Note: Descriptions are shown in the official language in which they were submitted.
% ~ ?
HOECHST AKTIENGESELLSCHAFT HOE 89/F 295 Dr. ~H/pe
Description
RNA with endoribonuclease activity for ~RNA of ripening
gene , the preparation thereof ~nd ~he u~e thereof in
plant~
Under certain condition~, RNA molecule6 are able, without
involvement of protein~, to catalyze reactions on other
RNA molecules or to eliminate autocatalytically fra~ments
from their own molecules. Thus, there is autocatalytic
deletion of an intron containing 413 nucleotides from the
3~ end of the 23s rRNA of Tetrahymena thermophila, and
conversion into a circular form. This takes place by a
series of phosphoester tran~fer reactions in which
guanosine cofactors are involved (Cech,`T.R., Nature 30,
578-583 (1983)). Dependiny on the RNA su~strate or the
chosen reaction conditions, the intron can function a~
~pecific ribonuclease, terminal transferase, phospho-
transferase or acid pho~phatase. Moreover, one RNA
molecule i5 able to carry ~ut a number of conversions
without it~elf being changed andt in this re~pect,
behaves like an enzyme. This is why the term ribozyme has
been coined for RNA molecules wit:h the~e properties.
SLmilar reactions without involvement of pr~teins have
also been demonstrated for ~ome Yiroidal RNAs a~d ~atel-
lite ~NA~. ~hus, self-procsssing appears to be an
essenti21 reaction for replication of avocado 6unb1o~ch
viroid (ASBV) ~Hutchins, C.J. et al. Nucleic A~id~ Res.
14, 3627-3640 (1986~), satellite ~NA of tobacco ring~pot
virus (~TobRV) (Prody, G.A. et al., Science 231, 1577-
1580 (1986)) and ~atellite ~NA of lucerne tran~ient
streak virus (sLTSV) (Forster A.C. et al., Cell 49, 211-
220 (1987)~. It i~ ~upposed that circular forms ~re
produced durin~ the replication of these RNAs and lead,
as templates, to the synthesis of RNA~ with exten ions.
~hese transcripts are cut to the ~orrect genome length by
the self-cataly~ed endonucleolytic reactions.
- 2 -
The structures of the RNA5 which presumably take the
latter in for the reaction have been described as hammer-
heads (Forster, ~.C. et al., Cell 49, 211-220 (1987);
Haseloff, J. e~ al., ~ature 334, 585-591 (1988)).
The cleavage ~i~es for th~e RNA enzymes are spe~ific and
must display particular 6tructural requirements for
processi~g to be able to occur.
It has now been found that ribozyme6 are able to attack
plsnt RNA coding for ripening enzymes and thus can be
used for i~fluencin~ the ripening proceRses in plants.
Regulation of the expression of the DNA coding ~or the
ripening enzyme polygalacturonase by antisense RNA has
been described by Smith C.J.S. et al. in Nature 334, 724
(1988). A fragment of the polygalacturonase cDNA is
placed in the opposite orientation in an expre~sion
vector. This vector plasmid i5 usled to transform, via E.
coli and Agrobacterium tumefacien~i, stalX ~egments of the
tomato. Expression o~ antisen~e ~NA can then be detected
in the leaves o~ the tomato plant. ~t is as~umed that the
antisense RNA attaches it~elf to the actual polygalac-
turonase RNA, leading to inactivation of the 12tter, with
the further consequence tha~ there is partial inhibi~ion
of polygalacturonase synthesi~.
Ribozymes which bind to sipening enzyme RN~ and are able
to cleave the latter at particular cleavage ~ite~ in th~
equenCe have now been developed for ~pecifically
influencing the ripeni~g process in plantsO It i~ po~-
~ible with the aid of the ribozymes according to the
invention ~or the synthesi~ of particular ripening
enzymes not ~ust to be partially i~hibited but to be
virtually completely inhibited, i.e. about 80~100%.
?.~2~
3 ~
Hence the invention relates to:
1. Genes or gene fragments encoding ribozymes~ and the
corresponding ribozyme RNA sequences and a process
for the preparation thereof.
2. Plants, plant cell~ and par~s or ~eeds of the plants
which con~ain th~ DNA or ~NA ~equence speci~ied
under 1.
3. The use of ribozymes for inhibiting the synthesis of
ripening enzymes in plants.
The invention i5 described in detail hereinafter, espe-
cîally in it~ pxeferr~d emhodiments. The invention i8
also defined in the claims.
~he ribozyme can be synthesized on ~he basis of ~he DNA
~equence of the ripening gene to be inhibited. In this
connection, it is pos~ible in principle to ~tart from any
DNA sequence coding for a plant ripening enzyme. Examples
of such plant ripening enzymes are polygalacturonase,
pectin estera6e and so-called ripening-related proteins.
It is preferable to choose a6 "starter" for the synthesis
at least 10 consecutive nucleotidles, in particular 14 to
~0 nucleotidPs, advantageously ~Erom the middle of the
cDNA sequence of the structural gene. It is particularly
advantageou~ to start from the cDNA seguence for p~ly-
qalacturonase (Griers~n, D. et al., NAR 14, 8595 (1986~),
pectin estera~e (Ray, 3. et al., Eur. J. Biochem. 174,
119 (1988)) and for ripening-related protein (Ray, J. et
al., NAR lS, 10~87 (1987)~ in each instance.
Chemical synthesi~ of ~ligonucleotides on the bAsi6 of
the cDNA sequence is carried out in such a ~ay that the
initial and final sequences of the oligonucleotides are
each composed of 5, preferably 7 to 10, nucleotides
which, taken t~gether, are complementary to a ~N~
sequence ~f the ripening enz~me to be inhibited and that
_
the initial and ~inal sequences of the oligonucleotides
are separated by an interpolated RNA sequence which is
composed partly of specific nucleotides predetermined for
the functionality of the ribozyme and partly of variable
nucleotides. ~he appearance of the ribozyme hybridized
with substrate RNA can be outlined as follows.
~-~NNNNNNNNNNNNGUC ~NNN~NNNNNNNNN-3 ~___sub~ rate ~NA
3-KKK~KKKKCA KKKKK~KK-5
A G
C G Gy
VY
y ~ ~ ribozyme
y ~ Loop
~L ,
... ..
where
N are nucleotides of the ~ubstrate RNA, A, C, G or T,
R are complementary nucleotide~3 to N in the ribozyme,
V are variable nucleotides in the ribozyme and
VL are variable nucleotides in the loop of the
ribozyme.
The number Of VL nucleotides in the loop can be 0-550. A
GU recognition sequence is preferably chosen as cleavage
5 ite in the substrate RNA.
The said oligonucl~otide~ are provided with an appro
priate linker. Linkers of this type pOS~e85/ for example,
cleavage sites for ~coRI, SalI, BamHI, HindIII, ~coRV,
SmaI, ~hoI, RpnI, preferably ~baI and PstI.
The synthesized oligonucleotides are cloned with he aid
of the vectors pUCl9, pUC18 or pBluescript (~tratagene,
HPidelberg, Product Information~, and ~equenced. The
verified oligonucleotide is cloned into an intexmedia~y
vector with a plant promoter, Examples o~ vectors of ~his
type ara the plasmid~ pPCV701 ~Yelten, J. et al. EMBO J.
~.J J.~ L
- 5 -
3, 2723-2730 (1984)), pNCN (Fro~m M. ~t al., PNAS B2,
5824-5826 t~985)) or pNO5 ~An G. et al., EMBO J. 4, 277-
276 ~1985)). The vector pDH51 (Pietrzak, M. et al.,
Nucleic Acids ~es. 14, 5857, (19B6)) with a 35S promoter
is preferably used.
After ~ubseguent transformation of E. coli, such as, for
ex~mple, E. coli ~C 1061~ D~1, DR1, GM48 or ~L-l, posi-
tive clones are identified by methods known per ~e
(Maniatis et al., Lab. Manual), 6uch as plasmid mini-
preparation and cleavage wi~h an appropriate restrictionenzyme.
These positive clone6 are then subcloned into a binary
plant vector. Plant vectors wbich can be employed are
pGV3850 (Zamb~ysk, P. et al., ENBO J. 2, 2143-2150
(1983)) or pOCU8 (Olszewski, N., Nucleic Acids Res. 16,
10765-10782, (1988)~. pOCA18 is preferably used.
The resulting binary plant vector,s which contain a plant
promoter with the attached DNA fragment for ribozy~e
production in the T-DNA are u~ed to trans~orm plants.
Techniques such as electroporation or microin~ection can
be employed for this.
Preferably employed is cocultivation of protoplasts or
transformation of small pieces of leaf ~ith Agrobacteria.
For this, the plant vector ~onstruct is transferred by
transfor~ation with purified DNA orl mediated by a helper
strain such as E. ~oli S~10 (S~mon R. et al.,
Biotechnology lr 784-791 (1983)), into Agrobacteri~m
tumefaciens such as ~2~2 with a Ti plasmid ~ia triparen-
tal ma~ing. Direct transformation and triparental mating
were carried out ~s described in "Plant ~olecular Biology
Manual" (~luwer Academic Publi~hers, Dordrecht (1988)).
It is possible in principle to transform all plants with
binary plant vectors carrying ribozyme DNA. Dicotyledo-
nous plants are preferred, especially productive plants
~2~
-- 6 --
such as, for example, fruit-bearing plants. Tomato,
strawberry, avocado and plants which bear tropical
~ruits, for example papaya, mango, but al~o peax, apple,
nectarine, apricot or peach, may he mentioned as ex-
amples. The described process is particularly preferablycarried out with ~he tomato. The transformed oell~ are
selected with the aid of a selec~ion medium, cultured to
a callus and regenerated to the plant on an appropriate
medium (Shain et ai., Theor. ~ppl. Genet. 72, 770-770
(1986); Masson, J. et al., Plant Science S3, 167-176
(1987), han et al., Plant Nol. Biol. 11, 551-559 (1988);
~cGranaham et al., Bio/Technology 6, 800-804 ~1988);
Novrate et al., Bio/Technol. 7, 1~4-159 ~1989~).
The resulting plant is altered by the transformation in
such a way thst the ribozymes are expressed in the cells,
which in turn has the effect that the ribozyme RNA not
only binds to the RNA complementary to the appropriate
ripening genes and brings about more ox less extensive
inhîbition of synthesis o~ the ri~penin~ enzyme, but that
the RNA complementary to the appropriate ripening ~enes
is ~pecifically cut at GUC seq~lences, which le~ds to
almost complete inhibition of synthesis of the relevant
ripening enzyme.
~ he formation of the ribozyme-specific ~econdary struc-
tural feature6 of the ribozyme RNA synthesized in the
transgenic plant in vivo was entirely unexpected, BO that
the observed i~hibition of ~ynthesis of the ripening
enzymes was ~ompletely surpri~ing.
The example~ which follow fierve to illustrate ~he invPn-
tion f~lrther.
- 7 -
~ample~
Unless indicated otherwise~ percentage data xelate to
wei~ht.
1. Cloniny of the oligonucleotides
The ~ynthesis of the oligonucleotides for ribozyme
expression was based on the cDNA seguence
a) 5' TG~TGGAGTCCATG~T~A 3~ Section of the polygalact.
cDN~ eequence according to
Grierson, D. et al.,
Nucleic Acids Re~. 14,
8595-8603
b) S' TAGCAAGTCCTGACCTAA 3' Section of the cDNA
sequence of pect~n estera~e
according to Ray, J., Eur.
J. Biochem. 174, 119-124
(1988)
c) 5' TGCTTTGTCCGATACAGT 3' Section of the cD~A
seg~ence of a xipening-
related ~rotein according
to Ray, J., Nucleic Acids
Re~ , 105~7 (1987)
The phosphoramidite method (~ngels J. et al., Advances in
Biochemical Engineering Biotechnology Volume 37 ed~: A.
~iech~er, Springer Vexlag, Berlin/Heid~lberg, 1988) wa~
u~ed to ~ynthesize the following oli~onucleotides in a
synth~sizer: -
fox a) 5' CTAGATGATACATGCTGATGAGTCCGTGAGGACGAAACTCCATCTGCA 3
3'TACTATGTACGRCTACTCAGGCACTCCTGCTTTGAGGTAG 5~
f or b) 5' -CTAGATTAGGTCAGCTGATGAGTCCGTGAGGAGGAAACTTGCTACTGCA-3'
3~-TAATCCAGTCGACTACTCAGGCACTCCTGCTTTGAACGATG-5~
2 ~
-- 8 --
for c~ 5' -CTAG~CTGTATCGCTGATGAGTCCGTGAGGACGAAACAAAGCACTGCA-3'
3'-TGACATAGCGACTACTCAGGCACTCCTGCTTTGTTTCGTG-5'
Th~ vector pDH51 (Pietrzak, M. et al., Nucleic Acids Res.
14, S857 19) was cut with the re~triction endonucleases
XbaI and P~tl, incubated with calf lntestinal phospha~a~e
(CIP), phenol-treated and precipitated ~Maniati~, ~ab.
Manual). The vector treated in thi way wa6 ligated with
a three-~old exce~ of phosphorylated oligonucleotid~s
and transformed into E. coli ~C1061. Positive clones were
identified by plasmid minipreparations and 3ubsequent
digestion with ~baI and PstI.
In addition, the ampicillin resistant transformed E. coli
cells (100 ~g of ampicillin per ml of LB m~dium) were
transferred to nitrocellulose membrane~ (Gene Screen
Plus~, NEN, Boston) and incubated on ~B medium containing
ampicillin at 37C for a further 14 hours. The colonies
were then disrupted in 0.5 M :NaOH and fixed. After
drying, it was possible to hybr.Ldize the filters wi~h
radiolabeled oligonucleotides. Positive clones produced
blackening on the film.
2. Subcloning of a 35S promoter gene fragment in pOCA 18
A 0.75 ~b EcoRI fr~gment was isolated from ~a~h of the
clones obtain~d a~ in 1. ~his fragment was inserted into
a pOCA 18 vector which had been cut with ~coRI, and was
transformed into E. coli MC1061. Positive clones were
identified by plasmid minipreparatio~s and, after subse-
guen~ hydr~ly~is with ~coRI, by the appearance of the
0.75 kb band.
3. Transformation of Agrobacterium tumefacien~
In order to bP able to transform plants, th~ construct
obtained as in 2. mu8t be tran~ferred into an Agrobac-
terium. This takes place ~ither by triparental mating or
directly. In the case of triparental mating, 100 ~1
2 ~
g
samples of bacteria from overnight cultures of E. coli
SM10, the E. coli ~C1061 carrying the construct, and
~grobacterium ~umefacien~ were 6pun down and taken up
together in 30 ~1 of LB medium. After 30 minutes ~t room
5 temperature, thi~ bacteria su~pension was placed on a
filter on an LB plate without antibiotics. ~he filt~r was
incubated at 37C for 12 h and then washad in 2.5 ml of
10 mM MgCl2. Aliguots were selected on LB plates contain-
ing rifampicin, ~etracycline and kanzmycin. Po~itive
colonies were identified by hybridizstion with 32P-labeled
DNA of the gen~ to be expr~ssed.
In the case of direct transformation of Agrobacteria, the
cells were cultured at 28C overnight in YEB medium (1~
yeast extract, 1~ peptone, 0.5~ NaCl) containing 25 ~g/ml
kanamycin and 100 ~g/ml rifampicin. After 16 hours, the
bacteria suspen~ion was diluted to an OD~50 of 0.1 and
incubatPd further at 28C until t!he OD550 was 0.5. 1 ml of
this culture was ~pun down and wal~hed with 1 ml of 150 mM
NaCl. After wa~hing, the precipitate wa~ resuspended in
600 ~1 of ice-cold 10 mM CaCl2 solution.
The pOCA lB vecto~ with ~he cloned 0.75 kb fragment was
isolated ~rom the E. coli clones obtained as in 2. after
disruption of the cell~ with 0.2 N NaOH/1% SDS, wa~
purified by CsCl density gradient centrifugation. 1 ~g of
~5 the plasmid DNA was added to the competent Agrobacteria,
and the Eppendorf tubes were placed on ice for 1 hour.
After 1 hour, the pla6mid solution was in~ubated at 37C
for 5 minutes, and 2 ml of YEB medium were added. The
cells were then incubated at 28C i~ a ~hakin~ incubator
overni~ht. Thereafter 100 ~1 ~ample~ were placed on YEB
plates containing 1~0 ~/ml rifampicin, 25 ~g/ml kanamy-
cin and 2.5 ~g/ml tetracycline. Colonies were found on
the plates after 2 day6 at 28C. Positive clones were
detected by hybridization wikh the appropriate 32P-labeled
oligonucleotides a~, b~ or c~.
For this, the trans~o~med Agrobac~eria were ~treaked on
2 Q
-- 10 --
Gene Screen Plus membranes and incubated on YEB plates
containing 100 ~g/ml rifampicin, 25 ~g/ml kanamycin and
2.5 ~g/ml tetracycline at 28C or 14 hour~. The mem-
branes wer~ then placad on 0.5 ~ NaOH for 2 minutes and
subseguently on 0.5 M Tris, pH 7.5, for 2 minutes. After
drying, prehybridization wa~ carried ~ut in 10~ dextran
sulfate/l M NaCl/l~ SDS at 55C for 2 hour6, and then the
radiolabeled oligonucleotidee a), b) or c~ were added.
The filter~ were incubated together with the radiolabeled
oligos at 55C overnight. After washing at 55Uc for 30
minutes each with lx SSC (0.15 M NaCl, 0.015 N sodium
citra~e, pH 7.0) and 2x with 0.2 x SSC, the positive
clones were identified by blackening of a film placed on
top.
4. Transformation of toma~oes
a) Protoplast transformation:
Tomato protoplasts (Plant ~ell Reports 6, 172-175
(1987)) are washed once with W5 solution (154 mM NaCl,
125 mM CaCl2 . 2 2 ~2~ 5 mM RCl, 5 mM ~lucose) and
once with MaMg solution (0.45 1~ mannitol, 25 mM MgCl2,
0.1~ 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.8)
from Sigma Chemie, Deisenhof~n, FR Germany). After
careful centrifugation at 600 rpm for 3 minutes, the
supernatant, apart from 0.5 ml, is aspirated offO To
this are added by pipette 50 ~g of calf thymus DNA,
10 ~g of the described pla~mid and 10 drops of 45%
polyethylene glycol (PEG 8000). After 10 minu~es, the
protoplasts are washed twice w~th W5 solution and
incubated in LC~ medium (Plant Cell Reports 6, 172-175
(1987)).
b) Transformation of ~mall pieces of leave~ with
Agrobacterias
Tomato l~aves are cut up small and placed on MS medium
(Murashige, T. et al., Physiol. Plant 15, 473 497
(1962)~ containing 2~ sucrose and 1 ppm plant growth
hormone zeatin (Serva Feinbiochemica GmbH & Co.,
7 ~ ~
Heidelberg, FR ~ermany) and incubated at 25C over a
period of 16 hours li~ht/day. Infection with Agrobac-
teria is carried out one day later. This entails the
small pieces of leaves ~eing briefly Lmmersed in a
dilute bacteria suspension (OD 0.15) of the trans-
formed Agrobacterium strain, replaced on the ~ame
plate and ~urther incubated under the same conditions.
On the third day, all ~he small pieces of leaves are
washed with 250 mg~l carbenicillin solution and placed
on a 2MS medium containing 1 ppm zeatin, 200 ~g/l
cefotaxime (Hoechst AG, Frankfurt), 200 mg/l carbeni-
cillin and 100 mg/l kanamycin. Regenerant~ were
transferred after approximately 20 more days to 2~S
medium containing 1 ppm zeatin, 200 mg/l cefotaxime,
200 mg/l carbenicillin and 100 mg/l kanamycin.
Detection of the ribozyme ~NA oE a transgenic tomato
plant which is directed against the ripening-related
protein RNA.
The total cellular RNA was isolated from a small piece of
leaf from a transgenic tomato plant which had been
transformed with the ribozyme-encoding gene directed
against the ripening-relate~ protein RNA. For thi~, the
leaf materi 1 was ground with a mortar and pestle under
liquid nitrogen. ~he powdered leaf material was ~ixed
with twice the volume o~ an extraction buffer (0.2 M
~odium acetate, 1~ SDS, 10 m~ EDTA), twice the volume of
phenol (equilibrated with extraction buf er3 and half the
volume of chloroform~isoamyl alcohol 24:1 (v~v)- After
very thorough mixing, the phases were ~eparated by
centrifugation and the phenolic extraction of the upper
aqueous phase W86 repeated. This aqueous phase was, af~er
mixing and separation by centrifugation, agai~ tran~-
ferred into a fresh Eppendorf tube and extracted with
chloroform~ isoamyl alcohol 24:1 (v/v). ~hen 1/3 volume
of 8 ~ LiCl was added to the aqueous phase, and the
mixture was maintained at 4C overnight. A ~recipitate
formed after centrifugation This was taken up in w~ter
2 ~
12 -
and, after addition of 0.3 M ~odium acetate (p~ 5.5)
(final concentration), washed with 2 1/2 vol~mes of
ethanol. After washing with 70~ ethanol and drying, the
precipitate was taken up in 50 ~1 of water.
To detect the specific expre~6ion of the ribozyme-encod-
ing gene, samples of about 4 ~g of total R~A from a non-
transformed wild-type tomato plant and from a transgenic
tomato plant were applied to a 1% agarose gel with 6%
formaldehyde. For the application, the ~NA was dried,
taken up in 50% formamide/6~ formaldehyde and heated at
60C for 15 minutes. The agarose gel was briefly washed
with H20 after the run and transferIed with 10 x SSC to a
Gene Screen Plus membrane. After 24 hours, the membrane
was washed with 2 x SSC, incubated at 80C for 2 h ~nd
dried.
Hybridization was carried out after 2 hour~' prehybridi-
zation with 1~ SDS, 1 M NaCl and 10~ dextran sulfate at
55C with radiolabeled oligonucleotide c) of the
ripening-related protein DNA.
Detection of the ribozyme-encodinla gene directed against
~he ripening-related protein RN~.
2 small pieces of leaf from he tran~genic tomato plant
or from a non-txans~ormed wild-type tomato were com-
minuted under li~uid nitrogen with a mortsr and pe~tle.
~he powder was placed in Eppendor~ tube~ and m~xed with
500 ~1 of 2 x CTAB buffer (2 x C~AB- 2% cetyltrLmethyl-
ammonium bromide, 100 mM Tris pH 8.0, 20 mM ED~, 1.4
NaCl, 1% polyvinylpyrrolidone ~W = 40,000) which h~d
previously been haated ~t 65C. Then 500 ~1 o
chloroform/isoamyl alcohol 24:1 (~/v) were added, and the
aqueous phase was ext~acted. The t~o phases were then
~eparated by centrifuga~ion. Ths aqueous phase was
pipetted into a new Eppendorf tube, and 100 ~1 of 5% CTAB
which had been heated at 65C wer~ added. Another cxtrac-
tion with chloroform~ieoamyl alcohol was c~rried ou~
7 ~J ~
13 -
before 500 ~1 o CTAB precipitating buffer (1% C~AB,
50 mM Tri~ pH 8.0, 10 mM EDTA) were added to the aqueous
phase. After cen~rifugation, ~he precipitate was dis-
solved in high-~alt TE (10 mM Tris pH 8.0, 1 m~ EDTA, 1
NaCl~ and precipitated with 2 1/2 volumes of ethanol.
After centrifugation, wa~hing and drying, th~ precipitate
was ~aken up in water and treated with 25 ~g/ml RNase A
(final concentration). ~he RNase was subsequently removed
by phenol treatment. ~fter renewed drying, the DNA was
taXen up in S0 ~1 of water.
Then 4 ~g of the DNA were hydrolyzed with the restriction
endonuclease Eco RI at 37C for 1 hour. The hydrolyzate
was fractionated on a 1% agarose gel. The gel was ~haXen
wi~h 0.4 N NaOH/0.6 M NaCl ~or 30 min and then with 0.5
M Tris Cl pH 7.5/1.5 ~ NaCl for 30 min. The si~e standard
used was a PstI hydrolyzate of ~-phage DNA.
The DNA was then trans~erred to a Gene Screen Plus
membrane with 10 x SSC via a cap.Lllary blot. The filter
was then dried and prehybridized with 1% SDS/l M NaCl/10~
dextran sulfate. Por the hybridization, the prehybridiza-
tion mix was mixed with a radiolabeled ~ample of
oligonucle~tide c) which had prelriously been boiled for
10 minutes.
~he ribozyme-encoding ~ene was detected by the appearance
of blackenin~ at about 0.~ kb on the X-r~y film placed on
top.
Detection of the in vitro activity of the ribozymes
The oligonucleotide which encoded a ribo~yme against the
ripening-related protain RNA was cloned into the Blue-
script ~ector which had been opened after hydrolysi~ withthe restric*ion Pndonucleases XbaI and ~tI. In parallel
with thisl a DNA fragment of the ripening-related pxotein
(nucleotide numbers 792-815 of DNA ~equence publiRhed by
Ray, ~. et al., ~ucleic Acids Res. 1~, 10587 ~1987)) was
~7
- 14 -
cloned into the SacI/RpnI cleavage ~ite in the same
vector. Both vectors were used in an in vitro R~A poly-
merase reaction a5 DNA templates for the RNA sy~thesis.
For this, in parallel, the vector carrying the ribozyme
gene and ~he vector c~rrying the DN~ fragment of the
ripening related protein gene were ~ut with the r2s~ric-
tion endonuclea~e SacI. 1 ~g ~amples of the opened
vectors were then incubated with 10 ~mol of each of the
nucle~tides ATP, GTP, CTP, U~P and (~-32P)UTP (5 . . . ) i~
50 mM ~EPES (p~ 7.5) and 10 U of T7 RNA polymerase at
37DC for 30 minutes. The ~NA was, after DNase treatment,
precipita~ed in ethanol.
The synthesized RNA of the vector carrying the ribozyme
gene and the RNA of the vector carryin~ the ripening-
related protein gene ~ragment were incubated together in50 mM Tris.Cl (pH 7.8) and 10 mM MgCl2 at 25C for
~ hours. The reaction products were then separated on a
5% denaturing polyacrylamide gel (8 M urea) and iden-
tified by autoradiography on an X-ray film. The auto-
radiogram shows that, in the presence of ribozyme RNA,the RNA transcript of the ripening-related protein gene
~ragment was cleaved.
Detection of the delayed ripening of transgenic tomatoes
Comparison of a non-tran~formed wild-type toma~o plant
with a transgenic tomato plant ~hich ~arrie~ the
ribozyme-encoding ~ene dire~ted against the ripening-
related protein RNA rsvealed that the to~atoes from the
transgenic plant ripened several days later. The rib~zyme
activity and the delay in ripening asfiociated therewith
has thu~ also been detected in the tomato fruit, ~hich
r presents the actual site of a~tion.
