Gene, 119 (1992) 199-205 Q 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

199

0378-l 119/92/$05.00

06607

Expression of a downstream gene from a bicistronic transcription unit in transgenic tobacco plants (Gene expression; hygromycin; kanamycin; resistance-encoding marker; translation)

gene; plant transformation;

Shigeru Iidaa3b, Ortrun and Ingo Potrykusb

W. Saulb, Katja

uDepartment

Scheidb,

Michael

Seipelb*, Chikara

Miyazaki”

of Biological Science and Technology, Science L’pliversity of Tokyo, Nodu-shi, Chiba. Japan: and b Institute for Plant Sciences, Federal Institute

of Tech~oiogy, Ziiri~h, Switzerland. Received

Mittelsten

recombinant DNA; selective

by A. Nakazawa:

Tel. ~41-~~25644~8

18 November

1991; Revised/Accepted:

10 April/13

April 1992; Received

at publishers:

18 May 1992

SUMMARY

We have constructed a set of plasmids carrying an artificial compact stop-start codon sequence, -TGATGTAACATGA, between an upstream open reading frame, terminating at one of the stop codons, and a downstream kanamycin-resistance (KmR)-encoding gene (npt11) initiating at the second ATG. These plasmids were introduced into tobacco protoplasts by direct gene transfer. The efficiency of expression of the downstream nptI1 gene was measured by scoring the number of KmR transformants. With a closer distance between the functional stop and start codons, a tendency to less efficient expression of nptll was found. The integration and expression of both genes as a bicistronic transcription unit were verified by Southernand Northern-blot analyses. A possible application of the compact stop-start codon sequence for insertional mutagenesis is discussed.

INTRODUCTION

Plant nuclear genes so far analyzed are expressed as monocistronic mRNAs. However, genes expressed from plant viral genomes show more variation in the organization of the transcription units (Gronenborn, 1987; Bonneville et al., 1988). In particular, the genes of the double-

Correspondence to: Dr. S. Iida, Department of Biological Science and Technology, Science University of Tokyo, Yamazaki, Noda-shi, Chiba 278, Japan.

Tel. (81-471)

* Present address: Institute Technology,

Hdnggerberg,

Abbreviations:

aa, amino

24-1501;

Fax (81-471)

for Molecular Zurich,

Switzerland.

acid(s);

bp, base pair(s);

mosaic virus; CTAB, cetyl triethylammonium transferase-encoding Km, kanamycin; nt, nucleotide(s); R, resistant;

gene; Hy, hygromycin

25-1841.

Biology,

Federal

Tel. (41-1)

bromide;

CaMV,

Institute

of

377 3401. cauliflower

hph, Hy phospho-

B; kb, kilobase

or 1000 bp;

nptll, neomycin phosphotransferase B-encoding gene; ORF, open reading frame(s); PEG, polyethylene glycol;

‘, sensitive.

stranded DNA virus CaMV and its relatives are tightly organized on the genome in what appear to be polycistronic tr~s~~ption units. Some genes, for example ORF I, II, III, IV and VI, are arranged such that the end of one ORF is directly in front of, or overlapping with, the start of the next ORF. The sequence between such ORFs are of the type UNATG or ATGA (Balazs et al., 1982; Hull et al., 1986). Polar effects observed in mutations in this region suggested that the genes are translated from a single polycistronic mRNA (Gronenborn, 1987; Ftttterer et al., 1990). It was proposed that a ribosome, after termination of synthesis of the upstream ORF, could reinitiate translation on the start codon of the downstream ORF, provided stop and start codons were in close proximity (the ‘relay race’ model). The reinitiation is regarded to proceed according to the scanning model (Kozak, 1989). In order to test its possible use for insertional mutagenesis we have examined the general applicability of this effect. In particular we have examined whether a compact

200

a

V B.H

?

HP

EH

HP

EH

pSHl913 V BH

E

P

pSHl914

nptll

HP

pSHt907 pSHl909 pSHI911

EH

P3SS

psHm8 pSHD10 pSHl912 E

V

ABH

EP

pSHl938

p

hph

HP

EH

npt2

P35S

T3!5S

500 bp

b -

pSHMS, pSHiSl0

P8t.l - soul CTG CAG CCA AGA TCT TOG AGG m

psHl907, pSHlaO3

CTG CAG CT0 AAG ATC TTG GAG GTG ATG -

pSHW1. pSHigl2 pSHl938

Fig. 1. Structure (P35S),

of plasmids.

(a) Physical

the CaMV 35s polyadenylation

derived from pABD1 (Paszkowski truncated BarnHI;

maps.

TC ACA AGC l-i.0

GAG CTG CAA GAT Cl-F GGA GGT GATGTA AC

TC ACA AGC TTG

All plasmids

are derivatives

signal (T35S) and the pUC18

of pDH51

sequence

(Pietrzak

ORF.

Plasmid

P, PstI; V, EcoRV. (b) Nucieotide

pSHI938

stop-start codon sequence 5 ’ -mTGmCA= inserted as the junction between two genes allows the expression of the downstream rzptI1 gene in a bicistronic transcription unit, with termination of the upstream ORF occurring at one of the three stop codons. To measure the efficiency of expression of the nptZZ gene, we scored num-

et al., 1986) consisting fragment

gene (Gritz and Davis,

was described

sequences

taining three different compact stop-start sequences were synthesized. All plasmids at the BglII site. The PstI, BglII and Hind111 sites are indicated by the overlines

m

(thick line). The HindHI-Hind111

et al., 1984). The unique Purl site within the hph structural

H, HindHI;

J&I C AIQ ATC ACA AGC lTG

~ CTG CA0 GAA GAT CTT GGA GGTGAT GTA AC m

5’ part of the hph gene (dhph) for the upstream B, BglII; E, EcoRI;

HIflC4lU TGTAAC BTs ATC ACA AGC -KG

previously

at the intercistronic

of the CaMV 35s promoter containing

the npfll

(Bilang et al., 1991). Restriction

regions.

gene is

1983) was used to construct

The oiig~~xyribonucleotides

the

sites: A, con-

including pSHI913 contain the identical sequence up to the AAGATCT while the stop and start codons are underlined.

bers of KmR transformants after introducing the various constructs into tobacco protoplasts by the method of direct gene transfer. In general, our results confirm those recently reported for the expression of bicistronic transcription units using the Agrobacterium system (Angenon et al., 1989; Koncz et al., 1989) and further extend information con-

201 cerning the influence of the distance start codons. The results suggest an alternative

between

between

the stop and

strategy for the use of

insertional mutagenesis in identifying new plant genes. Previous strategies have relied on introduction of a promoterless nptll gene into a suitable context for expression or a fortuitous fusion in frame with an expressed protein. Pro-

mid pSHI938 carries the complete hph gene terminating at the third stop codon, in addition to the complete nptll gene. Plasmid pSHI913 contains the nptll gene in a monocistronic transcription unit. The plasmids pSHI908, 910, 912 and 914 are identical to pSHI907, 909, 911 and 913, respectively, except that the Hind111 fragment containing the nptII coding region is inserted in the opposite orientation to the promoter.

viding stop codons in all three reading frames in front of the nptll gene would facilitate the expression of KmR after introduction into the plant genome without requirement for a fusion in frame to an expressed gene.

EXPERIMENTAL

the PstI and BgZII sites at the 3’ ends of the up-

stream ORFs (Fig. lb), which therefore terminate at each one of three different stop codons. All of them carry an upstream ORF of approximately 120 aa derived from the 5’ region of the hph gene and the complete nptII gene as the downstream ORF starting at the ATG indicated. Plas-

AND DISCUSSION

(a) Construction of plasmid vectors We have constructed a set of plasmids carrying the sequence 5’-TGATGTAACATGA between two ORFs (Fig. 1). The sequences of the three plasmids pSHI907, pSHI909 and pSHI911 are identical except for three nt

(b) Introduction of plasmids carrying bicistronic transcription units All plasmids were introduced into protoplasts of tobacco (Nicotiana tabacum cv Petit Havana line SRl; Maliga et al.,

lOO-

10 -

l-

50

200

800

3333

Km concentmtion

pSHl913

q

efficiency of the downstream with 40 pg calf-thymus

1987). The number dicated lines.

of KmR colonies

n

pSH1907

sequence)

together

pSHI911

pSHl909

Fig. 2. Expression

nptli gene in bicistronic

pSHl938 pSHI910,908,912,914

transcription

carrier DNA were used to transform

were scored

units. Plasmid

lo6 tobacco

after 5 weeks of culture in liquid medium

by the bars are the mean of at least three independent

Cg/mfj

experiments,

protoplasts containing

DNA (10 pg, cut at the two Bg1I sites in the pUC by direct gene transfer the indicated

and the range of the actual number

with PEG (Negrutiu

Km concentrations.

of KmR colonies

et al.,

The values in-

are shown by the thin

202

a

1

2

3

4

5

8

7

8

910

il

c

Fig. 3. Integration of the nptlf and hph genes into genomic DNA. Total DNA from transgenic plants was isolared with the CTAB method modified after Murray and Thompson (1980). DNA (IO pgjsample) was used for Southern-blot analysis following standard protocols. (a) Filter probed with the n$II gene (HindHI-Hind111 fragment of pSHI913). Lanes: 1 and 2, untransformed control plant; 3 and 4, transformant with pSH1913; 5-7,8-H& 11-13, three transformants with pSHI938; 14 and 15, 1 and IO pg of plasmid pSHI913; 16 and 17, 1 and 10 pg of plasmid pSH1938. 1, 3,5,8 and 11, undigested DNA; 2, 4, 7, 10, 13, 14 and 15, EiindIII digests; 6, 9, 12, 16 and 17, EcoRI digests. (b) Filter probed with the hph gene (BarnHI-BumHI fragment of

203

123456789

nt

Fig. 4. Expression

ofthe npcll gene from a bicistronic

et al., 1991). RNA (10 pg/sample) Hind111 fragment pSHI909;

of pSHI913).

5, transformant

transcript.

was used for Northern-blot Lanes:

with pSHI907;

1, transformant

Total RNA from transgenic analysis

following

with pSHI913;

6-9, four transformants

2, untransformed

pSHI938). Lanes: 1 and 2, untransformed plant; 3 and 4, transformant 11 and 12, 1 and 10 pg of plasmid pSHI938. 1, 3, 5, 7 and 9, undigested 9, transformant

fragment

with pSHI909;

of pSHI913).

plant;

previously

The filter was probed with the

3, transformant

with pSHI911;

(Mittelsten

Scheid

npcll gene (HindHI4, transformant

with

pg Hy/ml. Out of 16 such KmRHyR calli thus obtained, four independent transgenic plants were regenerated. (c) Molecular analysis of regenerated plants Southern-blot analyses revealed that the plants transformed with pSH1938 contained one to three copies of the introduced segment with both the nptll and the hph genes (Fig. 3a and b). Three plants regenerated after transformation with pSHI907, pSHI909 and pSHI911 also carried one to three copies in their genome, whereas one plant with pSHI907 appeared to have more than three copies of the introduced nptZZ gene (Fig. 3~). The transformants were also analyzed for the expression of both resistance genes at the transcription level. All four pSHI938 plants contained an mRNA hybridizing to the nptZZ gene with the size of 1800 nt as expected for a bicistronic mRNA (Fig. 4, lanes 6-9). An nptll transcript of approximately 1300 nt was detected in two plants transformed with pSHI911 and pSH1909 (Fig. 4, lane 3 and 4). A transformant with pSHI907 produced a comparatively high level of a shorter mRNA (Fig. 4, lane 5). Since the size is similar to the monocistronic transcript (Fig. 4, lane l), probably this is either due to a deletion of the dhph region or to fusion of the nptll gene with a strong plant promoter

with pSHI913; 5 and 6, 7 and 8, 9 and 10, three transformants with pSHI938; DNA; 2, 4, 6, 8, 10, 11 and 12: BumHI digests. (c) Filter probed with the nptll

Lanes: 1 and 11, 5 pg of plasmid

5 and 10, transformant

plants was isolated as described protocols.

with pSHI938.

1975) using the method of direct gene transfer (Paszkowski et al., 1984, modified according to Negrutiu et al., 1987). KmR transformants were selected at Km concentrations of 50, 200, 800 and 3200 pg/ml. The plasmids carrying the nptll gene downstream in the bicistronic transcription units gave KmR transformants less efficiently than pSH1913 carrying the monocistronic nptll gene (Fig. 2). The closer the distance between the stop and start codons in the intercistronic region was, the less transformants tended to appear (pSHI938 and pSHI911< pSHI907 < pSHI909), indicating less efficient expression of the nptll gene in close positioning of the two codons. Although such a tendency was previously noted (Angenon et al., 1989; Koncz et al., 1989) it is surprising that a distance effect was observed even in such a compact intercistronic sequence. A total of only three KmR colonies appeared, at the lowest Km concentration, with plasmids pSHI908, pSHI910, pSHI912 and pSHI914 which carry the nptZZ gene in the opposite orientation to the promoter. Single calli from transformation with pSHI909, pSHI907, pSHI911 and pSHI913 selected with 50 pg Km/ml were chosen to regenerate plants. Among the KmR transformants with pSHI938 (containing an active upstream hph gene) 25 relatively large calli were chosen to examine their ability to grow in the presence of 25

gene (HindHI-Hind111

standard

with pSHI911;

pSHI907;

6, untransformed

2 and 3 and 7 and 8, two transformants plant;

1-6, EcoRI digests;

7-11,

BarnHI

with pSH1907; digests.

4 and

204

a

b

Fig. 5. Resistant antibiotics

phenotype

of regenerated

(a), with 25 pg Hy/ml

plants.

Leaf sections

of in vitro-grown

(b) or with 50 ng Km/ml (c). Lanes:

plants were cultured

1, untransformed

4 weeks on A medium

plant; 2, transformant

with pSHI913;

(Caboche,

1980) without

3-5, three transformants

with pSHI938.

to render the expression of the nptll gene stronger (Fig. 3c, lanes 2 and 7). Similar observations were also reported recently for transgenic tobacco plants obtained by Agrobacterium-mediated transformation (Angenon et al., 1989). All transcripts with increased sizes also hybridized to the hph-specific probe (data not shown).

(d) Resistance level of regenerated plants The resistant phenotype of transgenic plants was confirmed by explantation of leaf sections on proliferation medium (A medium; Caboche, 1980) containing 50 pg Km/ml or 25 pg Hy/ml. Plants transformed with pSHI938 produced callus on both selective media, while plants containing pSHI913 were resistant to Km only (Fig. 5). However, the enzyme activity derived from the nptZZ gene on the bicistronic messengers was too weak to be detected in the NPTII gel assay (Reiss et al., 1984), whereas very strong enzyme activity was found in a plant with pSHI913 containing the monocistronic nptIZ gene (data not shown). We thus could not determine the molecular size of the translation products and could not rule out the possibility that some of the downstream nptll genes are expressed as fusion proteins with the upstream ORFs. However, we believe that it is unlikely at least in the case of the Hy-resistant plants with pSHI938, since the 3’-terminal region of the upstream hph gene is known to be important for its activity (Bilang et al., 1991).

(e) Conclusions (I) The results presented here indicate that a downstream nptll gene in bicistronic transcription units can be used for selection on the protoplast level in direct gene transfer experiments, although the marker gene is weakly expressed. (2) Constructs utilizing a compact stop-start codon sequence as described here, therefore, offer a new design for insertional mutagenesis into strongly expressed genes, independent of fusions in the correct reading frame.

ACKNOWLEDGEMENTS

We would like to thank the Friedrich-Miescher-Institute, Basel, where this project was initiated. We also thank Rosemarie Hiestand-Nauer for her help in DNA sequencing, Werner Arber, in whose laboratory some of the plasmids used were constructed, and Jerzy Paszkowski for his helpful discussions. This work was supported in part by a grant from the Japanese Society for the Promotion of Science and by grants from the Ministry of Education, Science and Culture in Japan.

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