absent in C. retnhardtii was not scored. Forseveral of the RFLPs shown, the length changesresponsible have been further localized (see Figure 6). of the rRNA gene operon is initiated 72 nucleotides upstream of the 16s rRNA and continues through the 23s gene (SCHNEIDERet at. 1985). The origins of the C. reinhardtii probes A-E used to detect the RFLP variation in the transformants are shown by dotted lines. B, BarnHI; H, Hindlll; K, KfnI; E, EcoR1. transformants occurred at frequencies of 2 and 5 X in the donor fragment. Colonies resistant to specti- 10-6, respectively, following bombardment, whereas nomycin wereselected, and tested forgrowth on no spontaneous isolates resistant to both antibiotics kanamycin and streptomycin. Spectinomycin resistant were detected in 4 X 10' unbombarded cells of either colonies appeared at frequencies of 36 to 59 and 0.3 CC-124 or CC-1852 plated directly on selective me- to 2.3 X when CC-130 cells were bombarded dium (Table 2). with P-228 and P-229 respectively (Table 2). Virtually Second, the plasmids P-228 and P-229,carrying spr all these spr isolates were bonafide transformants since markers from C. reinhardtii and C. smithii, respectively they carried both the donor kr+ and sr+ or the kr+ (Figure l), were used to transform C. reinhardtii strain alleles. In two experiments, only one spectinomycin CC- 130. This strain is sensitive to spectinomycin but resistant colony arose spontaneously on unbombarded carries kr and sr mutations flanking the spr mutation control plates containing a total of 1.4 X 1O9 CC- 130 Chloroplast rRNA Gene rRNA Chloroplast Transformation 879 cells (Table 2). This is not surprising since the fre- mycin and streptomycin resistance (Table 2). This quency of nuclear mutations resistant to spectinomy- suggests that integrationat this end of the donorDNA cin is much lower thanthe frequency of nuclear resulted from an exchange event in the 200 bp inter- mutations resistant to streptomycin (HARRIS1989). val (<3% of the 7-kb insert sequence) between the er Effects of chloroplast DNA sequence heterology mutation and the BamHI site in the 23s gene that on thetransformation frequency of chloroplast marks the end of the donor sequence. The majority rRNA genes: The extent of homology between the of the P-183 transformants had an er+ spr sr pheno- donor plasmid and recipient chloroplast genomes ap- type, which is consistent with an exchange event oc- pears to affect the frequency of rRNA gene transfor- curring in the 3.8-kb interval between the er and spr mation (Table 2). When the donor plasmid contained mutations. Transformants with the donor er spr sr an insert homologous to therecipient, except forthree phenotype following P-183 bombardment of C. rein- single base pairchanges thatconfer the antibiotic hardtii strains CC-124 and CC-125 were remarkably resistant phenotypes, the frequency of transformation constant in frequency regardless of whether spr-sr or was several to many fold higher compared to a donor er were used as the selectable markers (Table 2). plasmid containing a chloroplast DNA insert which Two phenotypic classes of spectinomycin resistant had some heterologous intergenic regionswith respect CC-130 transformants were obtained following bom- to the recipient genome (Table 2). For example, co- bardment with P-229 which carriesthe 16s rRNA transformation frequencies for sr and spr were two- gene with the spr marker and flanking RFLPs from to threefoldhigher when P-183 (chloroplast insert C. smithii. Seventeen of the 20 spr isolates had a kr+ from C. reinhardtii) was used with CC-124 (C. rein- spr srf phenotype indicating that they had received hardtii chloroplast genome)than with CC-1852 (C. most or all of the 16s gene from the donor. Two smithii chloroplast genome). An even larger increase transformants had a kr' spr sr phenotype suggesting was observed when CC-130 (C. reinhardtii chloroplast the occurrence of an exchange event within the 16s genome) was transformed to spectinomycin resistance gene between the spr and sr mutations, which are 649 with P-228 (chloroplast insertfrom C. reinhardtii) bp apart. One isolate was resistant to all three antibi- rather than P-229 (chloroplast insert from C. smithii). otics, as was the spontaneous spectinomycin resistant Transformationfrequencies using a specific donor mutant of CC-130 that grew on non-bombardedspec- plasmid and recipient cell combination were reasona- tinomycin control plates. The kr+ spr sr+ isolates were bly consistent between experiments (e.g., P-228/CC- presumed to be true transformants since the proba- 130, Table 2). However, transformation frequencies bility of three independent mutations occurringin the fordifferent plasmids varied considerably. Forex- 16s rRNA gene of a CC-130 kr spr' sr recipient cell ample, CC-130 cells bombarded with P-228 and se- is exceedingly low.Analysis of theRFLP markers lected for spectinomycin resistance yielded a 5-10- flanking the rRNA genes confirmed that they were fold higher transformation frequency than when CC- bona fide transformants (see below). Several hundred 124 cells were bombarded with P-183 and selected spectinomycin resistant CC-130 isolates, obtained fol- for streptomycin and spectinomycin resistance, even lowing transformation with P-228 which carries the though the recipient CC- 130 strain was not grown in 16s rRNAgene from C. reinhardtii with the spr FdUrd. This is probably not due to preferentialselec- marker but no RFLP differences, were analyzed for tion for a single marker (spr) rather than two closely the phenotypes of the flanking kr and sr loci. Over linked markers (spr sr) since bombardment of FdUrd- 89% of the spr transformants had a kr' spr sr+ phe- grown CC-125 with P-183 resulted in similar trans- notype, which would be expected if the resident 16s formation frequencies whether erythromycinor strep- rRNAgene were replaced by the homologous se- tomycin + spectinomycin selection was imposed quence on P-228. Therefore, thevast majority of CC- (Table 2). Although we cannot exclude differences 130 transformants (7549%) possess the unselected between recipientstrains or in the quality of the markers (krf and sr+) from the donor that flank the plasmid DNA preparations used, the largest differ- selectable spr allele in the 16s rRNA gene. ences in transformation frequency were observed be- Molecular analysis of rRNA gene transformants: tween physically different donorsequences containing Analysis of the P-183/CC-1852 and P-229/CC-130 the same selectable markers. rDNA transformants indicated that the major phe- Phenotypic analysis of rRNA gene transformants: notypic classin each case reflectedreplacement of The presence of unselected donor alleles allowed us much or all of the resident rRNA genes by sequences to estimate how much of the 23s and 16s rRNA introduced on the donorplasmid. This was confirmed genes were transferred from the donor to the recipi- by molecular analysis of RFLPmarkers (Figure 1) ent chloroplast genome. The er mutation in P-183 flanking the 16s and 23s rRNA genes from repre- (Figure 1) was transferred to 21%-29% of the CC- sentative transformants. SOUTHERN (1975) blot hy- 124 or CC-1852 transformants selected for spectino- bridizations of BamHI digests of total cellular DNA 880 S. M. Newrnan et al.

Probe: pUC8 + 7.0 kb Barn HI encoding chloroplast rRNA genes (P-59) Probe: pUC18 + 1 .I kb Kpn I-Em RI for 5' end of 16s rRNA gene Digest: Kpn I-Em RI Digest: BarnHl 1 2 3 4 5 6 7 8 9 1011~~ 1213141516 1 2 3 4 5 6 7 8 9 10 I112 1314 1.5-

8.1 - 1.I- L 7.0 - " i

8 er er ererer er + + + + + + 0.8- ac 'E.- g spectinomycin-streptomycin resistant transformants 0.7- P FIGURE2.-RFI.P analysis of P-183/CC-1852 transformants. probe: pUC18 + 1.4 kb Kpn I-Hindlll for 5' end of 23s rRNA gene Total cell DNA was prepared from the recipient strain CC-I852 Digest: Kpn I-Hindlll (I;lne 1). CC-227 from which the P-1 83 donor plasmid was derived I .4- (lane 2). six pr spr ST (lanes 34) and six er+ spr sr (I;ules 9-14) tr;~nsform;lnts.DSA samples were digested with RamHI, separated I .I- on ().X% ag;~rosegels and blotted to nitrocellulose before hybridi- zation with a cloned 1k1m 1 1/12 fragment from wild type C. ~~+sr++++sr+++++++ rpinhardtii (probe A, Figure 1). spectinomycin resistant transformants from 20 sr spr er+ or ST spr er P-183/CC-1852 trans- FIGURE3.-RFI.P analysis of P-229/CC-130 transformants. To- formantsrevealed that none contained the 8.1-kb talcell DNA from the recipient CC-I30 (lane I), CC-2477 from Bam 1 1/12 fragment typical of theC. smithii CC-1852 which the P-229 donor plasmid was derived (lane 2). I2 kr+ spr ST+ recipientstrain (Figure 2). In seven transformants (I;~nes3, 5-8, 10-16) and two kr+ SPT sr (lanes 4. 9) transformants (e.g., lanes 5, 8, 9) the Bam 1 1/12fragment was was isolated. digested with KpnI-RcoRI (top panel). Kpnl (middle indistinguishablefrom the 7.0-kb Bam 11/12frag- panel), or Kpnl-Hind111 (bottom panel) and fractionated on 1.0% agarose gels, blotted to nitrocellulose. Probes B and <:were used ment of the donor. Nine transformants (e.g., lanes 6, forthe 1.4/1.1-khKpnl-HindllIand0.4/0.6-kbKpnl-EcoRIRFLPs 7, 10, 13, 14)had a Bam 11/12fragment that was in the IGS-2JS spacer whereas probes D and E were used for the intermediate in length between the 8.1-kb recipient I .1/1.5-kb BroRI-Kpnl and 0.8/0.7-kbKpnl RFLPs upstream of and the 7.0-kb donor fragments. We determined that the 16s gene as shown in Figure I. the Bam 1 1/12 fragments of intermediate length are from the donor plasmid. Sequences homologous to actually chimeras, containing the RFLPs just beyond the pUC vectors were not detected in total cell DNA the 5' end of the 16s gene of the CC-1852 recipient preparations from the 40 rRNA gene transformants and the RFLPs from the 16s-23s spacer region of analyzed. These results indicate that the vectoris not the P-183 donor plasmid(see below). Three trans- stably integratedinto the recipient chloroplast (or formants (lanes 3, 4, 11) were mixed at the time of nuclear) genome noris the plasmid replicating auton- analysis, having both the donor Bam 11/12 fragment and a second fragment of intermediate lengthin vari- omously in thetransformants. The transformation able proportions. Analysis of a set of 12 single cell process is apparently limitedto sequences homologous clones from each of these three transformants dem- between the donor and recipient,since alterationsare onstrated that all three transformants were mixtures seen only in Ban1 11/12, while the adjacent BamHI of cells pure for one or the other RFLP type. All fragments (Bam 14/15 andBam 10) in the P-l83/CC- subclones were considered to be bona$de transform- 1852 transformants are identical in size to those of ants because they contained the appropriate pheno- the CC-1852 recipient strain (data notshown). typic markers and the donor form of at least one Detailed mapping of the exchangeevents in RFLP that was scored. One transformant (Figure 2, rRNA gene transformants: The length differences lane 12) had a Ban1 1 1/12 fragment smaller than the between the insert sequencein the transforming plas- 7.0-kb donor fragment. mids(P-183 and P-229) and the homologous se- Similar results were obtained when 20 representa- quences in therecipient chloroplast genomes (CC- tive P-229/CC-130 spr transformants were analyzed 1852 and CC-130) represent the sum of five easily in thesame way, with theexception that one spr scorable RFLPs (Figure1). Three or four RFLPs were transformant possessed a BamHI-Hind111 fragment analyzed in the transformants (Figures 3-5) to deter- identical in size to therecipient (data not shown). mine wherespecific exchange events occurred during Therefore, in everyhomoplasmic transformant but the process of integrating the donor fragment into one, both resident copies of the rRNA genes were the recipient genome. Twenty P-229 transformants replaced by all or part of the homologous sequences ofstrain CC-130 were analyzed for the 1.4/1.1-kb Chloroplast rRNA Gene Transformation 88 1

23s rRNA 16s rRNA kr+ sr 00

I "_" Ill- I - - - - -I - - - II recipient (CC-130) ' 1.4 '0.4' ' 1.1 I- 0.8 ' I H KKE E K KB 1 11 I11 IV RFLP

1.1 K0.6E E 1.5 """ " " """_ - donor insert (P-229) Ill I I I 1 I

Region of Exchange Resistance RFLP Number Class I II I11 IV genotype I I1 111 IV FIGURE4.-Diagrammatic interpretation H KKS E K KB of postulated exchange events necessary to -I ::,* :* : :I- generate the different classes of transformants + spr sr RR R R 1 A in the P-229/CC-130 experiment. Thedonor plasmid insert and recipient chloroplast ge- nome are labeled at thetop of the figure. Diagrams are presented for the origin of each class of transformants, with the antibiotic re- sistance genotype, pattern of RFLPs and num- + spr + DDD D 8 B bers of each class indicated to theright. Ab- 7 r breviations for restriction enzyme sites are identified in the legend to Figure 1. The size (kb) and location of the RFLPs that were scored are indicated and the positions of the H KKS E K KB antibiotic resistance markers are designated by 0. Recombinant restriction fragments that are 7 I+ spr sr XD R R 1 c intermediate in length between either RFLP type (classes C,D, E) are designated by X. One member of class B and onemember of class D were initially mixed for RFLPs 5' to the 16s gene. Single cell cloning resulted in isolates pure for the two RFLP types.

+ spr + XD D D 4 D

H KK5 E K KB

+ spr + XD R R 4 E

+ spr + DD R R 2 F

KpnI-Hind111 RFLP spanning the 5' end of the 23s recipient form of this RFLP and the adjacent 1.1-kb rRNA gene, and for the adjacent 1.1/1.5-kb EcoRI- KpnI-EcoRI RFLP were observed in eight transform- KpnI and 0.8/0.7-kb KpnI RFLPs at the 5' endof the ants (e.g., Figure 3, lanes 4 and 7; Figure 4, classes A, 16s gene (Figures 3, 4). C, E, F). In these transformants an exchange event Twelve transformants inherited both RFLPsin the occurred between the spr locus in the 16s gene and 5' region of the 16s gene from the donor,indicating the polymorphism immediately 5' to the gene in the that an exchange event occurred between the poly- KpnI-EcoRI fragment.Two transformants were morphism in the KpnI 0.7/0.8-kb fragment and the mixed for both the 1.1/1.5- and 0.8/0.7-kb RFLPs, BamHI site ca. 500 bpaway that marks the endof the containing donor and recipient forms of each restric- insert sequence (e.g.,Figure 3, top andmiddle panels, tion fragment (Figure 3, top and middle panels, lanes lanes 3 and 5; Figure 4, classes B and D). The 0.8-kb 14 and 15); subcloningof the initial colonies resulted 882 S. M. Newman et al.

23s rRNA 16s rRNA + ++ id! a I I Ill I I I I "_ """_ " recipient (CC-1852) 1.1 1 B L L 1.5 0'7K B I 111 IV RFLP

B _"" 1.4 E E- - 1.1- - - - -o.sKB - - donorinsert (P-183) t I Ill I I I I

0 00 -er -spr sr Region of ExchangeResistance RFLP ClassNumber FIGURE5.-Diagrammatic inter- I III IV genotype I 111 IV 8, H KKKE, E, K KB pretation of postulated exchange I events necessary to generate the dif- er spr sr spr er D R R 3 A ferent classesof transformants in the 7 P-183/CC-1852 experiment. Symbols are explained in the legend to Figure I; kick, k 4 2 Ai 4. (A) Recombinant restriction frag- ment longer than thedonor RFLP

8, H KKKE, E, K KB (class C) is indicated by X. The loca- I tionof the putative illegitimate ex- +sr spr D R R 6 B change event between multiple SDR 1 elements that generates this non-pa- rental form of the KpnI-Hind111 RFLP is designated by *. Two mem- bers of class D and one member of

8, H KKKE, E, K KB class E were initially mixed for RFLPs I 5' to the 16s gene. Single cell cloning c resulted inisolates purefor the two + X D D 1 spr sr spr RFLP types.

\ / sprersr D D D 3 D

E, H KKKE, E, K KE I 1 r sr+ spr D D D 7 E in only pure colonies of the two types. One of these kb KpnI-Hind111 (e.g., Figure 3, bottom panel, lanes transformants contained a donor and one an inter- 6 and 8) and 0.4/0.6-kb KpnI-EcoRI intergenic RFLPs mediate length formof the KpnI-Hind111 RFLP from (data not shown). This is indicative of an exchange the 16s-23s spacer (Figure 3, bottom panel, lanes 14 eventbetween the polymorphism in the 1.4/1.1-kb and 15). No evidence was observed for physical ex- KpnI-Hind111 fragment and theHind111 site marking change of donor and recipient chloroplast sequences the end of the insert. Only one transformant (kr+ spr between the adjacent 1.1/1.5-kbEcoRI-KpnI and 0.81 sr) had the recipient forms (Figure 3, bottom panel, 0.7 KpnI RFLPs immediately upstream of the 16s lane 4) of the intergenic RFLPs (Figure 4, class A). rRNA gene (Figures 3, 4). This genotype and RFLP patternis consistent with an When theRFLPs in the16s-23s rRNA spacer exchange event having occurred between the kr locus region of the P-229/CC-130 transformants were ana- and the polymorphism in the small KpnI-EcoRI frag- lyzed, a strong bias was observed for integration of ment near the 3' end of the 16s rRNA gene. The the donor form of the KpnI-Hind111 fragment. Ten putative kr spr sr transformant with the same pheno- kr+ spr sr+ transformants of CC-130 (Figure 4, classes type as the spontaneous spectinomycin resistant mu- B, F) contained the donor form of both the 1.4/1.1- tant isolated fromthe CC-130 control plates, also Chloroplast rRNA Gene Transformation 883 possessed the recipient forms of all four RFLPs. This pattern of inheritance typical of nuclear mutations, isolate, which was not included in the tabulation pre- indicating that the biolistic process is indeed muta- sented in Figure 4, very likely resulted from a spon- genic. The frequency of uniparentally inherited mu- taneous spectinomycin resistance mutation on one of tants resistant to high levels of streptomycin was also thebombarded plates ratherthan from adouble increased somewhat above the control levels in this exchangeevent with ends in the kr-spr and spr-sr experiment, suggesting that the biolistic process also intervals in the 16s gene. induces chloroplast mutations. Ninetransformants (Figure 4, classes C, D, E), Strategies for biolistic transformation of chloro- which possessed donor forms of the intergenic KpnI- plast rRNA genes: Our experiments have taken ad- EcoRI RFLP(data not shown), had KpnI-Hind111 vantage of the existence of multiple antibiotic resist- fragments that were intermediate in length (ca. 1.15 ance markers in the 16s and 23s rRNA genes and kb) compared to the donor andrecipient RFLPs (e.g., easily detectable RFLPs that distinguish the chloro- Figure 3, bottom panel,lanes 3 and 7) and were likely plast genomes of the interfertile C. reinhardtii and C. of recombinant origin. One transformant (kr+spr sr+) smithii strains. Both the P-183/CC-1852 and P-229/ was mixed for both the KpnI-Hind111 RFLP (Figure CC-130 donor/recipient combinations, which differ 3, bottom panel, lane5) and for theKpnI-EcoRI RFLP by diagnostic chloroplast RFLPs flanking the selecta- (data not shown), containing recipient and interme- ble markers, yielded transformants at frequencies of diate types of the former RFLP and donor andrecip- -2 X 10-6 (1-2 transformants perplate of bombarded ient forms of the latter. Although this isolate con- cells). In contrast, the recipientCC-130 strain yielded tained donor forms of the RFLPs (KpnI-EcoRI 1.1/ only one spr isolate (frequency 1.4 X lo-’) when 1.5 and KpnI 0.8/0.7) upstream of the 16s rRNA unbombarded cells were plated onspectinomycin and gene (and therefore is a bona fide transformant), it unbombarded cells of CC-1852 yielded no colonies may represent the only example of a transformant in resistant to spectinomycin plus streptomycin. Donor which an exchange event occurred betweenthe KpnI- plasmids containing two or more selectable markers Hind111 and KpnI-EcoRI RFLPs in the16s-23s obviate the problems of antibiotic resistance mutations spacer region. An additional exchange event between that arise either spontaneously or as a consequence of the KpnI-EcoRI RFLP and the kr locus needs to be particle gun bombardment. Analogous methods have postulated to account for its kr+ spr sr+ phenotype. recently been used to transform the of Single cell cloning of this isolate was not performed tobacco cells (SVAB, HAJDUKIEWICZand MALICA and therefore it is not included in Figure 4. 1990). Transformants, selected for a spectinomycin Comparable results were obtained when these resistance marker in the 16s rRNAgene, were scored RFLPs were analyzed in 14 er+ spr sr and 6 er spr sr for a flanking streptomycin resistance marker and a transformants of strain CC-1852 bombarded with P- novel restriction site constructed in the donor DNA. 183 (Figure 5). However one transformant with an As in our experiments with Chlamydomonas, integra- er+ spr sr phenotype (Figure 5, class C) contained a tion of donor sequencesoccurred by homologous non-parental 1.7-kb formof this RFLP thatwas larger replacement, followed by copy correction, and segre- than either the donorrecipient or restriction fragment gation to yield isolates homoplasmic for transformed (see BOYNTONet al. 1990a, Figure 5). Since the overall chloroplast genomes. length of the Bam 11/12 fragment in this transfor- Effect on transformation frequenciesof sequence mant is smaller than the recipient form by ca. 300 bp heterology in intergenic regions between donor and (Figure 2, lane 12), and the RFLPs at the 5’ end of recipient: When the plasmid DNA insert was com- the 16s gene are identical to the recipient, one or pletely homologous to the corresponding region of more illegitimate exchangeevents most likelyoc- curred in the cluster of SDR elements found in the the recipient genome (with the exception of the single 16s-23s spacer. base pair changes which confer the antibiotic resist- ance phenotypes, for example P-183/CC-124 and P- 228/CC-130), higher frequencies of transformation DISCUSSION were observed than when the donor DNA possessed Microprojectile bombardment can be mutagenic: several sequence heterologies dispersed throughout Bombardment of wild-type C. reinhardtii cells with intergenic regions of the plasmid insert, for example, tungsten microprojectiles results in over a 1000-fold P-183/CC-1852 and P-229/CC-130 (Table 2). These increase in the frequency of mutants resistant to low heterologies are primarily insertions and deletions of levels of streptomycin, which is characteristic of nu- 50-400-bp SDR elements (PALMERet al. 1985). Be- clear sr-I mutations (HARRIS 1989).In a second trans- tween the regions of heterology, both the codingand formationexperiment involving P-183/CC-124, 48 non-coding sequences are quite homologous, exhibit- isolates resistant only to lowlevels of streptomycin ing only ca. 2-4% mismatch over stretches of several weretested genetically. All exhibiteda biparental hundred base pairs (S. M. NEWMAN,unpublished 884 S. M. Newman et al. data). Sequence heterology between the donor DNA single particle (ARMALEOet al. 1990). Therefore the and the recipient chloroplast genome may reduce the chloroplast rDNA transformantsin this study presum- efficiency of pairing, and the probability of exchange ably represent the product of two or three distinct events leading to integration, thus decreasing the fre- kinds of recombination eventsfollowed by segregation quency of chloroplast rRNA gene transformation. of thetransformed genomes to yield homoplasmic Our results are similar to those from transformation single cell isolates. and recombination experiments in Saccharomyces cere- Preferential localization of exchange events dur- visiae (SMOLIK-UTLAUTand PETES1983), Escherichia ing integration of donor DNA: Exchange events coli (SHENand HUANC 1986) and mammalian cells leading to rRNA gene transformation are not ran- (WALDMAN andLISKAY 1987) where recombination domly distributed along the length of the donor and inspecific intervals is decreased by increasing the recipient sequences. This conclusion stems from the extent of sequence heterology. Althoughthe effect of following observations. (1) Recipient forms of all the donor/recipient heterology on chloroplast transfor- RFLPs flanking the rDNA region were observed in mation frequencies is striking, we have reason to only one (Figure 4, class A) out of 40 transformants believe that heterologies do not always alter the pat- examined from P-l83/CC-1852 and P-229/CC-130. tern of exchange events between the donor andrecip- Exchange events that generate restriction fragments ient sequences (see below). Furthermore, the RFLPs of intermediate length between the donor andrecip- generated by the sequence heterologies providephys- ient appeared to occur within -500 bp of the 5’ end ical markers critical for documenting unequivocally of the 23s rRNA genein the 1.4/1.1-kbK~nI-HindIII chloroplast rRNA gene transformation. RFLP (Figures 4, 5) or -500 bp from the 5’ end of Temporary heteroplasmic nature of transform- the 16s gene(see below). (11) Eighteen out of 20 CC- ants: A minority of the transformants were initially 130/P-229 transformants were phenotypically kr+ spr mixed for both forms of some RFLPs at the time the sr+, and, as a minimum, must represent replacement DNA of the clones was first isolated and analyzed (e.g., of virtually all of the resident 16s gene by the corre- Figure 3, top and middle panels, lanes 14 and 15). In sponding sequence in the P-229 plasmid (Figure 4). all cases subclones proved to be homoplasmic for one (111) The frequency of the spr sr transformants that of the two types of RFLPs. The failure todetect carriedthe unselected er marker(7/34) from CC- persistent heteroplasmons is consistent with the exist- 1852/P-183 (Table 2) was high considering that the ence of an efficient copy correction mechanism for er locus is only ca. 200 bp from the end of the 7.0-kb ensuring homoplasmicity of theinverted repeat in insert in P-183. Althoughthis distance represents only chloroplast DNA (MYERSet al. 1982; PALMERet al. 5% of the length from the BamHI site at the end of 1985) and the rapid segregation of chloroplast ge- the plasmid to the selectable spr marker, over 20%of nomes (BOYNTONet al. 1990b). Integration of donor the exchangeevents are observed in this interval. DNA into one copyof the invertedrepeat on the Collectively thesedata suggest thatthe exchange recipient chloroplast genome by homologous replace- events resulting in rRNA gene transformation do not ment of recipient sequences, which results in the for- occurrandomly over the regions of homology be- mation of transientheteroplasmic genomes, is fol- tween the donorinsert and therecipient chromosome. lowed by intramolecular copy correction that yields Since the positions of the length differencesrespon- homoplasmic molecules with either the donor or re- sible for each RFLP are reasonably well known, the cipient form of a given RFLP in both inverted repeat observedrecombination frequency between RFLPs elements. The transient heteroplasmic genome might can be determined for the transformants and com- also undergointermolecular copy correction,thus pared to the expected frequency of recombination spreading the introducedsequences to additional mol- (Figure 6). Foreach interval between scorable physical ecules that could then undergo intramolecular copy or geneticmarkers, a predicted recombination fre- correction. Novel polymorphisms arising following quency has been calculated based on the assumption recombination between donor and recipient RFLPs that one exchange event occurs (100% probability) in the inverted repeat are also efficiently copy cor- between the selectable marker and each end of the rected to the other repeat (Figure 3, bottom panel). donor insert and that the recombination frequency Less frequently, two integration events or two copy per unit lengthis constant. Substantial bias is observed correction events with different endpoints may occur for the exchange events to occur between the restric- in the same recipient cell and result in a colony that tion sites at the p1asmid:insert junction and the first is initially mixed for the diagnostic RFLPs. A prece- available marker which is eitherthe unselected er dent exists for multiple transformation events occur- mutation in the P-183/CC-1852transformation or ring within the nucleus of the same yeast cell bom- the polymorphism in the KpnI-Hind111 fragment barded with amixture of two different plasmids, spanning the 5‘ end of the 23s rRNA gene for theP- although most recipient cells are penetratedby only a 229/CC-130 experiments. A similar bias is also ob- Chloroplast rRNA Gene Transformation 885

23s 16s I- I- 0 00 0 0 Y) aN 00 0 Lo ? 91- P,? ? I vv yyI1 I I vyI VI,I (A) I B' er H K K spr Sr K K B

" " " - " - - 0.2 2.4 0.2 0.35 0.40.2 0.7 0.650.9 0.15 0.4 0.5 interval (kb)

7 12 0 1 0 0 n.a. 9 0 0 11 number events

5 55 8 9 5 16 n.a. 46 8 2620 Ye expected4nterval

21 74 0 5 0 0 n.a. 45 0 0 55 Yo observedlintervai

A " * :2 =z - " - 0.50.35 0.4 0.2 0.7 0.650.9 0.15 0.4 0.5 intelvd (kb)

109*0 0 1 2 6 0 0 12 number events

23 1623 19 9 33 25 34 6 1915 96 expeclednnterval

50 45 0 0 5 10 30 0 0 60 % obsetvednntervai FIGURE6.-Comparison of the expected and observed frequencies of exchange events for (A) P-183/CC-1852 and (B) P-229/CC-130 transformants. Relative positions of restriction sites, antibiotic resistance loci and size of heterologies responsible for the RFLPs are based on fine structure mapping and sequenceanalysis. The unique 0.5-kbKpnI fragment of C. smithii is included as partof the 680-bp polymorphism in the 16s-23s rRNA gene interval. Abbreviations areas in Figure 1 legend. The expected percent recombination per interval is calculated separately for the two halves of the map, from the BamHI sites to sr or spr for (A) and from spr to the BamHI or Hind111 sites for (B), as being directly proportional to the physical distance between pairs of genetic and physical markers. Events marked by the * occur within the polymorphism, generatinga RFLP of intermediate (ca. 1.15 kb) length (e.g., Figure 3, bottom panel). Number and percentof events occurring between the BamHI site and er are derived from phenotypicanalysis of 34 spr sr P-l83/CC-1852 transformants, although the RFLP patterns of only 20 isolates (6 er spr sr and 14 er+ spr ST) were determined. Since sr and spr alleles were used as the co-selectable markers in P-1831 (X-1 852 transformation experiments, recombination frequencies in that interval were not applicable (n.a.). served for integration events near the 5' end of the dissimilar DNA sequences and therefore branch mi- 16s rRNA gene. In both cases this is seen when the gration would not proceed into the vector. Presum- percent of exchange events expected in a given inter- ably resolution of any heteroduplex between donor val is compared to the percent of all exchange events and recipient sequences would occur in a region of that actually occur in that interval (Figure 6, A and homology near the insert:vector junction. This hy- B). Based on an equal probability of exchange per pothesis would explain why a KpnI-Hind111 RFLP of unit length, a strong preference for exchange at both intermediate size is observed in rDNA transformants ends of the inserts from P-183 and P-229 is observed of CC-130 generated with P-229 (Figure 4), but not compared to internal intervals nearest the selectable with P-183/CC-1852 (Figure 5). In the case of the P- markers. Asmall increase in the expectedus. observed 183/CC-1852 transformants (Figure 6A), no recom- frequency of exchange is also detected in the 2.4-kb bination between the -50 and -250 RFLPs in the interval from the er locus to the polymorphism im- 1.4-kb KpnI-Hind111 fragment is seen compared to mediately 5' to the 23s rRNA gene (Figure6A). The the 8% expected, whereas in the P-229/CC-130 trans- increases in the observed us. expected values for ex- formants (Figure 6B) recombination in this same in- change events at each end of the donor fragments terval isnow morethan two-fold higherthan ex- range from 2.1- to 4.2-fold. pected. The -50 and -250 RFLPs in the KpnI- Effect of heterology on the location of exchange Hind111 fragment (Figures 1, 6) may be close enough events: Heterology can have several distinct effects to the Hind111 end of the P-229 insert (but too far on the location of exchange events. Such events prob- from the BamHI end in the 23s rRNA geneof the P- ably cannot occur beyond the endof the donor insert 183 insert) to be involved in the resolution stepof the because of the lack of homology between vector and exchange event leadingto integration. This difference recipient DNA sequences (LIEB, TSAIand DEONIER in the frequency of exchangeevents for the same 1984). Heteroduplexes would not extend into these 0.35-kb region in the KpnI-Hind111 fragment clearly 886 S. M. Newman et al.

shows that position rather thansequence is responsible stream of the 16s rRNA gene. No physical evidence for the variation. Other polymorphisms (0.1-0.5-kb for exchange was observedbetween the adjacent additions and deletions of SDR elements) distributed RFLPs upstream of the 16s rRNA gene in either the throughout the rDNA region of the chloroplast ge- P-l83/CC-1852 or P-229/CC-130 transformants or, nome do not appear to stimulate theactive exchange with one exception, between the RFLPs separated by process that is observed near the insert:vector junc- clusters of KpnI sites in the 16s-23s intergenic spacer tions (Figure 6). region of these transformants. These RFLPs cosegre- The tendency for exchange events to occur repeat- gate (e.g., Figure 3, top and middle panels) and there- edly within a specific region may also be related to fore appear to betightly linked genetically. While the the distribution of heterologous and homologous se- tight linkage of these RFLPs may be the effect of quences. In those transformants with an intermediate heterology (see above) and not thecause of preferen- size KpnI-Hind111 RFLP, this fragment is always ca. tial recombination at vector:insert junctions,there 1.15 kb. If the polymorphism that generates the 300- may also be a strong selection for a mechanism in C. bp length variation in the 1.4/1.1-kb KpnI-Hind111 reinhardtii that reduces exchange eventsbetween SDR fragment is the sum of separate 50-bp and 250-bp elements in the chloroplast genome. This would min- polymorphisms, then an exchange event in the ho- imize the numberof deletion and inversion mutations mologous sequence between thosetwo polymorphisms arisingfrom illegitimate pairing and crossing over could generate asingle intermediate size classof 1.15- between these regions of homology present in most kb KpnI-Hind111 fragments containing the 50-bp pol- intergenic regions ($ BOYNTONet al. 1990b, PALMER ymorphism (Figure 6). et al. 1985). Such a mechanism would serve to increase Tracts of homology less than some minimum length the relativerecombination frequencies in those re- may notpromote efficient heteroduplexformation gions of the chloroplast genome lacking SDR elements needed for recombination. This would be manifested in sexual crosses andduring integration of donor as apparent linkage between adjacent RFLPs, when, fragments that generate chloroplast transformants. in fact, heteroduplexformation necessary for ex- Integration events involving the 0.9-kb region at change events could not be initiated. The minimum the 5’ end of the 16.3 rRNA gene located ca. 1.4 kb length of homologous DNA requiredfor efficient from the vector:insert junction occur at the expected recombination varies from 23 to 90 bpin prokaryotes frequency compared to the strong suppression of ex- (SHENand HUANG 1986) tobetween 200 and 400 bp change events observed for all other internal regions in eukaryotic nuclear DNA (BOLLAG, WALDMANand in commonbetween thedonor fragment and the LISKAY 1989), although recombination between ho- recipientgenome (Figure 6). This is likely not the mologous sequences as short as 20 bp has been ob- result of recombination suppression between tightly served in several systems (WATTet al. 1985). In ad- linked RFLPs promoting recombination in an adja- dition, single base pair changeswhich disrupt stretches cent region of homology, since the comparably sized of perfect homology can affect initiation of recombi- 0.7-kb interval at the 3’ end of the 16s gene, which nation (WALDMAN andLISKAY 1988) although prop- is also flanked by tightly linked RFLPs, shows strong agation of heteroduplexes through sequences of suppression of exchange events. The normal level of greater heterogeneity readily occurs (BOLLAG,WALD- exchange events occurring in the 0.9-kb interval up- MAN and LISKAY1989). Therefore in localized regions stream of the 16s gene in a region of recombination flanking the chloroplast rRNA genes of Chlamydo- suppression could result fromunique properties of monas, homologous sequences may notbe long the 400-bp region of heterology or could be related enough nor exhibitsufficient homology to permit t.he to transcription of the rRNA operon,which is initiated formation of heteroduplexes. At present we are un- 72 bp 5’ to the 16s gene(SCHNEIDER et al. 1985). sure of the extent and distribution of sequence ho- Transcriptionallyinduced exchange events are ob- mology required for heteroduplex formation initiat- served in thepromoter region of the S. cerevisiae ing chloroplast DNA exchange events in Chlamydo- nuclear rDNA repeat unit (VOELKEL-MEIMAN, KEIL monas. Clearly heteroduplexformation occurs and ROEDER1987). Since transformantscontaining frequently in the 200-bpinterval between the er the rDNA region from the recipient strain marker and the BamHI site marking the vector:insert do not appear to be favored over those having the junction of P-183 to generate er spr sr transformants donor promoter (Figures 4, 5), recombinants are not with CC-1852. However this short interval is adjacent being selected to maintain aparticular promoter- to a long region of homologous 23srRNA gene rRNA gene combination. Preferential localization of coding sequence which may enhance pairing. exchange events upstreamof the 16s rRNA gene and Exchange near the vector:insert junctions may also in a particular region of the 16s-23s rRNA spacer is be enhanced by recombinational suppression imme- also seen in 60 recombinant meiotic progenyfrom diately adjacent to theKpnI sites upstream and down- crosses of strains carrying the C. reinhardtii and C. C hloroplast rRNA Gene Transformation Gene rRNAChloroplast 887 smithii chloroplast genomes (S. M. NEWMAN,E. H. GRANT,D. M., N. W. GILLHAMand J. E. BOYNTON, 1980 HARRIS,N. W. GILLHAMand J. E. BOYNTON,in prep- Inheritance of chloroplast DNA in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 77: 6067-607 1. aration). In contrast, crosses involving C. reinhardtii HARRIS,E. H.,1989 The Chlamydomonas Sourcebook. Academic strains carrying different chloroplast antibiotic resist- Press, San Diego. ance markers in the 16s and 23s rRNA genes, show HARRIS,E. H.,J. E. BOYNTONand N. W. GILLHAM, 1987 remarkably uniform recombination over7-kb a region Chlamydomonas reinhardtii, pp. 257-277, in Genetic Maps 1987. containing the rRNA genes (HARRISet al. 1989), and ACompilation of Linkageand Restriction Maps of Genetically Studied Organisms. Vol. 4, editedby S. J. O’Brien. Cold Spring there seems to be no recombinational suppression Harbor Laboratory, Cold Spring Harbor, N.Y. between markers in the 16s and 23s rRNA genes. HARRIS,E. H., J. E. BOYNTON, N. W. GILLHAM,C. L. TINGLEand In conclusion, recombination events leading to ho- S. B. Fox,1977 Mapping of chloroplastgenes involved in mologous replacement of recipient sequences by chloroplast biogenesis in Chlamydomonas reinhardtii. cloned donor fragments during chloroplast transfor- Mol. Gen. Genet. 155: 249-265. HARRIS,E. H., B. D. BURKHART, N. W. GILLHAM J.and E. BOYN- mation opens the possibility of manipulating specific TON, 1989 Antibiotic resistance mutations in the chloroplast chloroplast sequences as a means of beginning to 16s and 23s rRNA genes of Chlamydomonas reinhardtii: cor- understand the mechanisms of chloroplast DNA re- relation of genetic and physical maps of the chloroplast ge- combination. RFLP differences between donor and nome. Genetics 123: 281-292. recipient have been shown to be useful for defining HOSLER, J. P.,E. A. WURTZ,E. H. HARRIS,N. W. GILLHAM and J. E.BOYNTON, 1989Relationship between gene dosage and the sites of exchange surrounding chloroplast genes gene expression in the chloroplast ofChlamydomonas reinhard- introduced by transformation. The extent and distri- tii. Plant Physiol. 91: 648-655. bution of heterologous and homologous regions be- LEMIEUX,C., AND R. W. LEE, 1987 Nonreciprocal recombination tween therecipient chloroplast genome and donor between alleles of the chloroplast 23s rRNA genein interspe- insert clearly influence the frequency, and probably cific Chlamydomonas crosses.Proc. Natl. Acad. Sci. USA 84: 4166-4170 the distribution, of exchange events between chloro- LEMIEUX, C., BOULANGER,J. C.OTIS and M. TURMEL, plast genomes. 1989Nucleotide seqeunce of the chloroplast large subunit rRNAgene from Chlamydomonasreinhardtii. Nucleic Acids This work was supported by National Institutes of Health grant Res. 17: 7997. GM-19427. S.M.N. was supported by National Institutes of Health LIEB, M., M. M. TSAIand R. C. DEONIER, 1984 Crosses between postdoctoral fellowship GM-12934. insertion and point mutations in lambda gene cl: Stimulation ofneighboring recombination by heterology.Genetics 108: LITERATURE CITED 277-289. ARMALEO, D., G.-N.YE, T. M. KLEIN,K. B. SHARK,J. C. SANFORD MANIATIS,T., E. F. FRITSCHand J. SAMBROOK, 1982 Molecular and S. A. JOHNSTON,1990 Biolistic nuclear transformation of Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Saccharomyces cerpuisiae and other fungi. Curr. Genet. 17: 97- Cold Spring Harbor, N.Y. 103. MYERS, A. M., D. M. GRANT, D. K. RABERT,E. H. HARRIS, J.E. BLOWERS,A. D., L. BOGORAD,K. B. SHARK and J. C. SANFORD, BOYNTONand N. W. 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