Expression of a mouse metallothionein gene in transgenic plant tissues

133

Gene, 77 (1989) 133-140 Elsevier GEN 02944

Expression of a mouse metallothionein

gene in transgenic plant tissues

(Recombinant DNA; pre-mRNA splicing; plasmid; regulatory signals; transcription)

polyadenylation;

RNA

maturation;

vectors;

intron;

exon;

Ri

VCronique Pautot”*, Ryszard Brzezinski b**and Mark Tepfer a a Laboratoire de Biologic Cellulaire, INRA - Centre de Versailles, 78026 Versailles Cedex, (France) and b Dkpartement de Microbiologic, Facult&de Mt?decine, Universitt de Sherbrooke, Sherbrooke, Qutbec (JlH 5N4 Canada) Received

by J.L. Slightom:

Revised:

12 December

Accepted:

2 August

1988

1988

15 December

1988

SUMMARY

Three gene constructions based on a mouse metallothionein I gene (mMT-I) were introduced into tobacco using an Ri plasmid vector system to test the effectiveness of animal gene regulatory signals in plant cells. No transcription from the native mouse gene was observed. In plant cells bearing chimeric mMT-I genes in which transcription was driven by the nopaline synthase promoter, neither polyadenylation nor splicing of mMT-I pre-mRNA was observed. Detailed comparisons of mMT-I sequences with those of known plant genes were carried out; slight differences in regions of known consensus sequences may be at least partly responsible for the non-recognition of mMT-I gene regulatory signals in plant cells, though other as yet unidentified, potentially necessary

sequences

may also be involved.

INTRODUCTION

Since techniques for introducing foreign genes into plants have been developed, genes from a wide range

to: Dr. M. Tepfer, Laboratoire

Correspondence lulaire,

INRA

(France)

- Centre

de Versailles;

Tel. (I)308330293 addresses:

(V.P.) Department

Pathology,

University

of California

(USA)

Biologie, Sherbrooke,

Tel. (714)787-3716;

Faculte

des

Quebec

037%1119/89/$03.50

Cedex,

Fax. (1)30833099.

* Current 92521

de Biologie Cel-

78026 Versailles

Sciences,

-

Riverside,

Universite

(JlK 2Rl Canada)

0 1989 Elsevier

Abbreviations:

and Plant

Riverside

CA

Dtpartement de

de

Sherbrooke,

Tel. (X19)821-7000.

Science Publishers

B.V. (Biomedical

bp, base pair(s);

Cm acetyl transferase; ing hGH;

of Botany

(R.B.)

of sources have been introduced into plants, primarily tobacco and petunia. In their native form, animal and yeast genes are generally not properly expressed in a plant-cell context. For instance, no

hGH,

1000 bp;

Km,

mMTI-I,

mouse

NOS, nopaline frame; T-DNA,

Division)

Ri, root

cut, gene encoding

human kanamycin;

growth

metallothionein synthase; inducing

DNA transferred

hormone;

mMT-I,

CAT; CAT,

hGH, gene encod-

Cm, chloramphenicol;

kb, kilobase

gene

encoding

I; NOS, gene encoding

nt, nucleotide(s); (plasmid);

or

mMT-I; NOS;

ORF, open reading

SV40,

simian

virus

40;

from Agrobacterium to plant cells.

134

tr~s~ription of either a yeast alcohol dehydrogenase gene (Barton et al., 1983) or mammalian interferon gene (Caplan et al., 1983) was observed in transgenic plants bearing these genes, while aberrant transcription was observed from a chicken ovalbumin gene (Koncz et al., 1984). Further studies on the effectiveness of animal promoters in plant cells have been based on the introduction into plants of chimeric genes placing a reporter gene under the control of the promoter studied. In studies of chimeric genes based on animal promoters and a CAT-coding sequence, An (1986) found no detectable CAT activity in transgenie plant cells bearing genes driven by the SV40 early promoter, the Herpes simplex thymidine kinase promoter and an mMT-I promoter (An, 1986). On the other hand, two Drosophila promoters, the copia long terminal repeat promoter (Ou-Lee et al., 1986) as well as the hsp70 promoter (Spena et al., 1985) do drive appropriate expression of reporter coding sequences in transformed plant cells. The consensus sequences that govern RNA maturation processes, such as splicing and polyadenylation of transcripts, appear to be common among plant and animal genes (Heidecker and Messing, 1986). Yet in the two cases where splicing of an animal pre-mRNA in transgeni~ plant cells has been studied, it was found that neither hGH (Barta et al., 1986) pre-mRNA nor globin pre-mRNA (Van Santen and Spritz, 1987a) are spliced correctly. In contrast, certain plant pre-mRNAs can be spliced by animal in vitro maturation systems (Van Santen and Spritz, 1987a). The results concerning recognition of animal polyadenylation signals are more complex. Barta et al. (1986) found that the hGH polyadenylation signal was not used in transformed plant cells. In a slightly different hGH construct, Hunt et al. (1987) have found significant polyadenylation at the normal hGH site, as well as elsewhere. In plants bearing constructs with 3’ regions of the adenovirus 5 Ela and the SV40 early genes, they found polyadenylation at sites either slightly shifted relative to the normal site (Ela) or at a presumed cryptic site weli upstream from the normal site (SV40). To further study the effectiveness of animal gene regulatory signals when introduced into higher plants, we have used a gene coding for mMT-I. Furthermore, development of genes that code for metallothionein in plants may make it possible to

modify the distribution in plants of pollutant heavy metals, such as cadmium, since it has been shown that a mammalian metallothionein is functional in a plant context (Lefebvre et al., 1987; Maiti et al., 1988). A genomic clone bearing the intact mMT-I gene was introduced into plant cells to study transcription from the mA4T-Z promoter. Two chimeric genes driven by the nopaline synthase promoter were also introduced into tobacco cells. In one chimer& gene a fragment bearing the complete coding sequence from the genomic mMT-I clone was used, to test the effectiveness of pre-mRNA splicing. Another gene based on the coding sequence and 3’-untranslated region derived from the mMT-I cDNA clone was also constructed and introduced into plants. In all three cases the Agrobacterium rhizogenes Ri plasmid was used as a vector (Tepfer and Casse-Delbart, 1987). We have found that the mMT-I promoter is not active in transformed tobacco root cells, which do not splice metallothionein pre-mRNA, and that the animal polyadenylation signals are not recognized. These results lend further credence to the idea that the known highly conserved consensus se-

’ ATG

pMT-I

MT-I

WIT-I

’ TGA

1

I RNOS Fig. 1. Genes (Brzezinski

ATG

introduced

MT-I

into plants.

et al., 1987) as present

gene in pVPl0,

which

and the w&T-I

genomic

(A) Intact

of the promoter

ofp~EOlO5 coding

mMT-I

gene

in vector pVP8. (B) Chime&

is composed

regions ofthe NUS cassette

3’Nl.X

TGA

(Simpson

region.

and 3’

et al., (1986)

(Cj Chimeric

genes in

pVP4, in which the mMT-Z cDNA coding region plus 3’ region (Mbikay signals.

et al., 1981) is under

the control

Solid bars are mMT-Z sequences,

NOS. The bent arrows (AATAAA).

The introns

indicate

of NOS 5’ and 3’ open bars are from

sites of polyadenylation

are shown at half scale.

signals

135

quences involved in pre-mRNA maturation are not sufficient to direct proper maturation, and that other flanking sequences must be involved.

EXPERIMENTAL

AND DISCUSSION

(a) Construction

of intermediate

described by Brzezinski et al. (1987). This vector, pVP8, also bears, as an internal control, a constitutive gene conferring Km resistance to plant cells subcloned from pNEO105 (Simpson et al, 1986). The latter gene is under control of the NOS promoter and 3’ regions. A second intermediate vector, pVP10, bears the chimeric gene shown in Fig. lb, in which the mMT-I coding region including introns, from which mMT-I polyadenylation signals had been removed by BAL 31 exonuclease digestion, has been cloned between the NOS promoter and NOS 3’ regions from pNEO105.

vectors

The inte~ediate vector pVP8 bears an intact copy of the mouse m&U-I gene, shown in Fig. la, which was subcloned from the genomic clone (LmtI)

A 1

B 2

1

C 2

3

E

D 3

4

4

56

F 5

6

1.4 kb 1.4 kb

0. 8 kb

Fig. 2. Expression of genes ~troduced into plants. Total RNAs were extracted from root cultures bearing the genes shown in Fig. 1. Denatured RNAs were separated on a 1.0% agarose-1.2 M formaldehyde gels (Lehrach et al., 1977), transferred to Amersham Hybond-N membranes and hybridized to “2P-labeled probes, as described by Feinberg and Vogelstein (1983). Size of transcript (0.8 and 1.4 kb) was estimated by interpolation of positions of RNAs of the BRL RNA ladder visualized on the ethidium bromide-stained gel. Panel

Probe

Lane

Source of RNA

&4T-Z cDNA

1 2 1 2 3 4 3 4 5 6 5 6

control transformed roots, Cd2 + -treated roots bearing gene in Fig. 1A, Cd2+ -treated control transformed roots, Cd” -treated roots bearing gene in Fig. 1A, Cd* + -treated control transformed roots roots bearing gene in Fig. 1B control transformed roots roots bearing gene in Fig. 1B control transformed roots roots bearing gene in Fig. 1C control transformed roots roots bearing gene in Fig. 1C

Ri T-DNA fragment Hind111 32 r&&l--Z cDNA w&T-Z intron 1 mA4T-ZcDNA NOS 3’ region

136

The third intermediate vector, pVP4, bears the chimeric gene shown in Fig. lc, based on the mMX-I cDNA clone pM135 (Mbikay et al., 1981). This chimeric gene is composed of the NUS promoter,

(2) Splicing of mMT-I pre-mRNA Northern experiments were also carried out on root cultures having incorporated pVP 10, which bears a chimeric gene in which the genomic mMT-I

followed by the mMT-I coding sequence without introns, the mMT-I 3’ region and the NOS 3’ region.

coding region, including of the NOS promoter. obtained

(b) Plant tr~sformation Stem segments of tobacco (Nicotiana tabacum var. Xanthi XHFDS) were inoculated as described elsewhere (Tepfer and Casse-Delbart, 1987) and for each bacterial strain several independent genetically transformed root clones were obtained and maintained in culture. Correct insertion of the vectors bearing the genes of interest was verified by Southern-blot experiments carried out on DNA extracted from axe& root cultures, using the corresponding inte~ediate vector as probe (not shown). (c) Analysis of transcripts in transformants (I/ Expression of the in&et mMT-I gene Since ~~sc~ption of metallo~ionein genes is induced in animals by the presence of heavy metals (Durnam and Palmiter, 1981), we have studied transcripts in transformants bearing the intact &T-I gene in the absence or presence of an inducing heavy metal (10 uM CdSO, for 12 h). Total RNA was isolated (Chirgwin et al., 1979), purified (Gligin et al., 1974) and analyzed by electrophoresis in formaldehyde (1.2 M) denaturating 1 y0 agarose to gels (Lehrach et al., 1977). After transfer Hybond-N nylon membranes, blots were hybridized with 32P-labeiled probes (Feinberg and Vogelstein, 1983). As shown in Fig. 2A, when total RNA extracted from Cd2+ -treated roots is probed with the isolated mMT-I cDNA, no metallothionein transcript was detected, while RNA transcribed from T-DNA ORF 15 (Slightom et al., 1986), also present in the transformed plants, was detected in all cases (Fig, 2B), showing that Cd” treatment had no effect on expression of the T-DNA gene. The chimeric Km-resistance gene in these transformed roots was functional, as the root clones bearing pVP8 were resistant to 100 pg Km/ml (data not shown).

when

introns, is under the control Fig. 2C shows the results

the mA4T-I

probe. A 1.4-kb transcript

cDNA

was used

as

was detected, which is the

size expected if introns are not excised, while a correctly spliced transcript would be approx. 0.8 kb. When a probe specific for mMT-I intron 1 was used (Fig. 2D), the same 1.4-kb transcript was detected, confming that intron 1 was not excised. Even after much longer exposures of these blots, no weak bands that could correspond to partially or totally spliced transcripts were detected (not shown). (3) Effectiveness of mMT-I polyadenylation signals The question of the efficiency of the mMT-I polyadenylation signaIs was addressed in Northern experiments on root cultures having incorporated pVP4. In this case, when an isolated fragment corresponding to the mMT-I cDNA was used as probe, a 0.8kb transcript was detected (Fig. 2E), the size expected if the NOS polyadenylation signals were used; a shorter transcript of 0.65 kb would have been expected if the mMT-I signals were recognized. Longer exposure of these blots failed to reveal such a shorter transcript. This was confirmed (Fig. 2F) by probing with a fragment isolated from the NOS 3 ’ region, which also hybridized with the 0.8-kb transcript, demonstrating that the NOS polyadenylation sequences, 3 ’ to mMT-I polyadenylation signal, were used. (d) Comparison *+f nzn/iT-I sequences

and plant consensus

Since these experiments were initiated, increasing numbers of higher plant and animal genes have been sequenced, allowing much improved comparisons of regions presumed to control gene expression. We have compared appropriate regions of the mMT-I gene (Glanville et al., 1981) with consensus sequences observed in controlling regions of plant genes; in some cases we have also sought individual pIant genes whose sequences are as similar as possible to mMT-I sequences in the consensus regions. Joshi (1987) has derived consensus sequences

137

differences between the plant consensus and met-I sequences. In the region of the TATA box, the G in the position TATA + 3 in the mMT-I promoter is extremely unusual in plant genes, as it was only observed in a soybean nodulin gene (Table I). At the

from plant genes in three regions involved in ,,.the control of transcription and translation, the TATA box, the site of initiation of transcription and the ATG translation start codon. We have compiled the yO nucleotide usage in these regions of the genes studied by Joshi to better evaluate the importance of

TABLE

I of 5’ region of mMT-I gene with plant genes

Comparison

TATA box region %G

11

11

14

10

0

0

0

0

3

1

5

%A

18

18

38

9

3

97

9

94

47

95

30

71

I1

in plant

%C

34421647

1

1

0

1

0

1

5

8

29

genes a

%T.‘

31

29

32

34

96

1

90

5

53

1

63

16

I5

Plant consensus

b

T

C

A

C

TATATATAG

G

A

G

Nucleotide I

frequencies

m MT-I ’

CGACTATAAAOAG

Wheat

CAACTATAAATAG

gliadin“

Soybean

nod&n

C

*

C

T

Trauscription

T

T

1

A

T

A

%G

18

14

10

6

6

8

8

frequencies

%A

18

20

22

7X

18

24

43

%C

37

31

49

4

21

45

21

genes R

%T

21

35

18

12

49

22

22

Plant consensus

h

CTCa;TCA

/

mMT-I” Soybean

A

start point region

Nucleotide in plant

T

AGCHTCA hspt 7Sd

CTCGTCA

Translation

ATG start codon

Nucleotide

’ %G

22

18

I3

19

frequencies

c %A

31

30

51

66

39

48

in plant /

genes a

Plant consensus

513

region 0

0

100

81

13

17

100

0

0

13

12

13

%C

13

29

25

1

47

28

0

0

0

0

71

12

%T

35

23

11

14

9

11

0

100

0

6

5

59

TAAACAA

T

G

cc;I:

mMT-1’

CTCGBAA

T

G

GAC

Maize actind

TTGAGAA

T

G

G

Maize waxyd

ATCGGCA

T

G

GCG

a Nucleotide compiled

44

’

frequencies

(as shown for each nt position

by Joshi (1987). For the transcription

’ The most frequent nt are underlined ’ Sequence

nucleotides

is either greater

from Glanville

d Plant genes cited for comparison

T

shown below) were calculated

start point, only the 49 genes where it has been localized

are shown; the TATA box, transcription

if the frequency

ofmMT-I

in the “plant consensus”

C

than 50% or greater

et al. (1981). Boxed nucleotides are from Joshi (1987).

start point and the translation

for the 79 plant genes

precisely

than twice that of the next most frequent

are very rare at these positions

were used.

ATG start codon are overlined; nucleotide.

in plant genes (frequency

-Z 10%).

138

transcription start point, 22 bp downstream from the mMT-I TATA box, it is surprising that the first transcribed nt is a G, but this is also observed in 6% of the plant genes examined. Similarly, the G 2 nt upstream from the ATG start codon is unusual, but is also found in 5 y0 of the plant genes considered. It has recently been shown that the mMT-I start codon is correctly recognized in plants (Maiti et al., 1988). We have carried out similar comparisons at the intron-exon splice boundaries, using the plant sequences analysed by Brown (1986), though we have not considered presumptive branch points, since neither the mMT-I branch points nor those of plant introns (with very few exceptions) have been identified with certainty (Table II). The 5’ splice junctions of the m.MT-i gene are not particularly divergent from the plant sequences; indeed this is not surprising as the plant and animal consensus sequences are C/AAG : GTAAGT vs. identical, essentially

TAELE

C/AAG : GTA/GAGT, respectively, where the colon represents the splice site (Brown, 1986). The situation at the 3’ splice junction is more complex. In the region of nt -5 to - 15, plant introns are much more purine-rich; the majority of plant genes having five or more purines in this position. The absence of purines in this region of the mMT-I introns may be significant. Of the 177 plant introns studied by Brown (1986), none is completely purine-free in the region of nt -5 to -15, as are the corresponding regions of the m&lT-I introns. (e) Conclusions In plants bearing the intact mMT-I gene, we observed no detectable co~esponding transcript (Fig. 2A). These results are in agreement with those of An (1986), who observed no CAT activity in plant cells bearing a chimeric cat gene driven by the mMT-I

II

Comparison

of splice junctions

of mMT-2 gene and plant genes

5’ exon-intron

splice junction

: 100

Nucleotide

%G

20

11

72

frequencies

%A

33

55

11 :

0

in plant

%C

33

10

Ii

:

0

genes a

%T

IS

24

c

A

Plant consensus

b

6:

13

6

65

11

0

70

55

16

23

7

20

8

11

10

19

11

49

1

0

G:

0

99

GTAAGT

A mMT-I

intron

1’

CCG:GTAAGA

mMT-I

intron

2”

AGA:GTGAGT

3’ intron-exon

splice junction

Nucleotide

%G

I2

14

16

15

14

19

20

23

23

18

11

50

2

0

frequencies

%A

18

17

13

30

19

21

19

23

20

25

I1

20

5

100

100

in plant

%C

23

16

20

14

14

13

19

I5

15

13

10

14

67

0

0

genes a

%T

47

53

51

44

53

47

42

38

41

44

68

16

27

0

0

1CTTTTTT

xTTTG5;AG:G

1’

TCTTTCT

CCTCCC

A

G:G

mMT-I intron 2”

TCCTCCT

TCTTCT

A

G:G

Plant consensus mMiT-I intron

a Nucleotide

b

frequencies

of ptant genes compiled b The most frequent most frequent c Sequence

(as shown for each nt position by Brown (1986). Intron-exon

nucleotides

nucleotide.

are shown. If underlined,

Intron-exon

of the corresponding

in the “plant consensus” junctions

junctions

regions

frequency

are indicated

of in&on-exon

are indicated

: 60

0 : IS

: 14 : 11

shown below) were calculated

from the 177 introns

by colons.

is either greater

than 50% or greater

than twice that of the next

by colons.

splice junctions

(colons)

of MT-1

are from Glanvilie

et al. (1981).

139

promoter. Minor differences between the mMT-I sequences and plant genes in transcriptional control regions, such as the G at TATA + 3 (Table I) may be significant in this regard, but a more likely hypothesis would be that plants lack the trans-acting factors that activate metallothionein gene expression in animals in response to heavy metals or glucocorticoids (Durnam and Palmiter, 1981). The inability of plant cells to excise mMT-I introns (Fig. 2,C and D) is perhaps more surprising. The consensus sequences of plant and animal 5’ splice junctions are identical (Brown, 1986), and more recently it has been shown that the complementary sequence of IJl RNA, which is implicated in the recognition of the 5 ’ splice site, is identical in plants and animals (Van Santen and Spritz, 1987b). Presumably the difllculties lie in the correct recognition of the 3’ site, where significant differences exist, particularly in the region of nt -5 to -15 to the 3 ‘ splice site, where plant introns are more pm-me-rich. The hexanI~cleotide AAUAAA is usually implicated in the control of polyadenylation of premRNAs, though this sequence is not always present in plant genes (Hunt et al., 1987). Other poorly characterized signals downstream from the site of polyadenylation may also play a role in both animal (McDevitt et al., 1984) and plant (De Pater et al., 1987) genes. It is possible that the lack of activity of the mMT-I polyadenylation signals in the gene which we have studied could be due to the absence of such downstream regions. The presence of the NOS 3’ region could also have prevented recognition of the met~lo~ione~n polyadenylation signal, since Barta et al. (1986) found no polyadenylation at the izGH site when the latter was followed by the NOS 3’ region, while Hunt et al. (1987) found significant use of the hGi7 site when it was followed by an rbcS 3’ region. Indeed, little is known about how plants select among the multiple polyadenylation sites found in the 3’ region of many plant genes (Heidecker and Messing, 1986).

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