Proc. Natl. Acad. Sci. USAVol. 92, pp. 8149-8153, August 1995Plant Biology

Targeted gene inactivation in petunia by PCR-based selection oftransposon insertion mutants

(reverse genetics/dTphl/transposable element)

RONALD KOES*t, ERIK SOUER*, ADELE VAN HOUWELINGEN*, LEON MUR*, CORNELIS SPELT*,FRANCESCA QuArrROCCHIO*, JOHN WING*, BERRY OPPEDIJK*, SHARLIN AHMED*, TAMARA MAESt,TOM GERATSt, PIETER HOOGEVEEN*, MARTINA MEESTERS*, DAISY KLOOS*, AND JOSEPH N. M. MOL*Department of Genetics, Vrije Universiteit, Institute of Molecular Biological Sciences, Biocentrum Amsterdam, de Boelelaan 1087, 1081 HV Amsterdam, TheNetherlands; and tDepartment of Genetics, University of Ghent, Ledeganckstraat 35, B9000 Ghent, Belgium

Communicated by Marc C. E. Van Montagu, University of Ghent, Ghent, Belgium, May 22, 1995

ABSTRACT Establishment of loss-of-function pheno-types is often a key step in determining the biological functionof a gene. We describe a procedure to obtain mutant petuniaplants in which a specific gene with known sequence isinactivated by the transposable element dTphl. Leaves arecollected from batches of 1000 plants with highly active dTphlelements, pooled according to a three-dimensional matrix,and screened by PCR using a transposon- and a gene-specificprimer. In this way individual plants with a dTphl insertioncan be identified by analysis of about 30 PCRs. We foundinsertion alleles for various genes at a frequency of about 1 in1000 plants. The plant population can be preserved by selfingall the plants, so that it can be screened for insertions in manygenes over a prolonged period.

offspring and found to be due to insertions of dTphl elements(E.S., A.v.H., D.K., J.N.M.M., and R.K., unpublished data).Thus, the high incidence of mutations in the W138 line ismainly due to transposition of dTphl elements.We show how plants with a dTphl insertion in a specific gene

can be identified in a large plant population by a polymerasechain reaction (PCR) assay. Such plants are heterozygous forthe insertion allele unless the insertion occurred in a previousgeneration. After selfing, progeny homozygous for the inser-tion allele are obtained that can be analyzed for an alteredphenotype. The screened plant population is maintained byselfing all plants, so that semipermanent libraries are obtainedthat can be screened at any time. These libraries are open tothe scientific community.

The current DNA methodology allows the identification andcloning of genes, without prior knowledge about their bio-chemical function. For example, many genes have been iden-tified by sequence homology with genes from other species oron the basis of their expression pattern. As the programs onsequencing of randomly selected cDNA clones and wholegenomes proceed, the number of cloned genes with unknownfunction is rapidly increasing (e.g., refs. 1 and 2).One way to determine the function of a gene is to inactivate

it. In plants, inhibition of the expression of specific genes hasbeen achieved by antisense RNA and cosuppression. Bothmethods are based on transcription of a transgene causing adownregulation of the mRNA level of homologous endoge-nous genes (for review, see refs. 3-5). Antisense RNA andcosuppression have been successfully employed to establishthe function of genes involved in flower pigmentation (6), fruitripening (7), flower development (8, 9), and carotenoid bio-synthesis (10). However, these methods have features that limittheir general use (see Discussion), and alternative strategiesare therefore required.Here we present such an alternative strategy for petunia. It

is based on inactivation of a gene by insertion of the trans-posable element dTphl (11-14). Petunia flowers of the lineW138 have a variegated pigmentation pattern due to muta-bility of the anl gene (15, 16). In W138 offspring, new unstablemutations are found at relatively high frequency due to theactivity of transposable elements (17, 18). Analysis of apolymorphic restriction fragment in the dfrC gene of W138identified a 284-bp insertion sequence, designated dTphl, withfeatures of a transposable element (19). A mutable allele of theflower pigmentation gene rt contained a dTphl insertion,indicating that dTphl can inactivate a gene and cause amutable phenotype (6). Ten mutable alleles of the pigmenta-tion genes an3 and anl3 were recently isolated from W138

MATERIALS AND METHODSPlant Materials. Petunia plants of the line W138 were grown

in a greenhouse. Large plant populations consisted of multiplefamilies, with a maximum size of 25 plants, each originatingfrom a single seed capsule. Only one seed capsule of eachparental plant was grown. Because W138 plants harbor atransposon insertion in the flower pigmentation gene anl,-90% of the plants had spotted flowers while -10% hadevenly colored red flowers resulting from a sporogenic rever-sion event.DNA Isolation. Large pools of leaves were ground in liquid

nitrogen to ensure thorough mixing, after which excess mate-rial was discarded. DNA was isolated essentially as described(20) and treated with RNase. After addition of an equalvolume of 200 mM Tris-HCl, pH 7.5/50 mM EDTA/2 MNaCl/2% (wt/vol) N-cetyl-N,N,N-trimethylammonium bro-mide and 15 min of incubation at 65°C, the solution wasextracted with chloroform/isoamyl alcohol (24:1, vol/vol). TheDNA was precipitated with 2-propanol and dissolved in 10mMTris/1 mM EDTA, pH 8.0.

Detection of dTphl Insertion Alleles. For detection ofinsertion alleles we used the oligodeoxynucleotide primer outl(5'-GGGAATTCGCTCCGCCCCTG-3'), which is comple-mentary to the terminal inverted repeat ofdTphl, and a primercomplementary to the gene to be knocked out. Primers wereobtained from Isogen Bioscience, Amsterdam. PCR was car-ried out in 50 ,ul containing 50 mM Tris HCl, pH 9.0, 4 mMMgCl, 0.01% gelatin, 25 pmol of outl, 25 pmol of the gene-specific primer, and -0.1 j,g of plant DNA, using 30 cycleseach consisting of 30 sec at 94°C, 30 sec at 55°C, and 1 min at72°C. Amplification products were size-separated by electro-phoresis in 1% agarose gels, blotted to Hybond-N membranes,and hybridized to a gene-specific probe (usually a cDNA) todetect insertion products.

tTo whom reprint requests should be addressed.

8149

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

6, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995)

Thin-Layer Chromatography (TLC). One petal limb wasboiled in 1 ml of 2 M HCl for 20 min. Dihydroflavonols andflavanones were extracted with 150 jl of ethyl acetate and 2,ul were applied on Kieselgel F254 TLC plates (Merck). Afterseparation in chloroform/methanol/formic acid (90:9:1),spots were visualized in UV light.

RESULTSScreening for dTphl Insertions with a PCR-Based Assay.

Rare insertions of a transposable element into a gene can bedetected by PCR using a gene-specific and a transposon-specific primer (11-13). Only if a transposon is inserted intothe gene will a suitable template be generated that will beexponentially amplified in PCR.To test whether plants with new dTphl insertions can be

identified among a population of wild-type plants by PCR, wescreened 1000 W138 plants for insertions in an3, the geneencoding the flavonoid biosynthetic enzyme flavanone 3-hy-droxylase (F3H). One leaf was taken from each plant andpooled in 10 groups of 100 leaves. DNA was isolated from eachpool and subjected to PCR analysis using an an3-specificprimer and a transposon-specific primer, outl, complementaryto the terminal inverted repeat of dTphl. This allows detectionof dTphl insertions irrespective of their orientation. A rangeof amplification products was obtained (Fig. LA Left), most ofwhich were also observed when the outi primer alone was used(data not shown). Because the pattern varied between differ-

A first round last round(pools ofr 00 plants) (single plants)

1 2 3 4 5 6 7 8 9 10 12 1 4 1 5 7 X 9 10 2 x 4 9 h 7 x 9 10

2~~~~~~~

0.9 S'_..''.'' ,SD.6_

ckb

I.51.2

I,-{115

wild-type

AACCAATGCATGITGAATACrACr

TSl)

T3631-1TIR TIR

CAOOGGGCGGAoCrACT -//- CCrGGCrCCoCCCCrO

AACCAATCCATG1TGA CATGTTGAATACTACT

TSI) TSOI

bp

FIG. 1. Identification and characterization of an an3 insertion byone-dimensional screening. (A) Single leaves were taken from apopulation of 1000 W138 plants and pooled in 10 groups of 100. dTphlinsertions in an3 were identified by PCR analysis using one gene-specific primer and the dTphl primer outl. (Left) Ethidimum bromide-stained amplification products. (Center) Amplification products hy-bridizing to an an3 cDNA probe (21). The strong signal in pool 7originates from multiple plants harboring the mutable an3-s205 allele.The 400-bp fragment detected in pool 5 represents a new insertionevent. The single plant harboring this insertion allele was identified byrescreening the plants in pool 5 in two additional rounds. (Right)Amplification products generated from single plants in the final roundof screening. Lane 9 contains amplification products from the singleplant (T3631-1) harboring the dTphl insertion in an3. (B) PCR analysisof the an3 insertion allele present in plant T3631-1. The diagram at thebottom shows the position of the two an3-specific primers used togenerate an3 amplification products. The triangle shows the positionof the dTphl element in the an3-t3631 insertion allele. (C) Sequenceanalysis of the dTphl insertion in plant. Sequence of only the ends ofthe dTphl element is shown. TSD, target-site duplication. TIR,terminal inverted repeat of dTphl.

ent DNA samples, the products obtained with the outl primeralone presumably derived from loci containing two closelyspaced dTphl elements or one dTphl copy close to a sequenceresembling the outl primer. After blotting and hybridizationwith an an3 probe, we detected an3-specific amplificationproducts for 2 pools (Fig. 1A Center). Pool 7, which gave thestrongest signal, contained 15 plants homozygous for thedTphl insertion allele an3-s205, previously identified by phe-notype. The 400-bp fragment detected in pool 5 represented anew insertion event. To identify the plant containing this newallele, pool 5 was split into 10 batches of 10 plants. PCRanalysis showed that the 400-bp an3 fragment could be am-plified from only 1 batch of 10 plants (data not shown). PCRanalysis of the 10 individual plants showed that the 400-bp an3fragment originated from a single plant, T3631-1 (Fig. LARight, lane 9).To confirm that plant T3631-1 indeed contained a dTphl

insertion, part of the an3 gene was amplified with two gene-specific primers. PCR on wild-type W138 plants (plantsT3612-2, T3629-1, and T3631-2) generated a single 1.2-kbamplification product, whereas plant T3631-1 yielded a 1.5-kbproduct (Fig. 1B) that hybridized to a dTphl probe (data notshown), in addition to the 1.2-kb product. Cloning and partialsequencing of the 1.5-kb product confirmed that a 284-bpdTphl element had inserted in the first intron of the an3 gene,creating an 8-bp target-site duplication (Fig. 1C), like thosefound for other dTphl insertions (refs. 6 and 19; E.S., A.v.H.,D.K., J.N.M.M., and R.K., unpublished data).To establish whether insertions could also be isolated for

other genes, we screened the same plant population forinsertions in the genes myb27 and myb92. For myb27 a dTphlinsertion allele was detected that was assigned to a single plantin two additional rounds of screening. For myb92, however, noplants with a dTphl insertion could be found (Table 1).

Identification of dTphl Insertion Alleles in a Single Roundof Screening. The repeated rounds of screening become therate-limiting step when one wants to screen for insertions inmultiple genes. We therefore switched to a more efficient leafpooling system, as illustrated in Fig. 2. From a population ofplants, leaves are collected and pooled three times, each timeaccording to another pattern (blocks, rows, and columns). Inthis way material from each plant is represented in a uniquecombination of a block, a row, and a column.To test the feasibility of such an approach, three sets of

W138 plants, of about 1000 plants each (populations 2-4,Table 1), were screened for insertions in different genes.Typical results of the PCR analyses are shown in Fig. 3. For thegene myb92 one strongly hybridizing fragment was detectedconsistently in the three dimensions of the screen (column 9,block E, row II). In addition, a number of weakly hybridizing

Table 1. Number of insertion alleles identified by PCR assay

No. of insertion alleles

Gene Pop. 1 Pop. 2 Pop. 3 Pop. 4

an3 1 - 1myb27 1 2 1myb92 0 1 1 0mybbl 1 0jafl3 1ap2a 1 2 2sha4 1sha3 1sha6 2es87 - 1 3

Each of the four populations consisted of about 1000 plants.Populations (Pop.) 1 was screened one-dimensionally; populations 2-4were screened three-dimensionally. The number of insertion allelesfound is given; 0, no insertion allele found; -, not tested.

B

8150 Plant Biology: Koes et aL

Dow

nloa

ded

by g

uest

on

Aug

ust 2

6, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995) 8151

columns (I-rn)

- - _m- w 5- =ml 1 1 1 11 1 1 1 1 1 1

123456

1-1VI

04rA.

A

blocks (A-D)

FIG. 2. Three-dimensional system for pooling of leaves. The whitedots represent plants as they are usually positioned in a greenhouse.Single leaves of a plant are taken three times-one to be pooledaccording to the blocks A-D, one to be pooled according to thecolumns I-III, and one to be pooled according to the rows 1-6. In thisway leaf material of a single plant is represented by a uniquecombination of a block, a column, and a row.

fragments showed up irregularly in the different lanes. Thesefragments most likely originated from somatic insertion eventsgiving rise to chimeric plants. PCR and sequence analysis ofDNA from the plant on position 9-E-II confirmed that it washeterozygous for a wild-type and a dTphl insertion allele ofmyb92 (data not shown). For the gene es87 four differentlysized fragments were detected, each consistently in a block, arow, and a column (Fig. 3B). This indicated that there werefour plants in the population each harboring a dTphl insertionat a different position in the gene. For three plants we couldconfirm that they were heterozygous for the wild-type es87allele and a mutant allele harbouring a dTphl insertion (datanot shown). For the fourth allele we could not prove this,because the dTphl insertion mapped outside the sequencedregion of es87 and, thus, could not be amplified with twoes87-specific primers.We screened the three plant populations (populations 2-4)

for dTphl insertions into eight additional genes-an3, myb27,mybl, jafl3, ap2a, sha3, sha4, and sha6-and also isolatedinsertion alleles for these genes (Table 1).

Do dTphl Insertions Inhibit Gene Expression? To testwhether the dTphl insertions led to gene inactivation and amutant phenotype, we analyzed 2 plants (V2221-14 andT3631-1) harboring a dTphl insertion in an3. Plant V2221-14was identified by three-dimensional screening (population 4,Table 1) and was heterozygous for a wild-type allele (An3+)and an allele with a dTphl insertion in the second exon(an3-v2221; data not shown). Because this plant also harboredthe unstable anl-w138 allele, the following two phenotypesmight be expected in the progeny in a 3:1 ratio: anl unstable/An3+ and anl unstable/an3 unstable. In anl unstable/An3+flowers, Anl+ reversion events lead to a random pattern ofevenly colored sectors and spots, whereas in anl unstable/an3unstable flowers reversions of one gene (anl or an3) lead tosectors displaying revertant spots of the other gene. Thus, inthe latter case revertant spots are confined to certain sectorsof the flower (22). Among 23 progeny plants, 17 had thespotting pattern typical for the unstable anl-w138 allele (Fig.4A Left), whereas 6 plants had the spotting pattern predictedfor the anl/an3 double mutant (Fig. 4A Right). PCR analysisconfirmed that plants of the first class were An3+/An3+ orAn3+/an3-v2221, whereas plants of the last class were an3-v2221/an3-v2221. TLC (Fig. 4B) showed that flowers from thefirst class accumulated dihydroquercetin, whereas flowersfrom the latter class accumulated eriodictyol, confirming thatan3 expression was blocked. Closer inspection of an3-v2221/an3-v2221 flowers showed that they contained two types ofrevertant spots: spots with diffuse boundaries and spots withsharp boundaries. The gene anl controls transcription ofmultiple structural anthocyanin genes and controls flowerpigmentation in a cell-autonomous way (23). As a consequenceAnl+ revertant sectors exhibit sharp boundaries. An3+ rever-tant sectors, on the other hand, have diffuse boundaries (E.S.,

A an I-mut an I -mut/an3-mut

rows blocks columns1 2 3 4 6 57 8 910 ABOCDDEF G HGKL _m E2>>5:>H ?MXX

.0 S.pB m. .4

B rows blocks columns1 2 3 4 6 S 7 8 9 10 A B C D E F G H K L - E 2> 5 5 X X

address4-C-III,,

9-A-VI- * *

8-K-1"1W... .....

7-F-Ilr .:- .

prim. -> _

FIG. 3. Identification of insertion alleles by three-dimensionalscreening. The position (address: row-block-column) in the three-dimensional matrix of plants with an insertion allele is given at left.prim., Primer. (A) Identification of a dTphl insertion allele of the genemyb92. (B) Identification of four insertion alleles of the gene es87.

enodictyol

a1fn3

dihydroquercetin

(4t1/

anthocyanin

FIG. 4. Phenotype caused by a dTphl insertion into the gene an3.(A) Phenotype of an An3+ (Left) and an an3-mutable flower (Right),both in an anl-mutable (anl-w138) background. (B) TLC analysis offlavonoids accumulating in anl-mutable/An3+ and anl-mutable/an3-mutable flowers. The action of the genes an3 and anl in the antho-cyanin pathway and the positions of purified eriodictyol and dihy-droquercetin spots are indicated in the diagram on the left.

A

address

9-E-II-B an I -muit/ an I -mutJ

An3+ an3-mnut

Plant Biology: Koes et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

6, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995)

A.v.H., D.K., J.N.M.M., and R.K., unpublished data) presum-ably due to cell-to-cell diffusion of pathway intermediates.These data show that the dTphl insertion in an3-v2221 causeda reversible gene inactivation.

Plant T3631-1 was heterozygous for an an3 allele with adTphl insertion in the first intron of the gene (Fig. 1). Becausethis plant harbored a second mutation (petals and anthersfused) causing male sterility, we analyzed the activity of thean3-t3631 alleles by crossing to an an3 tester line (W62) thathas a complete deletion of the an3 gene (E.S., A.v.H., D.K.,J.N.M.M., and R.K., unpublished data). All 46 progeny plantshad fully pigmented flowers whether they did or did notcontain the an3-t3631 insertion allele (data not shown). Ap-parently the insertion of dTphl in the first intron did notinactivate an3.

DISCUSSIONSite-Selected Transposon Mutagenesis. We describe a

method to select transposon insertion mutants for specificgenes by a PCR assay. If leaf material is pooled according toa "three-dimensional" matrix identification of single plantsamong a population of 1000 plants requires about 30 PCRs(Figs. 2 and 3). We prefer this one-step three-dimensionalscreening over the three repeated rounds of one-dimensionalscreening, as employed in Fig. 1, because (i) it is less laborious;(ii) it is less liable to detect false positives due to somaticinsertions of dTphl, as such sectors rarely expand to all theleaves of a plant and, thus, do not produce signals in all threedimensions of the screen (see Fig. 3); and (iii) it directlyidentifies a single plant. Because of this last feature the plantsthemselves are not needed once DNA has been isolated. Whenall plants are selfed and the seeds are stored one plantpopulation can be screened many times over a prolongedperiod. We have now constructed three such libraries com-prising a total of 4000 plants. A similar method was used toestablish permanent transposon insertion libraries in Caeno-rhabditis elegans (14).

Detection of Phenotypes. We hope to use transposon inser-tion alleles to establish loss-of-function phenotypes for anumber of genes (e.g., see Table 1; data on specific phenotypeswill be published elsewhere). We have noticed, however, thatheterozygous plants do not always give progeny with a mutantphenotype. Because this phenomenon is of general interest,these cases are discussed below.The analysis of two insertion alleles of an3 showed that

dTphl insertion in the second exon (an3-v2221) causes geneinactivation, whereas insertion into an intron (an3-t3631) doesnot. Also dTphl insertions into introns of the genes mybl,myb27, and myb92 did not inactivate the genes, as determinedby RNA gel blot analysis (L.M. and C.S., unpublished results).This is in line with the observation that dTphl insertions inanthocyanin genes that were identified by screening for phe-notypes were usually in the coding sequence of the genes (E.S.,A.v.H., D.K., J.N.M.M., and R.K., unpublished data) and inone case in the TATA box of the gene (6). Tcl insertions inintrons of C. elegans genes do not generally cause geneinactivation (13). For such alleles a knockout allele can beisolated by PCR screening for subsequent deletion derivatives(14). In petunia, however, dTphl-induced deletions seem tooccur at low frequency, if at all (E.S., A.v.H., D.K., J.N.M.M.,and R.K.; unpublished data), and therefore it is more conve-nient to isolate additional insertion alleles.Other insertion alleles failed to give a visually recognizable

phenotype, even though the insertions were in the protein-coding sequence of the gene and caused alterations in themRNA of myb92 (L.M. and C.S., unpublished data) and ap2a(T.M. and T.G., unpublished data). This phenomenon may beinherent to reverse genetic strategies. One possibility is thatthe mutant phenotype cannot be detected visually or can be

seen only under specific environmental conditions (e.g., ref.24). Alternatively, the function of the mutated gene may beredundant. This has also been observed in mice and yeast withloss-of-function alleles for regulatory proteins (e.g., refs. 25and 26). In such cases it may be necessary to isolate insertionalleles for multiple related genes and construct double mutantsbefore a phenotype is obtained (e.g., ref. 27).Comparison of Gene Inactivation Strategies. Until now,

transgene-based methods (antisense RNA and cosuppression)were the only way to selectively inhibit the expression of a plantgene with unknown function. Gene inactivation by transposoninsertions has several advantages over the transgene-basedmethods.

(i) Transgene-induced inhibition of an endogenous gene israrely complete (4). Generally, gene inactivation occurs only insome of the transgenic plants and even in such a plantinactivation does not occur in all cells, resulting in unpredict-able patterns (28-30) the extent of which is influenced byexternal and internal factors (31). In transposon insertionmutants the extent to which a gene is inactivated can often bepredicted on the basis of the integration site or from RNA blotanalyses and it will be similar in each homozygous plant.Transposon insertion mutants may, however, also be patterneddue to somatic excision of the transposon, but stable recessivederivative alleles where transposon excision created a typicalfootprint can be isolated in a second step.

(ii) Transgenes can suppress endogenous genes that shareonly 80% DNA homology (31), and homologous regions of 55bp and perhaps smaller are sufficient (32). This complicatesanalyses of genes with conserved domains [e.g., MADS (37)box genes and myb-homologous genes], as it is virtually im-possible to establish whether a phenotype is caused by inac-tivation of a single gene. In contrast, transposon insertions actin a completely gene-specific fashion.

(iii) Transgenes generate (semi) dominant phenotypes. As aconsequence, lethal phenotypes are difficult if not impossibleto recognize. Transposon insertions generally create recessivemutations, and lethality of a mutation can be recognized by theabsence of homozygotes for the insertion allele in progeny.When the mutation causes sterility, the heterozygotes can beused for further genetic analyses.

(iv) Cosuppression and antisense RNA strategies are toolabor-intensive to inactivate large numbers of genes, as thisrequires for each gene that suitable DNA constructs be madeand that at least 10-20 transgenic plants be raised. Oncepermanent transposon insertion libraries are established, itmay take relatively little effort to find insertion mutants for acertain gene.

General Applicability. Our results suggest that differentgenes in the genome of petunia are hit by dTphl with roughlysimilar frequencies ("1 insertion per 1000 plants screened;Table 1), suggesting that virtually any gene in the petuniagenome can be inactivated by a dTphl insertion. The strategydescribed here may also be used for other plant species,provided that defined insertion sequences are available. Forimportant model plant species such as maize and snapdragon,highly active endogenous transposable elements are known(33), and they can also be used as a mutagen in heterologousplant species (for review, see ref. 34). Endogenous transpos-able elements of Arabidopsis do not seem to transpose fre-quently enough to be useful (35); however, the available libraryof T-DNA insertions (36) may provide an equally good alter-native. The permanent petunia libraries of dTphl insertionsare in principle open to the scientific community. Researcherswho wish to screen for mutants are kindly invited to contact us.

Because the work presented in this paper appeared beneficial to theresearch of many people, such a true collaboration was spontaneouslyestablished that it was impossible to trace back the contribution of eachindividual. Thus, the order of authors is more or less arbitrary. We are

8152 Plant Biology: Koes et aL

Dow

nloa

ded

by g

uest

on

Aug

ust 2

6, 2

021

Proc. Natl. Acad. Sci. USA 92 (1995) 8153

indebted to Joop Meyer, Wim Bergenhenegouwen, and FredSchuurhof for the photographic work. F.Q., J.W., A.v.H., and E.S.were supported by grants from the Netherlands Organization forChemical Research, with financial aid from the Netherlands Organi-zation for the Advancement of Research (F.Q. and J.W.) and theNetherlands Technology Foundation (A.v.H. and E.S.). T.G. wasfunded by a Human Capital and Mobility grant (ERBCHBGCT920097) and T.M. by the Vlaams Instituut voor de Bevordering van hetWetenschappelijk-Technologisch Onderzoek in de Industrie.

1. Hofte, H., Desprez, T., Amselem, J., Chiapello, H., Caboche, M.,et al. (1993) Plant J. 4, 1051-1062.

2. Sasaki, T., Song, J., Koga-Ban, Y., Matsui, E., Fang, F., et al.(1994) Plant J. 6, 615-624.

3. Matzke, M. & Matzke, A. J. M. (1993) Annu. Rev. Plant Physiol.Plant Mol. Bio. 44, 53-76.

4. Kooter, J. M. & Mol, J. N. M. (1993) Curr. Opin. Biotechnol. 4,166-171.

5. Flavell, R. B. (1994) Proc. Natl. Acad. Sci. USA 91, 3490-3496.6. Kroon, J., Souer, E., de Graaff, A., Xue, Y., Mol, J. & Koes, R.

(1994) Plant J. 5, 69-80.7. Hamilton, A. J., Lycett, G. W. & Grierson, D. (1990) Nature

(London) 346, 284-287.8. Angenent, G. C., Franken, J., Bussher, M., Colombo, L. & van

Tunen, A. J. (1993) Plant J. 4, 101-112.9. van der Krol, A. R., Brunelle, A., Tsuchimoto, S. & Chua, N.-H.

(1993) Genes Dev. 7, 1214-1228.10. Bird, C. R., Ray, J. A., Fletcher, J. D., Boniwell, J. M., Bird, A. S.,

Teulieres, C., Blain, I., Bramley, P. M. & Schuch, W. (1991)BioTechnology 9, 635-639.

11. Ballinger, D. G. & Benzer, S. (1989) Proc. Natl. Acad. Sci. USA86, 9402-9406.

12. Kaiser, K. & Goodwin, S. F. (1990) Proc. Natl. Acad. Sci. USA 87,1686-1690.

13. Rushforth, A., Saari, B. & Anderson, P. (1993) Mol. Cell. Biol. 13,902-910.

14. Zwaal, R. R., Broeks, A., van Meurs, J., Groenen, J. T. M. &Plasterk, R. H. A. (1993) Proc. Natl. Acad. Sci. USA 90, 7431-7435.

15. Bianchi, F., Cornelissen, P. J. T., Gerats, A. G. M. & Hogervorst,J. M. W. (1978) 53, 157-167.

16. Doodeman, M., Boersma, E. A., Koomen, W. & Bianchi, F.(1984) Theor. Appl. Genet. 67, 345-355.

17. Doodeman, M., Gerats, A. G. M., Schram, A. W., de Vlaming, P.& Bianchi, F. (1984) Theor. Appl. Genet. 67, 357-366.

18. Gerats, A. G. M., Beld, M., Huits, H. & Presscott, A. (1989) Dev.Genet. 10, 561-568.

19. Gerats, A. G. M., Huits, H., Vrijlandt, E., Marafia, C., Souer, E.& Beld, M. (1990) Plant Cell 2, 1121-1128.

20. Dellaporta, S. J., Wood, J. & Hicks, J. B. (1983) Plant Mol. Biol.Rep. 1, 19-21.

21. Britsch, L., Ruhnau-Brich, B. & Forkmann, G. (1991) J. Biol.Chem. 267, 5380-5387.

22. Wijsman, H. J. W. (1986) Theor. Appl. Genet. 71, 291-296.23. Quattrocchio, F., Wing, J. F., Leppen, H. T. C., Mol, J. N. M. &

Koes, R. E. (1993) Plant Cell 5, 1497-1512.24. Schinkel, A. H., Smit, J. J. M., van Tellingen, O., Beijnen, J. H.,

Wagenaar, E., van Deemter, L., Mol, C. A. A. M., van der Valk,M. A., Robanus-Maandag, E. C., te Riele, H. P. J., Berns,A. J. M. & Borst, P. (1994) Cell 77, 491-502.

25. Rudnicki, M. A., Braun, T., Hinuma, S. & Jaenisch, R. (1992)Cell 71, 383-390.

26. Braun, T., Rudnicki, M. A., Arnold, H.-H. & Jaenisch, R. (1992)Cell 71, 369-382.

27. Rudnicki, M. A., Schnegelsberg, P. N. J., Stead, R. H., Braun, T.,Arnold, H.-H. & Jaenisch, R. (1993) Cell 75, 1351-1359.

28. Van der Krol, A. R., Lenting, P. E., Veenstra, J., Van der Meer,I. M., Koes, R. E., Gerats, A. G. M., Mol, J. N. M. & Stuitje,A. R. (1988) Nature (London) 333, 866-869.

29. Van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. M. &Stuitje, A. R. (1990) Plant Cell 2, 291-299.

30. Napoli, C., Lemieux, C. & Jorgensen, R. (1990) Plant Cell 2,279-289.

31. van der Krol, A. R., Mur, L. A., de Lange, P., Gerats, A. G. M.,Mol, J. N. M. & Stuitje, A. R. (1990) Mol. Gen. Genet. 220,204-212.

32. Cannon, M., Platz, J., O'Leary, M., Sookdeo, C. & Cannon, F.(1990) Plant Mol. Biol. 15, 39-47.

33. Gierl, A. & Saedler, H. (1992) Plant Mol. Biol. 19, 39-49.34. Balcells, L., Swinburne, J. & Coupland, G. (1991) Trends Biotech.

9, 31-36.35. Tsay, Y.-I., Frank, M. J., Page, T., Dean, C. & Crawford, N.

(1993) Science 260, 342-344.36. Feldmann, K. A. (1993) Plant J. 1, 71-82.37. Schwarz-Sommer, Zs., Huijser, P., Nacken, W., Saedler, H. &

Sommer, H. (1990) Science 250, 931-936.

Plant Biology: Koes et aL

Dow

nloa

ded

by g

uest

on

Aug

ust 2

6, 2

021