Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation

153

Gene, 53 (1987) 153-161 Elsevier GEN 01963

Promoter cassettes,

antibiotic-resistance

genes, and vectors for plant transformation

(Recombinant DNA; kanamycin; hygromycin; multiple restriction sites; tobacco; Agrobacterium; plasmids)

Steven J. Rothstein, Kristine N. Lahners, Richard J. Lotstein, Nadine B. Carozzi, Susan M. Jayne and Douglas A. Rice CIBA-GEIGY

Corporation, Research Triangle Park, NC 27709 (U.S.A.)

Received

20 August

Revised

26 November

Accepted

1986 1986

24 December

1986

SUMMARY

We have constructed a set of plant transformation vectors, promoter cassettes, and chimeric antibioticresistance genes for the transformation and expression of foreign genes in plants sensitive to Agrobacterium infection. The different vectors allow for either concurrent or consecutive selection for kanamycin and hygromycin resistance and have a number of unique restriction sites for the insertion of additional DNA. The promoter cassettes utilize the CaMV 19s and CaMV 35s promoters and are constructed to allow for the easy insertion of foreign genes. The cloned gene can then easily be inserted into the transformation vectors. We have utilized the promoter cassettes to express the hygromycin-resistance gene either from the CaMV 35s or the CaMV 19s promoters, with both chimeric resistance genes allowing for the selection of hygromycin-resistant tobacco plants.

INTRODUCTION

The transformation of defined sequences of DNA into plant cells and the subsequent expression of the Correspondenceto: Dr. S.J. Rothstein, Box

12257,

Research

Triangle

CIBA-GEIGY Park,

NC

Corp., P.O.

27709

(U.S.A.)

Tel. (919)541-8500. Abbreviations: transferase

aa, amino

protein;

CaMV, cauliflower tin;

salts;

naphthalene II protein;

or 1000 bp; MS,

and

Skoog

medium;

NAA,

acid; NPTII,

neomycin

phosphotransferase

nt, nucleotide(s);

transferred

Km, kanamy-

shoot-inducing

E. coli DNA polymerase DNA,

phospho-

bp, base pair(s);

tobacco

MSBN, acetic

hygromycin

mosaic virus; Hy, hygromycin;

kb, kilobase

(1962)

acid; AphIV,

BAP, benzylaminopurine;

PolIk,

Murashige

Klenow

I; R, resistance;

DNA; Ti plasmid,

(large) fragment Tc, tetracycline;

tumor-inducing

plasmid.

of T-

transferred DNA is a crucial tool in plant molecular genetics. The first system developed for plant transformation utilized the naturally occurring ability of A. tumefaciens to infect many dicotyledonous plants and to insert a portion of its resident Ti plasmid into the plant’s genome. While direct DNA transformation has been demonstrated for a number of plant species (Krens et al., 1982; Paszkowski et al., 1984), the Agrobacterium system is still extremely useful as it does not require specialized tissue-culture techniques . Transformation of plants using Agrobacterium requires two regions on the Ti plasmid: the T-DNA region which is actually transferred into the plant genome (Chilton et al., 1977), and the pTi virulence genes required, along with Agrobacterium chromoso-

0378-l 119/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

154

mal genes, for the transfer of the T-DNA (Klee et al., 1982). Some of the T-DNA genes code for the synthesis of opines unique to Agrobacterium-induced plant tumors, and for the production of the phytohormones auxin and cytokinin whose synthesis causes the tumorous phenotype typical of an Agrobacterium infection (Gartinkel et al., 198 1; Klee et al., 1982). Manipulation of the wild-type Ti plasmid system for genetic experiments is difticult due to the large size of the Ti plasmid and the abnormal phenotype of the transformed plants. Two developments have allowed the construction of Ti plasmid-derived vectors into which it is possible to easily insert foreign genetic material. First, the virulence functions were found to operate in tram on a T-DNA region inserted into other plasmids or even on the Agrobacterium chromosome (Hoekema et al., 1983; de Framond et al., 1983). Second, the only essential regions on the T-DNA for its transfer and insertion into the plant’s genome are fairly small regions of DNA called the T-DNA border sequences (Yadav et al., 1982; Zambryski et al., 1982). Therefore, it has been possible to develop binary vectors in which a resident Ti-plasmid derivative supplies the necessary virulence function, but lacks its own indigenous T-DNA region, making a disarmed helper A. tumefaciens (Bevan, 1984; An et al., 1985). A second plasmid, which contains the foreign DNA to be transferred into the plant inserted between the two T-DNA borders, can be transferred into the disarmed A. tumefaciens helper strain. Upon infection of plant tissues, the chimeric T-DNA region in the second plasmid will be excised, transferred and integrated into the plant host genome. This second plasmid can be relatively small and contain convenient restriction endonuclease sites for the easy insertion of the DNA to be transferred. The transformation and expression of foreign genes also requires the availability of positive selectable markers and plant-regulatory elements. While the wild-type T-DNA transforms plants to hormone independence, this is not a suitable selectable marker for the regeneration of normal plants. Resistance to kanamycin by expression of the bacterial NPTIIcoding gene in plant tissues has been shown to be a reliable screen for the selection of Km-resistant plant tissue (Herrera-Estrella et al., 1983; Fraley et al., 1983; Bevan et al., 1983). More recently, resistance to the antibiotic Hy by the expression of bacterial

AphVI-coding gene has also been used to select transformed plant tissues (Van der Elzen et al., 1985; Waldron et al., 1985). A number of different plant regulatory elements can be used to express foreign genes. These can be isolated from the plant genome itself, from genes present on T-DNA, or from plant viruses. In this communication, we present the construction of a set of binary Ti plasmid-derived vectors which contain chimeric genes which, when expressing NPTII or AphIV in plant tissue, confer resistance to either Km or Hy, or to both. We have also constructed promoter cassettes containing either the CaMV 19s or CaMV 35s promoter which have been designed so that DNA fragments can be easily inserted into our binary vector and their expression controlled by the CaMV promoters. A HyR gene was cloned into these promoter cassettes and shown to function in tobacco plants.

MATERIALS

AND METHODS

(a) In vitro mutagenesis

and nucleotide sequencing

The DNA to be mutated was first cloned into M13mp18 (Norrander et al., 1983). Single-stranded phage DNA was isolated and oligodeoxynucleotide mutagenesis was carried out following the protocol of Zoller and Smith (1984). Putative mutations were analyzed by nucleotide sequencing (Sanger et al., 1977), with the correct clone being inserted into pUC19 (Norrander et al., 1983). (b) Plant transformation

Four-week-old in vitro shoot tip cultures of Nicotiana tabacum cv. Coker 176 grown in MS medium (Murashige and Skoog, 1962) without hormones (26” C, 16/8 photoperiod, 4000 lux) were used as source material for leaf disc transformation. Experiments were performed essentially as described by Horsch et al. (1985) with the following modiflcations: nurse culture plates were omitted and the leaf discs were plated directly on non-selective MSBN medium (MS salts, B, vitamins, 3 y0 sucrose, 1 pg BAP/ml, 0.1 pg NAA/ml, pH 5.8, 0.8 % phytagar) after a lo-min incubation with overnight cultures

155

CHIMERIC T-DNA LEFT BORDER

TnJ KmR GENE T-DNA

RIGHT BORDER

Bal

UN/WE

RESTRICTION SUE:

Hindc Sal I, Xba I, BarnHI, kpn1, WI, EcoRI

Tn903 KmR GENE

Fig. 1. The structure of pCIB10. The plasmid vector pCIBl0 was constructed starting with the wide-host-range plasmid pRK252 obtained from Dr. D.R. Helinski (University of California, San Diego, CA) in the following fashion. pRK252 codes for a TcR gene that is located on a SmaI-Sal1 restriction fragment. This gene was replaced with a Kmu gene from Tn903 (Oka et al., 1981) by first digesting pRK252 DNA with SmaI + Sal1 and making the Sal1 end flush with PolIk (Boehringer Mannheim). A 1050-bp BarnHI fragment from plasmid ~368 (S.J.R.; unpublished) carrying the TrQ03 KmR gene was also blunted and inserted into the SmaI + SalI-cleaved pRK252. The unique EcoRI restriction site in this plasmid was then changed to a Bg/II site using a synthetic oligodeoxynucleotide adapter. The remaining components of pCIB 10 were first assembled in pBR322 and then all inserted into this BglII site. These other components consist ofthe following: a 400-bp EcoRI-EcoRV T-DNA left border fragment isolated from the nopaline Ti plasmid pTiT37 (Yadav et al., 1982), a right T-DNA border fragment isolated as a EcoRV-SstII fragment from pTiT37 (Bevan et al., 1983), and a plant-expressible chimeric KmR gene that utilizes the nopaline synthase regulatory elements to control transcription of the KmR gene were isolated from the plasmid Bin6 (Bevan, 1984) as a 1.3-kb SstII-EcoRI fragment. The polylinker region used was from the plasmid pUCl8 (Norrander et al., 1983). The DNA between the T-DNA left and right borders is normally the part of the plasmid transferred to the plant genome. In this region are the chimeric KmR gene for selection of transformation in plants and a polylinker region for insertion of other genetic material. The Tn903 KmR gene is a selectable marker in bacteria. The unique restriction sites present in the polylinker useful for the insertion of foreign genes are listed. (*Even though there are two Hind111 sites, one is in the Tn903 KmR gene which must be regenerated to make a functional gene.) Dark box, T-DNA borders; hatched box, chimeric Tn5 KmR gene; stippled box, Tn903 KmR gene.

of Agrobacterium. Dishes were placed at 26°C and kept in the dark for three days. Discs were then transferred to MSBN medium containing 500 pg cefotaxime/ml and either 75 pg Km/ml or 20 vg Hy/ml and incubated at 26°C in the light. Discs were subcultured weekly onto fresh antibiotic-containing media. After four to tive weeks, shoots were observed on the edges of the discs in those cases where the appropriate resistance gene was present. No shoots proliferated from control

discs plated on media containing either Hy or Km. When these shoots were approx. 8 mm tall they were removed from the leaf discs and transferred to rooting medium (MS medium without hormones, containing the appropriate selective antibiotic) in GA-7 containers (Magenta Corp., Chicago, IL). After approx. four weeks, regenerated plantlets were transferred to sterilized potting soil and into the greenhouse.

156

RESULTS

AND

after infection by A. tumefaciens. The following sites in the polylinker region of pCIBl0 are unique: EcoRI, SstI, KpnI, BamHI, XbaI and SalI. (The Hind111 site can also be used since the extra Hind111 site in the Tn903 KmR gene has to be reconstructed correctly to get Km-resistant bacteria.) Transfer of pCIB 10 and its derivatives into A. tumefaciens strain LBA4404, is facilitated by using the E. coli-mobilizing strain S17-1, which has the mobilization functions of plasmid RP4 integrated into the chromosome (Simon et al., 1983). This procedure involves introducing plasmid pCIB 10 into strain S 17-1 by DNA transformation and then its conjugative transfer into A. tumefaciens, without the complications of transfer of the RP4 plasmid. When the plasmid vector DNA is analyzed after growth in the Agrobacterium strain LBA4404, we do occasionally see the insertion of extra DNA into the plasmid sequences. It is, therefore, necessary to confirm the

DISCUSSION

(a) Construction

of the Ti cloning vector pCIBl0

A broad-host-range Ti plasmid-derived cloning vector was constructed for use in the transformation of dicotyledonous plants. The vector uses the binary system approach, in which virulence functions necessary for transfer of T-DNA are supplied in trans by the helper plasmid pLA4404 (Ooms et al., 1981; Hoekema et al., 1983). The vector pCIBl0 (Fig. 1) contains the RK2 replicon from plasmid pRK252 (which is a derivative of plasmid pRK290). It is capable of replication in a variety of Gram-negative bacteria, including Escherichia coli and A. tumefaciens. The details of the construction of pCIBl0 are given in Fig. 1. DNA fragments inserted into the polylinker region are transferred to the plant genome PROMOTER

CASSETTES

Plosmtd

Tranrlo,,onal

pCIB706

A AGC

,/I\

H,ndm

EcDR’ /4 _Srrl

&I

Sal1

XbbI

SolI Eo\mHl

Smo’l

Kpk 1

Ssil

Frame

TGG TGG ACT CTA GAG

I\

I

S;hI

Readtng

GAT CC BomH I

XboI

E;oRI

~01~A odd,l,on s,te

355 promoter pCIB710

H\nd,I

SphI

Prt I

Sal I

-

Xba I

Barn HI

19s promoter-

SmaI

Km1

sst I

EcoRl

POIY A add,t,on site

ATG

CC ATG GGT CGA

pCIB771

Sol I

NCOI

Fig.

2.

Promoter

from plasmid

cassettes

for expression

pABD1 (Paszkowski

1983) to make pCIB701.

(see Paszkowski

University

of California,

et al., 1984). The CaMV

into PstI + S&-cleaved

Vi. pCIB710

was constructed

fragment

by isolating

contains

fragment

fragment

corresponding

to a Sal1 site by using a synthetic

pCIB701

to make pCIB706.

and the CaMV poly(A)-addition

it into BarnHI-cleaved

the BarnHI

translation

pUCl9

start codons

between

create an NcoI site (CCATGG) and a NcoI-XbaI cassettes

synthetic

the start of transcription

sites in pCIB706

adapter

site.

It is possible

were inserted

site although

to make translation

frames of the useful restriction

site sequences

(Norrander

site et al.,

as well as the CaMV

pLVll1

(from

in the CaMV sequence

and the resulting

(bp 6485-7643

PstI-Sal1

S. Howell, (Hohn et al.,

fragment

in the CaMV sequence;

was

Hohn et al.,

site for insertion

of foreign genes was inserted pCIB710,

between

The resulting

plasmid,

site. pCIB771

was made by using in vitro mutagenesis fragment

into PstI + XbaI-cleaved

there is approx.

from pBR327

C

also codes for the first 26 aa of the CaMV gene

at the ATG start codon in the CaMV gene VI. The PstI-NcoI

oligodeoxynucleotide

and pCIB771.

and the BumHI

AlC

0amH I

site (Covey et al., 1981: Ode11 et al., 1985) and inserting

site). A new BarnHI

site at bp 7483 via in vitro mutagenesis.

have the same CaMV poly(A)-addition

codon and reading restriction

(thus destroying

and poly(A)-addition

fragment

pUC19

from plasmid

to bp 5306-5851

from pLVll1

1982), which codes for the CaMV 35s promoter the promoter

some sequences was isolated

Xbo I

the CaMV poly(A)-addition

into BarnHI-cleaved

oligodeoxynucleotide

This promoter

an 1150-bp BglII fragment

by first inserting

fragment,

19s promoter

San Diego, CA) as aPstI-Hind111

1982). The Hind111 site was changed

was constructed

on a BumHI-BglII

It should be noted that this inserted

sequences

inserted

of foreign genes. pCIB706

et al., 1984), located

CTC TAG AGG

containing

pCIB706

fusions using pCIB706

and pCIB771;

to

the gene VI promoter

to obtain pCIB771.

200 bp of extra DNA upstream

are shown. The ATG initiation

does not have any ATG

All these

from the poly(A)-addition

the ATG translation

codon in pCIB771

initiation

is part of the NcoI

157

plasmid structure prior to using the bacterial strains for plant transformation. (b) Construction of CaMV promoter cassettes and cbimeric HyR genes A promoter cassette pCIB706, containing the CaMV 19s (gene I/I) promoter and CaMV polyadenylation signals was constructed (Fig. 2). The restriction sites SalI, XbaI and BumHI can be used to insert genetic material between the promoter and poly(A)-addition site. The coding sequence for the first 26 aa of the CaMV gene VI-coded protein is still present, so that this construct can be used to make translational fusions to the beginning of the gene VI-coded protein. Alternatively, as described below for the chimeric HyR gene, foreign genes can be inserted into one of the available restriction sites and the excess genetic material deleted in vitro so that protein translation of the foreign protein can be correctly initiated. We have also constructed a deletion derivative, pCIB771, which has an NcoI restriction site located at the ATG start codon of gene VZ,as shown in Fig. 2. After insertion of DNA fragments into any of these promoter cassettes, the resulting chimeric gene can then be inserted into pCIB 10 by cleaving with either ZZindIII, SphI or PstI at one end and either SmaI, KpnI or SstI at the other. (SstI can be used for this purpose only when the SstI site present in the gene VI protein-coding region is deleted.) Another promoter cassette was constructed containing the CaMV 35s promoter and poly(A)addition site. The CaMV 35s promoter initiates at bp 7435 (Odell et al., 1985), transcribes the entire genome and terminates at approx. bp 7620 the second time around to create an RNA slightly larger than genome length (Covey et al., 198 1). This RNA is normally involved in the replication of the virus, although there is evidence that it might serve as a messager (Dixon and Hohn, 1984). The 35s promoter and poly(A)-addition site are present on an 1150-bp BgZII fragment (bp 6485 to bp 7643 ; see Fig. 2), and this fragment was inserted into the BamHI site of pUC19. A BumHI site (GGATCC) was then created following bp 7483 via in vitro mutagenesis to make pCIB7 10 (Fig. 2). There are no ATG start codons between this BumHI site and the start point of transcription.

This 35s promoter cassette was also inserted directly into Ti-vector pCIBl0 by cleaving both plasmids with Sal1 + EcoRI and ligating the DNA together to make pCIB770 (Fig. 3). DNA can then be inserted into the BumHI site and directly transferred into plant cells without further manipulation. These promoter constructs were used to make chimeric HyR genes. The HyR gene present on a BumHI fragment from plasmid pLG90 (a gift from L. Gritz) was inserted into the BumHI site of plasmid pCIB706, containing the CaMV 19s promoter cassette. In this BumHI fragment, an extra ATG start codon lies just upstream from the correct start site for the HyR gene. In vitro mutagenesis was used to delete the extra genetic material (from the gene VI-coding sequence as well as from the HyR gene). The resulting plasmid, pCIB7 13 (not shown) has the start codon for the HyR gene positioned at exactly the same site as that for the gene I’Z polypeptide. To make a chimeric HyR gene using the CaMV 35s promoter, the same BumHI fragment from pLG90 was inserted into the BumHI site of pCIB7 10 to make pCIB709 (not shown). The resulting chimeric gene retains the extra upstream ATG start codon, which has been shown to have an inhibitory effect on translation in other systems (Fraley et al., 1983; Bevan, 1984). The chimeric HyR genes from pCIB709 and from pCIB713 were inserted into the plasmid vector pCIBl0 as described in Fig. 3 to make pCIB715 and pCIB717, respectively. These vectors therefore code for resistance both to Hy and Km. Having two positive selectable markers can be useful to ensure the efficacy of a plant-transformation procedure. To construct a vector that codes only for Hy resistance, the chimeric KmR gene can be deleted from pCIB10. The chimeric HyR gene with the 19s promoter was then inserted into this deletion derivative to make pCIB743 (Fig. 3) which has unique restriction sites between the T-DNA borders for SalI, XbuI, SstI and KpnI. (c) Expression of hygromycin resistance in tobacco Plasmids pCIB 10, pCIB715 and pCIB717 were transformed into the E. coli strain S 17-1 and then mated to the A. tumefuciens strain LBA4404 as described in MATERIALS AND METHODS, section b. Tobacco leaf disc transformations were done (see MATERIALS ANDMETHODS, section b) andover 100

158

Plasmad

pCIB715 BornHI H\ndlU SphI

PrtI

Sol1

pClB717

I EroRI

Promoter -

pCIEi743

I

HtndllY. KpnI, 5511

Kpnl

Sri I

EcoRI

PO&A oddnhons,le

H”@

I EcoRI

EcoRI

BanHI SmoI

-I95

SrrI

I PII1

PrlI

HsndIQ Sphl

KpnI

wb A addwn ale

H”R

I EcoRl Sphl

PSI1 BomHI SmaI

--19SPromol.?r-

Hsnd,il

EcoRl

XboI

,,,,a PstI

Eco RI

Hnndlll*. SolI. XboI, KpnI. SrtI

Born HI

Ps11

Sal1 Xbd E.&i1 SmI

pCIE770

K+d Srtl EroRl

BarnHI lor exprarrmn, MmdlU’,S.lI. XbaI. Kpn I. Srt I. EcoRI

1 Barn HI H,ndm

SphI

PstI

Sol1

XboI

Fig. 3. Plant transformation constructed

by inserting

gene sequence],

coding

a BumHI

translation HindHI-St1 chimeric

between

of the T-DNA

digesting

pCIB713

and pCIB710

The translation

as the wild-type

HyR genes from pCIB709

the chimeric

was then cloned

into the deleted cassette

resistance

gene present

on each vector

the chimeric

pCIB717

inserted

and pCIB770 pCIB770

pCIBl0

from pCIB710

for expression

are each identical

has a unique BumHI

from the CaMV

should therefore

pCIB743

respectively.

was constructed The DNA between

was replaced

pCIB715

plasmid isolated

digested

and the unique restriction to pCIBl0

outside

and pCIB717

into pCIBl0

can be used for insertion region. However,

the

the BclI site near the ‘stuffer’

HyR gene was isolated Biolabs,

digested

by

Beverly,

pCIB770

was

with St1 + XbaI.

sites are listed. (*As in the case of pCIBl0

the polylinker

cloning

by first removing

with Hind111 + Sal1 to give pCIB743. fragment

was

have the same

with a 300-bp BarnHI-EcoRI

KmR gene. The chimeric

as a XbaI-SstI

respectively.

This involved

the SmaI site to a Sal1 site using a Sal1 linker (New England

second Hind111 site in the Tn!W3 KmR gene, but the Hind111 site in the polylinker pCIB715,

and pCIB709,

and that for the AphIV protein

CaMV gene VI protein. and pCIB713,

pCIB10.

HyR genes were

see Gritz and Davies (1983), for the HyR

to make pCIB707

HyR gene from pCIB713.

1986), thus deleting

the promoter

EroRI

from pCIB 10. The chimeric

of the AphIV polypeptide

in pCIB 10 and the EcoRI site in the polylinker

and Rothstein,

by inserting

gene was deleted.

511

codon for the gene VI protein

into Hind111 + &?I-cleaved

with Hind111 + SmaI and changing

MA). This fragment

pCIB706

initiation

t&I

were derived

[a gift from Dr. L. Gritz;

leader in this plasmid the chimeric

and then inserting

present

from ~443 (Gatenby

vectors

pLG90

to give pCIB713.

from these plasmids

fragment

The chimeric

the ATG translation

into pCIBl0

KmR gene from pCIBl0

constructed

from plasmid

site and 5’-nontranslated

by inserting

fragments

right border

plant transformation

fragment

by in vitro mutagenesis

initiation

were constructed

Several

for the HyR gene, into BumHI-cleaved

The excess DNA material deleted in pCIB707

Smal

vectors.

there is a

of foreign genetic material.)

in pCIB743

site in which a foreign gene having its own ATG translation

the chimeric

initiation

KmR

codon can be

35s promoter.

Hy-resistant plants were regenerated. No Hyresistant plants were found in control experiments using Agrobacterium strains which lacked the chimeric HyR gene. To verify the presence and expression of the chimeric HyR in the transformed plants, RNA and DNA were isolated from five resistant plants transformed with pCIB715 (containing the 35s promoter construct) and two resistant plants transformed with pCIB717 (containing the 19s promoter construct). DNA blot hybridization analyses of the inserted DNA using the isolated HyR gene as a probe showed that the DNA was inserted as expected and was present at one to five copies per genome (not shown).

Total RNA was probed with either the HyR gene or with a pea chlorophyll a/b-binding protein gene. As can be seen in Fig. 4a, plants transformed with the CaMV 35s promoter construct contain considerably higher levels (at least ten-fold) of HyR gene transcripts than plants transformed with the CaMV 19s promoter construct. However, there is far less HyR gene RNA present when the 35s promoter is used to initiate transcription when compared to the amount of chlorophyll u/b-binding protein RNA present in the same leaf material (Fig. 4b). This latter gene codes for one of the most abundant transcripts found in leaftissue. (Note that the transcript made from the 19s promoter is approx. 200 nt longer, due to the

b

J 717

715 hh 12345121 -r_1c

@P

+

OL

_I,.,-_-

it-1.5kb e-1.3 kb

1

c t

P

&

a

Fig. 4. Northern analysis of the HyR gene transcripts. Total cellular RNA was isolated by homogenizing leaf tissue (1 g) in a Brinkman polytron and pelleting the RNA twice through a CsCl cushion (Glisin et al., 1974). This material was electrophoresed through a 1.8% agarose, 2.2 M formaldehyde gel and the RNA blotted onto nitrocellulose and analyzed by hybridization to the DNA probe of interest (Maniatis et al., 1982). The heterologous gene for the pea chlorophyll u/b-binding protein (obtained from Dr. Sean Hemmingsen, Kingston, Ont.) was hybridized and the filter washed at 60°C rather than 68°C for the homologous HyR gene. (Panel a) 20 pg of total RNA were loaded in each lane. Five different plants transformed with pCIB715, two plants transformed with pCIB717 and a nontransformed plant were analyzed. Note, as expected, the HyR gene transcript coded for by pCIB717 is approx. 200 bp longer than that coded for by pCIB715. The film was exposed for 20 h prior to developing. (Panel b) 20 pg of the same RNA sample as in (panel a) were loaded in each lane and the filter was probed using the pea chlorophyll u/b-binding protein gene. The film was exposed for 3 h prior to developing.

presence of the extra DNA in the terminator fragment used in this construct.) We we did not analyze enough plants to ensure that position effects did not cause this disparity, it does appear that the 35s promoter is considerably more eficient than the 19s promoter in tobacco. Alternatively, the differ-

ences in the 5’ and 3’ ends of the transcripts could alter RNA stability. To determine the levels of Hy resistance of the transformed plants, leaf discs from resistant plants were plated on MSBN medium containing various concentrations of Hy. All the plants containing

160

either the 19s or the 35s promoter chimeric HyR gene grew on 100 pg hygromycin/ml and one of the plants, containing the 3% promoter HyR chimeric gene, would grow on as much as 500 pg Hy/ml. Plants containing the HyR gene using the 35s promoter grew marginally better than plants containing the 19s promoter; however, this growth advantage was certainly not as great a difference as that found in the steady-state mRNA levels. This may be due to lower translational efficiency because of the extra ATG start codon in the 35s construct or some other structural feature of the RNA coded for by this construct. Alternatively, a plateau in the level of resistance might exist, such that increased levels of enzyme expression do not result in increased levels of Hy resistance.

and Steve Howell for the gifts of plasmids used in the various constructions. We would also like to thank Georgia Helmer and Mary-Dell Chilton for advice and reading the manuscript and Jane Latta and Cindy Harden for the typing of the manuscript.

REFERENCES An, G., Watson, plants. Bevan,

M.W.,

antibiotic

Flavell,

R.B. and

resistant

transformation.

M.P. and Nester,

Chilton,

Chilton,

of higher

M.-D.:

gene as a selectable

Nature

A chimeric

marker

for plant cell

304 (1983) 184-187.

M.W.: Binary Agrobacterium vectors

Bevan,

for plant transfor-

Nucl. Acids Res. 12 (1984) 8711-8721.

M.-D.,

Montoya,

Drummond,

A.L.,

incorporation

(1) We have constructed vectors which can be used to transform any plant that can be infected by Agrobacterium. These vectors contain genes that can be used to select for plants resistant to Km or Hy, or both. Each vector has a number of unique restriction sites for the insertion of foreign DNA that will subsequently be transformed into the plant. (2) Promoter cassettes derived from the CaMV 19s and 35s promoters have been constructed. The DNA to be expressed can be inserted into unique restriction sites located between the promoter and poly(A)-addition site. The resulting chimeric gene can be excised easily and inserted into one of the plant-transformation vectors. These promoter cassettes can be used to construct either transcription or translation fusions with the inserted gene. (3) The plants transformed to Hy resistance had more HyR gene RNA when that gene was expressed from the CaMV 35s promoter than when the CaMV 19s promoter was used. While this is presumably due to the greater transcriptional efficiency of the 35s promoter, other explanations are possible.

S., Gordon,

EMBO J. 4 (1985) 277-284.

mation.

(d) Conclusion

B.D., Stachel,

E.W.: New cloning vehicles for the transformation

molecular

M.H.,

Gordon,

Merlo,

M.P.

of plasmid

and

D.J.,

Sciaky,

Nester,

DNA into higher

E.W.: plant

basis of crown gall tumorigenesis.

D.,

Stable

cells: the

Cell 11 (1977)

263-271. Covey, S.N., Lomonossoff, of cauliflower transcripts.

mosaic

G.P. and Hull, R.: Characterization DNA sequences

which encode

major

Nucl. Acids Res. 9 (1981) 6735-6747.

de Framond,

A., Barton,

new vector strategy

K.A. and Chilton,

M.-D.: Mini-Ti:

for plant genetic engineering.

a

Biotechno-

logy 1 (1983) 262-269. Dixon, L.K. and Hohn, T.: Initiation flower

mosaic

evidence

virus genome

from

deletion

of translation

of the cauli-

from a polycistronic

mutagenesis.

mRNA:

EMBO

J. 3 (1984)

R.B., Sanders,

P.R., Flick,

2731-2736. Fraley,

R.T., Rogers,

J.S., Adams,

S.G., Horsch,

S.P., Bittner, M.L., Brand, L.A., Fink, C.L., Fry,

J.S., Galluppi,

G.R., Goldberg,

Woo, S.C.: Expression Natl. Acad. Gartinkel,

N.L. and

genes in plant cells. Proc.

Sci. USA 80 (1983) 4803-4807.

D.J., Simpson,

don, M.P. and Nester, tine structure

S.B., Hoffmann,

of bacterial R.B., Dean,

L.W., White, F.F., Gor-

E.W.: Genetic

map of the T-DNA

analysis

of crown gall:

by site directed

mutage-

nesis. Cell 27 (1981) 143-153. Gatenby,

A.A. and Rothstein,

ATP-synthase

/?-subunit

S.J. Synthesis fusion

and binding to the inner membrane. Glisin,

V., Crkvensakov,

isolated

by cesium

R. and chloride

ofmaize

chloroplast

in Eschenkhia coli

proteins

Gene 41(1986)

Byus,

241-247.

C.: Ribonucleic

centrifugation.

acid

Biochemistry

13

(1974) 2633-2637. Gritz,

L. and Davies,

tance: ACKNOWLEDGEMENTS

We would like to thank Steve Lombardi for synthesis of oligodeoxynucleotides and Mary-Dell Chilton, Mike Bevan, Linda Gritz, Jurek Paszkowski,

J.: Plasmid-encoded

the sequence

of hygromycin

gene and its expression

hygromycin

B resis-

B phosphotransferase

in Escherichia coli and Saccharomyces

cerevisiae. Gene 25 (1983) 179-188. Herrera-Estrella,

L., DeBlock,

J.P., Van Montagu, dominant 987-995.

selectable

M., Messens,

M. and Schell, markers

E., Hernalsteens,

J.: Chimeric

genes

as

in plant cells. EMBO J. 2 (1983)

161 Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A.: A binary plant vector strategy based on separation of the vir and T region of the Agrobacferium tumefaciens Ti plasmid. Nature 303 (1983) 179-180. Hohn, T., Richards, K. and Lebeurier, G.: Cauliflower mosaic virus on its way to becoming a useful plant vector. Curr. Topics Microbial. Immunol. 96 (1982) 193-236. Horsch, R.B., Fry, J.E., Hotfmann, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T.: A simple and general method for transferring genes into plants. Science 227 (1985) 1229-1231. Klee, H.J., Gordon M.P. and Nester, E.W.: Complementation analysis of Agrobacterium tirmefaciens Ti plasmid mutants affecting oncogenicity. J. Bacterial. 150 (1982) 327-331. Krens, F.A., Molendijk, L., Wullems, G.J. and Schilperoort, R.A.: In vitro transformation of plant protoplasts with Ti plasmid DNA. Nature 296 (1982) 72-74. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Murashige, T. and Skoog, S.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15 (1962) 473-497. Norrander, J., Kempe, T. and Messing, J.: Construction of improved M 13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26 (1983) 101-106. Odell, J.T., Nagy, F. and Chua, N.-H.: Identification of DNA sequences required for activity of the cauliflower mosaic virus promoter. Nature 313 (1985) 810-812. Oka, A., Sugisaki, H. and Takanami, M.: Nucleotide sequence of the kanamycin resistance transposon Tn903. J. Mol. Biol. 147 (1981) 217-226.

Ooms, G., Hooykaas, P.J.J., Moolenaar, G. and Schilperoort, R.A.: Crown-gall plant tumors of abnormal morphology induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA functions. Gene 14 (1981) 33-50. Paszkowski, J., Shillito, R.D., Saul, M., Mandak, V., Hohn, T., Hohn, B. and Potrykus, I.: Direct gene transfer to plants. EMBO J. 3 (1984) 2717-2722. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5468. Simon, R., Priefer, U. and Ptihler, A.: Vector plasmids for in vivo and in vitro manipulation of Gram-negative bacteria. In Piihler, A. (Ed.), Molecular Genetics of the Bacteria-Plant Interaction. Springer-Verlag, Berlin, 1983, pp. 98-106. Van den Elzen, P., Townsend, J., Lee, K.Y. and Bedbrook, J.: A chimeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol. Biol. 5 (1985) 299-302. Waldron, C., Murphy, E.B., Roberts, J.L., Gustafson, G.D., Armour, S.L. and Malcolm, S.K.: Resistance to hygromycin B. Plant Mol. Biol. 5 (1985) 103-108. Yadav, N.S., Vanderleyden, J., Bennet, D.R., Barnes, W.N. and Chilton, M-D.: Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc. Natl. Acad. Sci. USA 79 (1982) 6322-6326. Zambryski, P., Depicker, A., Kruger, K. and Goodman, H.: Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J. Mol. Appl. Genet. 1 (1982) 361-370. Zoller, M.J. and Smith, M.: Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA 3 (1984) 479-488. Communicated by J.L. Slightom.