Recombinant DNA comprises promoter and terminator base sequences respectively upstream and downstream of an inverted base sequence complementary to a substantial sequence of bases in polygalacturonase mRNA. The antisense mRNA produced thereby delays softening of fruit, in particular tomatoes.

A very convenient source of DNA for use as the base sequence for 
transcription is provided by DNA that gives rise to polygalacturonase 
mRNA. 
The required antisense DNA can be obtained by cutting with restriction 
enzymes an appropriate sequence of such (mRNA-polygalacturonase producing) 
DNA; and then cloning the cut DNA into a vector containing upstream 
promoter and downstream terminator sequences, the cloning being so carried 
out that the cut DNA sequence is inverted with respect to its orientation 
in the strand from which it was cut. 
In the new vector, the strand that was formerly the template strand becomes 
the coding strand, and vice versa. The new vector will thus produce RNA in 
a base sequence which is complementary to the sequence of 
polygalacturonase mRNA. Thus, the two RNA strands are complementary not 
only in their base sequence but also in their orientations (5' to 3'). 
As source of the polygalacturonase DNA base sequence for transcription, it 
is convenient to use a cDNA clone such as pTOM6. The base sequence of 
pTOM6 is set out in FIG. 1. A cDNA clone such as pTOM6 may be obtained 
from the mRNA of ripening tomatoes by the method described by Slater at 
al, Plant Molecular Biology 5, 137-147. In this way may be obtained 
sequences coding for the whole, or substantially the whole, of the mRNA 
that is translated into PG. Suitable lengths of the cDNA so obtained may 
be cut out for use by means of restriction enzymes. 
An alternative source of DNA for the base sequence for transcription is the 
PG gene. It differs from that in the cDNA of, eg. pTOM6, in that introns 
are present. The introns are not transcribed into mRNA (or, if so 
transcribed, are subsequently cut out). The major part of the PG gene has 
been deposited as gTOM23 with the National Collections of Industrial and 
Marine Bacteria, Aberdeen, under Accession No. 12373. When using the PG 
gene as the source of the base sequence for transcription it is preferred 
to use primarily exon regions. 
A further way of obtaining a suitable DNA base sequence for transcription 
is to synthesize it ab initio from the appropriate bases, for example 
using FIG. 1 as a guide. 
Recombinant DNA and vectors according to the present invention may be made 
as follows. A suitable vector containing the desired base sequence for 
transcription (for example PTOM6) is treated with a restriction enzyme to 
cut the sequence out. The DNA strand so obtained is cloned (in reverse 
orientation) into a second vector containing the desired promoter sequence 
(for example CAMV 35S or the PG gene promoter sequence) and the desired 
terminator sequence (for example the 3' end of the nopaline synthase gene, 
the nos 3' end). 
It is often preferred, in applying the invention, to use the promoter of 
the PG gene. Use of this promoter, at least in tomatoes, has the advantage 
that the production of antisense RNA is under the control of the same 
system that controls PG mRNA. Thus whatever factors tend to produce the 
latter will tend at the same time to produce the former to interfere with 
it. 
Vectors according to the invention may be used to transform plants as 
desired, to make plants according to the invention. Dicotylodenous plants, 
such as the tomato, may be transformed by Ti plasmid technology, for 
example as described by Bevan (1984) Nucleic Acids Research, 12, 8711-721. 
Such transformed plants may be reproduced sexually, or by cell culture. 
The degree of production of antisense RNA in the plant cells can be 
controlled by suitable choice of promoter sequences, or by selecting the 
number of copies, or the site of integration, of the DNA sequences 
according to the invention that are introduced into the plant genome. In 
this way, for example, it may prove possible to delay softening of 
tomatoes for a greater or lesser period after ripening. 
The following Examples and Experiments illustrate the invention and how to 
carry it out. Examples relate to the manufacture of vectors according to 
the invention: Experiments relate to preparation of starting materials. 
All cloning procedures are performed under standard conditions as 
described by Maniatis et al (1982) "Molecular Cloning", Cold Spring Harbor 
Laboratory. Vectors for which an NCIB Accession number are given have been 
deposited at the National Collections of Industrial and Marine Bacteria, 
Torry Research Station, PO Box 31, 135 Abbey Road, Aberdeen AB9 8DG, 
Scotland. 
EXPERIMENT 1 
Construction of the plasmid pPH1 
A. Isolation of the nos 3' end. 
10 .mu.g of pWRECK2-CAMV-CAT-1 (NCIB Accession No. 12352) is digested with 
PvuI in order to linearize the DNA, under conditions recommended by the 
manufacturer. The completeness of digestion is analyzed by running an 
aliquot of the reaction of 0.8% agarose gels. The reaction is stopped by 
extraction with phenol/chloroform. DNA is precipitated with ethanol and 
dried under vacuum. The cohesive ends are removed by incubation of the 
linearized DNA with T4 polymerase at 37.degree. C. for 30 minutes. The 
enzyme is inactivated by incubation at 65.degree. C. for 15 minutes. The 
reaction volume is increased by the addition of HindIII buffer and HindIII 
enzyme is added. The reaction is carried out for 2 hours at 37.degree. C. 
The 250 bp PvuI/HindIII fragment is isolated from agarose gels by 
electroelution. DNA is phenol/chloroform extracted, precipitated with 
ethanol and resuspended in water. 
B. Linearization of pUC18. 2 .mu.g of pUC18 (plasmid commercially available 
eg. from Amersham) DNA is digested with Sphl under conditions recommended 
by the manufacturer. The reaction is stopped by extraction with 
phenol/chloroform. Cohesive ends are removed by treatment with T4 
polymerase for 30 minutes at 37.degree. C. The buffer volume is increased, 
and HindIII is added. The mixture is incubated for 2 hours at 37.degree. 
C. The reaction is stopped by extraction with phenol/chloroform. DNA is 
precipitated with ethanol and resuspended in water at 100 ng/ml. 
C. Cloning of nos. 3' end into pUC18 to give pNOS1. 
1 .mu.l of pUC18 prepared under (B) is ligated with 100 ng of nos 3' end 
prepared under (A) in a total of 15 .mu.l in the presence of T4 ligase. 
Incubation is carried out for 24 hours at 16.degree. C. An aliquot of the 
ligation is transformed into competent TG2 cells. An aliquot of the 
transformation mix is plated onto ampicillin and Xgal containing plates. 
White colonies are picked, and the DNA examined by restriction analysis. 
Molecules containing the nos 3' end are characterized by the presence of a 
260 base pair HindIII/BamHl fragment. These plasmids are called PNOSl. 
D. Preparation of the CaMV 35S promoter 
The CaMV promoter is obtained by digestion of pWRECK2-CAMV-CAT-1 (NCIB 
Accession No. 12352) with Scal for 2 hours at 37.degree. C. An aliquot of 
the restriction digest is analyzed by electrophoresis on agarose gels. The 
reaction is stopped by extraction with phenol/chloroform. After ethanol 
precipitation and resuspension in water, the DNA is cut with Hphl for 2 
hours at 37.degree. C. The cohesive ends are removed by treatment with T4 
polymerase under standard conditions. The CaMV promoter 629 base pair 
fragment is isolated by agarose gel electrophoresis and subsequent 
electroelution. 
E. Linearization of pNOSl 2 .mu.g of pNOS1 is cut with Sstl at 37.degree. 
C. for 2 hours. After completion of the reaction, T4 polymerase is added 
in order to remove the cohesive ends. The reaction is stopped by 
extraction with phenol/chloroform. Then DNA is precipitated with ethanol 
and resuspended in water at 100 ng/.mu.l. 
F. Cloning of CaMV 35S promoter into pNOS1 
PNOS1 Prepared as under (E) is ligated to CaMV35S promoter fragment 
prepared under (D) under standard conditions using T4 ligase. The reaction 
is carried out for 24 hours at 16.degree. C. An aliquot of the ligation 
mixture is transformed into competent TG2 cells, and Plated onto 
ampicillin containing Xgal plates. DNA is isolated from transformants and 
analyzed by restriction with NcoI and HindIII. Molecules containing the 
CaMV 35S promoter in the correct orientation are characterized by the 
presence of a 920 base pair fragment. These plasmids are called pPH1. 
EXPERIMENT 2 : Preparation of Plasmid pCB1 
A. Isolation of a PG promoter fragment 
Genomic clones are isolated from a partial Sau3A library of Ailsa Craig 
tomato DNA cloned into EMBL3 (Bird et al, in preparation). PG clones are 
isolated from the genomic library by screening with both the complete 
PTOM6 cDNA insert, and the isolated 5' Pstl/HindIII fragment from PTOM6 
(Grierson et al, NAR 1986). Several overlapping clones are isolated and 
the transcription start site of the PG gene located by S1 mapping 
experiments (Bird et al in preparation 1987). The pG promoter can be 
located on a 1.6 Kb HindlII fragment which also contains part of the PG 
coding information. 
B. Insertion of a SpeI site into the PG promoter 
In order to be able to manipulate the PG promoter sequence conveniently 
(ie. the DNA 5' to the transcription start) a Spel site is introduced by 
site directed mutagenesis using standard protocols. The HindIII fragment 
is isolated from genomic clone gTOM23 (NCIB Accession No. 12373), and 
cloned into the HindIII site of (commercially available vector) M13 mp19. 
After annealing with a suitable mismatch primer and extension using DNA 
polymerase, the mixture is transformed into competent TG2 cells. Phages 
are plated and duplicated filters were prepared for hybridization to the 
labelled mismatch primer. Putative clones are identified by hybridization 
under increasingly stringent conditions, isolated and the generation of 
the Spel site is determined by direct DNA sequence analysis. The promoter 
fragment is isolated from one isolate by restriction with Spel and 
HindIII. This fragment is then cloned into pUC19 (commercially available 
plasmid) cut with HindIII and XbaI. The promoter fragment is then 
transferred into Bin19 (Bevan, Nucleic Acid Research, 1984, 12, 8711-8721) 
cut with BamHl and HindIII. This plasmid is called pCB1. 
EXPERIMENT 3: Preparation of Plasmid pPH2 
A. Isolation of the PG promoter fragment from pCB1 
5 .mu.g of pCBl (prepared as in Experiment 2) is cut with HindIII for 2 
hours at 37.degree. C. The mixture is phenol/chloroform extracted and DNA 
precipitated with ethanol. After re-suspension in water the cohesive ends 
are filled in using DNA polymerase under standard conditions at room 
temperature for 15 minutes. The polymerase is inactivated by heating to 
65.degree. C. for 15 minutes. The DNA is then treated with BamHl for 2 
hours at 37.degree. C. The PG promoter fragment is then by electroelution 
isolated by agarose gel electrophoresis as a HindIII/BamHl 1.45 Kb 
fragment. 
B. Preparation of pPHl for insertion of the PG promoter fragment 
5 .mu.g of pPHI (prepared as in Experiment 1) is cut with Ncol for 2 hours 
at 37.degree. C. under standard conditions. The DNA is purified by 
phenol/chloroform extraction. The cohesive ends are filled in using DNA 
polymerase I Klenov fragment for 15 minutes at room temperature. The 
volume is increased and BamHI added. The mixture is incubated for 2 hours 
at 37.degree. C. The mixture is then separated on agarose gels, and the 
large fragment of approximately 3 Kb isolated by electroelution. 
C. Cloning of the PG promoter into the large fragment from pPH1. 
10 .mu.g of pPHl prepared as in Experiment 1 is ligated with the PG 
promoter fragment as prepared in A under standard conditions for 24 hours 
at 16.degree. C. An aliquot of the ligation mixture is used to transform 
competent TG2 cells. Aliquots of the transformation mixture are plated 
onto L plates containing ampicillin and Xgal. Colonies are picked and 
examined for the presence of the PG promoter DNA by electrophoresis to 
detect an increase in the size of the vector and by direct DNA sequence 
determination. This plasmid is called pPH2. 
EXAMPLE 1: Preparation of Plasmids pJR10 and pJR11 
A. Preparation of the antisense DNA 
A 730 base pair Hinfl fragment covering the 5' untranslated region, the 
putative leader sequence, and a substantial portion of the PG coding 
region is isolated from pTOM6. 5 .mu.g pTOM6 is restricted with Hinfl for 
2 hours at 37.degree. C. The reaction is stopped by extraction with 
phenol/chloroform, and ethanol precipitated. Cohesive ends are removed by 
treatment with T4 polymerase under standard conditions. DNA is purified by 
phenol/chloroform extraction and ethanol precipitation. 
B. Cloning of the antisense DNA into pPHl 
The DNA fragment as isolated in Example 1.A is cloned into the Smal site of 
pPHl. pPH1 DNA is restricted with Smal under standard conditions for 2 
hours at 37.degree. C. After incubation for 2 hours bacterial alkaline 
phosphatase is added in order to prevent self-ligation of pPHl during 
subsequent cloning steps. The reaction is stopped by extraction with 
phenol/chloroform. DNA is precipitated and resuspended in water. An 
aliquot of the DNA as prepared in (A) is ligated under standard conditions 
to Smal cut pPHl. Aliquots of the ligation mixture are transformed into 
competent TG2 cells, and plated onto ampicillin containing plates. 
Recombinants are analyzed by restriction digestion with HindIII. Both 
antisense (pPH10) and "sense" (pPH11) constructs are isolated. 
EXAMPLE 2 : Preparation of Plasmids pPH20 and pPH21 
A. Cloning of the antisense DNA into pPH2 
DNA as isolated in Example 1A is cloned into pPH2 cut with HincII. The 
plasmid containing the PG sequence in the antisense orientation is called 
pPH20, the plasmid containing the PG DNA in the sense orientation is 
called pPH21. 
EXAMPLE 3 : Transformation of Tomato Plants 
Transformation of tomato plants is achieved using a modification of the 
leaf disc transformation protocol published by Bevan et al, EMBO Journal 
4, 1921-1926, 1985. Transformed tomato plants are analyzed for the 
presence of the antisense constructs by Southern hybridization to genomic 
DNA. Expression of sense and antisense RNA is monitored by dot blot and 
Northern hybridizations. Firmness of the fruit is investigated using 
general physiological methods. The presence of antisense PG constructs in 
the cells of the ripening tomatoes is associated with firmness in the 
tomato being maintained for a longer period. 
There follows a second series of Examples and Experiments which have been 
carried out according to the same general scheme. 
EXPERIMENT 11 
Construction of the plasmid pJR1 
A. Isolation of the nos. 3' end 
This was carried out according to the method of Experiment 1 above. 
B. Removal of the CaMV 3' end from pDH51 
2 .mu.g of pDH51 (Pietrzak et al, (1986) Nucleic Acids Research 14, 
5857-5868) was digested with SphI at 37.degree. C. for hours under 
standard conditions. The reaction was stopped by extraction with 
phenol/chloroform. DNA was precipitated with ethanol and resuspended in 
water. Cohesive ends were removed by treatment with T4 polymerase for 30 
minutes at 37.degree. C. The buffer volume was increased, and HindIII was 
added. The mixture was incubated for 2 hours at 37.degree. C. The 
resulting 3.2 Kb fragment was isolated after gel electrophoresis on 
agarose gels by electroelution. The DNA was extracted with phenol and 
chloroform, precipitated with ethanol and resuspended in water. 
C. Cloning of nos 3' end into pDH51 to give pJR1 
1 .mu.l of pDH51 prepared under (B) was ligated with 100 ng of nos 3' end 
prepared under (A) in a total of 15 .mu.l in the presence of T4 ligase. 
Incubation was carried out for 24 hours in 16.degree. C. An aliquot of the 
ligation was transformed into competent TG2 cells. An aliquot of the 
transformation mix was plated onto ampicillin and Xgal containing L- 
plates. White colonies were picked, and the DNA examined by restriction 
analysis. Molecules containing the nos 3' end were characterized by the 
presence of a 260 base pair HindIII - BamHI fragment. These plasmids were 
called pJR1. 
EXPERIMENT 12 
Construction of plasmids pDHCl and pDHC4 
A. Isolation of a 730 bp HinfI fragment from pTOM6 
PTOM6 (NCIB accession No. 12351) was treated with HinfI for 2 hours at 
37.degree. C. under standard conditions. The 730 bp HinfI fragment (FIG. 
3) was isolated after separation on agarose gels. The cohesive ends of 
this fragment were filled in with DNA polymerase Klenov fragment 
A. The DNA was phenol extracted and ethanol precipitated. 
B. Linearization of pDH51 
1 .mu.g pDH51 was treated with SmaI for 2 hours at 37.degree. C. under 
standard conditions. The reaction was stopped by phenol extraction. The 
linearized vector was then precipitated with ethanol, washed and 
resuspended in water. 
C. Cloning of the pTOM6 HinfI fragment into pDH51 
The isolated HinfI fragment from pTOM6 (A) and the linearized vector (B) 
were ligated overnight under standard conditions. The ligation mix was 
used to transform competent TG2 cells. The transformation mix was plated 
onto ampicillin-containing plates. Clones were selected, DNA isolated and 
analyzed by digestion with BamHI and HindIII restriction enzymes. Plasmids 
were identified, and were named pDHC1 and pDHC4. pDHC1 contains the HinfI 
fragment in the antisense orientation; pDHC4 contains the HinfI fragment 
in the sense orientation. 
EXPERIMENT 13 
Construction of plasmid pCBl 
A. Isolation of a PG promoter fragment This was carried out as described in 
Experiment 
B. Insertion of a Spel site into the PG promoter fragment. 
This was carried out as described in Experiment 2B above. The resulting 
plasmid was called PCB1. 
EXPERIMENT 14 
Construction of plasmid pJR2 
A. Isolation of the PG promoter fragment from pCBl 
This was carried out as described in Experiment 3A. 
B. Preparation of pJR1 for insertion of the PG promoter fragment. 
5 .mu.g of pJRI (constructed in Experiment 11) was cut with NcoI for 2 
hours at 37.degree. C. under standard conditions. The DNA was purified by 
extraction with phenol and chloroform. The cohesive ends were filled in 
using DNA polymerase I Klenow fragment A for 15 minutes at room 
temperature. The volume was increased and BamHI added. The mixture was 
incubated for 2 hours at 37.degree. C. The mixture was then fractionated 
on agarose gels, and the large fragment of approximately 3 kb isolated by 
electroelution. 
C. Cloning of the PG promoter into the large fragment from pJR1 
PJR1 prepared as in B above was ligated with the PG promoter fragment 
prepared in A under standard conditions for 24 hours at 16.degree. C. An 
aliquot of the ligation mixture was used to transform competent TG2 cells. 
Aliquots of the transformation mixture were plated onto L plates 
containing ampicillin and Xgal. Colonies were picked and examined for the 
presence of the PG promoter DNA by electrophoresis on agarose gels in 
order to detect an increase in the size of the vector and by direct DNA 
sequence determination. Plasmids containing the PG promoter were called 
pJR2. 
CONSTRUCTION OF ANTISENSE AND SENSE pG VECTORS 
A series of antisense and sense (control) vectors containing different 
portions of the pG cDNA and pG gene were constructed for use in 
regenerating transgenic plants. The vectors produced are summarized in 
Table 1. The vectors constructed are based on pJR1 and pJR2. DNA fragments 
have been inserted into these vectors both into the antisense (A) and 
sense (B) orientations. Expression cassettes contained in these vectors 
were then transferred to Bin19 (Bevan (1984) Nucleic Acids Research, 12, 
8711-8721) for transformation of tomato plants. 
TABLE 1 
______________________________________ 
Name of 
Vectors Name of antisense 
sense PG fragment 
based on 
vector vector (see FIG. 2) 
______________________________________ 
pJR1 pJR16A pJR16S 740 bp HinfI 
pJR36A pJR36S fragment a 
pJR56A pJR56S fragment b 
pJR76A pJR76S fragment c 
pJR2 pJR26A pJR26S 740 bp HinfI 
pJR46A pJR46S fragment a 
pJR66A pJR66S fragment b 
pJR86A pJR86S fragment c 
______________________________________ 
EXAMPLE 10 
Construction of PG antisense vectors pJR16A and pJR16S 
pJR16A 
A. Isolation of a 740 bp pG antisense fragment 5 g pDHCl was cut with KPnI 
and PstI at 37.degree. C. for 2 hours under standard conditions. The 740 
bp KpnI - PstI fragment was isolated after agarose gel electrophoresis by 
electroelution. The fragment was extracted with phenol and chloroform and 
ethanol precipitated. The fragment was then resuspended in 10 .mu.l TE. 
B. Preparation of pJR1 
1 .mu.g pJR1 (from Experiment 14) was cut with KpnI and PstI at 37.degree. 
C. for 2 hours. The reaction was stopped by extraction with phenol and 
chloroform. The DNA was precipitated with ethanol, washed and dried. The 
vector was resuspended in 20 .mu.l TE. 
C. Ligation of the PG antisense fragment and pJR1 
The products of (A) and (B) above were ligated at 16.degree. C. for 24 
hours under standard conditions. The ligation was used to transform 
competent TG2 cells and the mixture was plated onto ampicillin-containing 
plates to select transformed cells. Single colonies were grown up to 
prepare plasmid DNA. The DNA was analyzed for the presence of 500 bp 
HindIII fragment. A clone containing this fragment was identified and 
called pJR16A. 
pJR16S 
D. Isolation of a 740 bp PG sense fragment 5 .mu.g pDHC4 was cut with KpnI 
and PstI at 37.degree. C. for 2 hours under standard conditions. The 740 
bp fragment produced was isolated after agarose gel electrophoresis by 
electroelution. The fragment was extracted with phenol and chloroform and 
precipitated with ethanol. The fragment was then suspended in 10 .mu.l TE. 
F. Ligation of the PG sense fragment to pJRI 
The products of (D) were ligated at 16.degree. C. for 24 hours under 
standard conditions. The ligation mix was used to transform competent TG2 
cells and the mixture was plated onto ampicillin containing plates. Single 
colonies were grown up to prepare plasmid DNA. The DNA was analyzed for 
the presence of a 900 bp HindIII fragment. A suitable clone was identified 
and called pJR16S. 
EXAMPLE 11 
Transfer of pJR16A and pJR16S to Bin 19 
A. Isolation of the 1600 bp expression cassettes 
pJR16A and pJR16S were cut with EcoRI at 37.degree. C. for 2 hours under 
standard conditions. An aliquot of the reaction mixture was separated by 
agarose gel electrophoresis to check that the reaction had gone to 
completion. It was then heated to 65.degree. C. for 15 minutes in order to 
inactivate the enzyme. The DNA was then cut partially with a small amount 
of HindIII in order to give all the possible EcoRI/HindIII partial 
digestion fragments. The EcoRI - HindIII fragment of approximately 1600 bp 
consisting of the 35S CaMV promoter, the PG insert sequences and Nos 3' 
end (expression cassette) was isolated after agarose gel electrophoresis 
by electroelution. The fragment was extracted with phenol and chloroform, 
and precipitated with ethanol. The fragment was washed, dried and 
resuspended in 10 .mu.l TE. 
B. Preparation of Bin19 for cloning 
5 .mu.g Bin19 DNA was cut with EcoRI and HindIII at 37.degree. C. for 2 
hours under standard conditions. The reaction was stopped by phenol and 
chloroform extraction, and DNA was precipitated with ethanol. The vector 
prepared in this fashion was resuspended in 20 .mu.l. 
C. Ligation of Bin19 to PG expression cassettes 
The products of (A) and (B) were set up for ligation. Aliquots of the PG 
antisense and sense cassettes were ligated to Bin19 at 16.degree. C. for 
24 hours under standard conditions. The ligation mixes were used to 
transform competent TG2 cells which were plated on L agar containing 
Kanamycin and Xgal. Recombinant colonies were identified by their white 
color. A number of these were picked from each ligation reaction and used 
to prepare plasmid DNA. The DNA was analyzed for the relevant restriction 
pattern by cutting with EcoRI and HindIII. 
EXAMPLE 12 
Construction of PG antisense vectors pJR26A and pJR26S 
pJR26A 
A. Isolation of a 740 bp PG antisense fragment 
5 .mu.g PDHCl were cut with KpnI at 37.degree. C. for 2 hours under 
standard conditions. The cohesive ends of the molecule were filled in with 
T4 DNA polymerase under standard conditions. The reaction was stopped by 
heating at 65.degree. C. for 15 minutes. The DNA was then also cut with 
PstI at 37.degree. C. for 2 hours under standard conditions. The resulting 
740 bp KpnI (blunt) - PstI fragment was isolated after agarose gel 
electrophoresis by electroelution. The fragment was extracted with phenol 
and chloroform, and precipitated with ethanol. The fragment was then 
resuspended in 10 .mu.l TE. 
B. Preparation of pJR2 
1 .mu.g PJR2 (from Experiment 14) was cut with HincII and Pstl at 
37.degree. C. for 2 hours under standard conditions. The reaction was 
terminated by extraction with phenol and chloroform and precipitated with 
ethanol. The purified vector was resuspended in 20 .mu.l TE. 
C. Ligation of PG fragments to pJR2 
The products of (A) and (B) above were ligated at 16.degree. C. for 24 
hours under standard conditions. The ligation mix was used to transform 
competent TG2 cells. The transformation mix was plated onto 
ampicillin-containing plates and incubated at 37.degree. C. overnight. 
Transformed colonies were grown up and plasmid DNA was prepared for 
analysis. A clone was identified which contained a 2 Kb EcoR1 - Hindlll 
insert. This clone was called pJR26A. 
pJR26S 
D. Isolation of the 740 bp PG fragment 
5 .mu.g pDHC4 were cut with KpnI at 37.degree. C. for 2 hours under 
standard conditions. The cohesive ends of the DNA were filled in with T4 
DNA polymerase. The reaction was stopped by heating to 65.degree. C. for 
15 minutes. The DNA was then also cut with PstI. The resulting 740 bp 
fragment was isolated after agarose gel electrophoresis by electroelution. 
The fragment was extracted with phenol and chloroform and precipitated 
with ethanol. It was then resuspended in 10 .mu.l TE. 
E. Ligation of the PG sense fragment to pJR2 
The products of (B) and (D) above were ligated at 16.degree. C. for 24 
hours under standard conditions. The ligation mix was used to transform 
competent TG2 cells, plated onto ampicillin containing plates and 
incubated at 37.degree. C. overnight. Transformed single colonies were 
grown up and plasmid DNA was prepared. The DNA was analyzed for the 
presence of a 1.6 Kb EcoRI - HindIII fragment. A clone was identified and 
called pJR26S. 
EXAMPLE 13 
Transfer of pJR26A and pJR26S to Bin19 
A procedure essentially the same as described above in Example 11 was used 
to subclone the 2.6 Kb EcoRI - HindIII partial fragments from pJR26A and 
pJR26S into Bin19 cut with EcoR1 and HindIII. Recombinants were identified 
by their white color reaction after plating onto L-agar plates containing 
kanamycin and Xgal. Recombinants were characterized by restriction 
digestion with EcoR1 and HindIII. 
EXAMPLE 14 
Construction of vectors pJR36A, pJR36S (fragment a) and pJR46A and pJR46S 
(fragment b) 
A. Isolation of fragments (a) and (b) 
5 .mu.g pDHC4 (from Experiment 12) was cut with KpnI and BamHI at 
37.degree. C. for 2 hours under standard conditions. The 500 bp fragment 
was isolated after agarose gel electrophoresis by electroelution, 
extracted with phenol, chloroform and resuspended in 20 l TE. The KpnI - 
BamHI fragment was then cut with HindIII. The cohesive ends of the 
fragment were filled with T4 DNA polymerase. The resulting fragments: a) 
199 bp HindIII - KpnI (blunt ended) and b) 275 bp HindIII - BamHl (blunt 
ended) were isolated after agarose gel electrophoresis by electroelution, 
extracted with phenol and chloroform, and resuspended in 10 l TE. 
B. Preparation of pJR1 
1 .mu.g pJR1 (from Experiment 11) was cut with SmaI at 37.degree. C for 2 
hours under standard conditions. The reaction was stopped by extraction 
with phenol and chloroform, and Precipitated with ethanol. The vector was 
then resuspended in 20 .mu.l TE. 
C. Ligation of fragment (a) into pJR1 
pJR36A and pJR36S 
Fragment (a) from (A) above was ligated to SmaI cut pJR1 (from (B) above) 
at 16.degree. C. for 24 hours under standard conditions. The ligation 
mixture was used to transform competent TG2 cells which were then plated 
onto ampicillin-containing plates. Transformed colonies were grown up and 
used for plasmid DNA preparation. EcoRI/PstI double digests identified 
those clones containing fragment (a) inserts. The EcoRI - PstI inserts of 
these clones were isolated and subcloned into M13 mp8 which had been cut 
with EcoRI and PstI. DNA sequence analysis was carried out in order to 
ascertain the orientation of the insert (a). Clones obtained from this 
experiment were called pJR36A and pJR36S, according to the orientation of 
the insert. 
D. Ligation of fragment (b) into pJR1 
PJR56A and pJR56S 
Fragment (b) from (A) above was ligated to SmaI cut pJR1, from (B) above, 
at 16.degree. C. for 24 hours under standard conditions. The ligation 
mixture was used to transform competent TG2 cells which were then plated 
onto ampicillin containing plates. Transformed colonies were grown up and 
used for plasmid DNA preparation. EcoR1/PSEI double digests identified 
those clones containing fragment (b) inserts. The EcoR1 - pstl inserts of 
these clones were isolated and subcloned into M13 mp8 which had been cut 
with EcoRI and PstI. DNA sequence analysis was carried out in order to 
ascertain the orientation of the insert (b). Clones obtained from this 
experiment were called pJR56A and PJR56S, according to the orientation of 
the insert. 
EXAMPLE 15 
Transfer of pJR36A/S and pJR56A/S to Bin19 
A. Preparation of expression cassettes containing fragments (a) and (b) in 
pJR1 
5 .mu.g each of pJR36A, pJR36S, pJR56A and pJR56S were cut separately with 
EcoRI and HindIII at 37.degree. C. for 2 hours under standard conditions. 
The resulting four fragments containing (a) 930 bp and (b) 1000 bp were 
isolated separately after electrophoresis on agarose gels by 
electroelution. The fragments were extracted with phenol and chloroform, 
and precipitated with ethanol. The four fragments were then resuspended in 
10 .mu.l TE. 
B. Preparation of Bin19 
Bin19 was cut with EcoRI and HindIII for 2 hours at 37.degree. C. under 
standard conditions. The reaction was stopped by addition of phenol and 
chloroform. After extraction the DNA was precipitated with ethanol, and 
resuspended in 20 .mu.l TE. 
C. Ligation of the fragments to Bin19 
The four EcoRI - HindIII fragments isolated in A were set up for separate 
ligation reactions using Bin19 prepared as described in B under standard 
conditions. The ligation mixtures were used to transform competent TG2 
cells which were plated onto L agar containing kanamycin and Xgal. After 
incubation overnight, recombinant colonies were identified by their white 
color. A number of the clones were picked from each separate ligation and 
were used to prepare DNA. The DNA's were analyzed for the presence of a 
EcoRI - HindIII fragment of the appropriate size for the insertion of the 
expression cassettes to Bin19. 
EXAMPLE 16 
Construction of pJR46A, pJR46S (fragment a) and pJR66A, pJR66S (fragment b) 
A. Preparation of pJR2 
1 .mu.g pJR2 (from Experiment 14) was cut with HincII at 37.degree. C. for 
2 hours under standard conditions. The reaction was terminated by 
extraction with phenol and chloroform. The vector was precipitated with 
ethanol, washed and resuspended in 20 .mu.l TE. 
pJR46A and pJR46S 
B. Ligation of PG fragment (a) to pJR2 
Fragment (a) from Example 14(A) above was ligated to HincII cut pJR2 from 
(A) above at 16.degree. C. for 24 hours under standard conditions. The 
ligation mixture was used to transform competent TG2 cells which were then 
plated onto ampicillin containing plates. Transformants were picked, grown 
up and used to prepare plasmid DNA. Plasmid DNA was cut with both EcoRI 
and PstI. DNA from clones which contained inserts were restricted with 
EcoRI and PstI. The EcoRI - PstI inserts were isolated after agarose gel 
electrophoresis by electroelution and subcloned into M13mp8 which had been 
cut with EcoRI and PstI. DNA sequence analysis was used to ascertain the 
orientation of the inserts (a). Clones were obtained from this experiment 
were called pJR46A and pJR46S, according to the orientation of the insert. 
pJR66A and pJR66S 
Fragment (b) from Example 14(A) was ligated to HincII cut, from A above, 
separately at 16.degree. C. for 24 hours under standard conditions. The 
ligation mixture was used to transform competent TG2 cells which were then 
plated onto ampicillin containing plates. Transformants were picked, grown 
up and used to prepare plasmid DNA. Plasmid DNA was cut with both EcoRI 
and PstI. DNA from clones which contained inserts were restricted with 
EcoRI and PstI. The EcoRI - PstI inserts were isolated after agarose gel 
electrophoresis by electroelution and subcloned into M13 mp8 which had 
been cut with EcoRI and PstI. DNA sequence analysis was used to ascertain 
the orientation of the inserts (b). Clones were obtained from this 
experiment were called pJR66A and pJR66S, according to the orientation of 
the insert. 
EXAMPLE 17 
Transfer of pJR46A, pJR46S, pJR66A and pJR66S to Bin19 
A. Preparation of expression cassettes containing fragments (a) and (b) in 
pJR2 
5 g of each of pJR46A, pJR46S, pJR66A and pJR66S were cut separately with 
EcoRI and HindIII at 37.degree. C. for 2 hours under standard conditions. 
The resulting four fragments of approximately 2.5 kb were isolated 
separately after gel electrophoresis by electroelution. The fragments were 
extracted with Phenol and chloroform, and precipitated with ethanol. The 
four fragments were then resuspended in 10 .mu.l TE. 
B. Ligation of expression cassettes into Bin19 
Aliquots containing the four fragments from (A) were ligated to Bin19 DNA 
prepared as described in Example 15 (B) in separate ligation reactions 
under standard conditions. The ligation mixtures were used to transform 
competent TG2 cells. The transformation mixture was plated onto L-agar 
plates containing kanamycin and Xgal. After overnight incubation 
recombinant colonies were identified by their white color. A number of 
clones for the separate experiments were picked and DNA was prepared. The 
DNAs were analyzed for the presence of the appropriate EcoRI - HindIII 
fragments. 
EXAMPLE 18 
Construction of PG vectors pJR76A and pJR76S 
A. Isolation of fragment (c) 
10 .mu.g gTOM 23 (a genomic clone containing the PG gene, NCIB No 12373) 
was cut with HindIII and BamHl. The 1.98 Kb fragment was isolated after 
agarose gel electrophoresis by electroelution. The cohesive ends of the 
fragment were filled in with T. DNA polymerase. 
B. Ligation of fragment (c) to pJR1 
The products from (A) above and Example 14 (B) (ie. pJR1 cut with SmaI) 
were ligated at 16.degree. C. for 24 hours under standard conditions and 
the mixture used to transform competent TG2 cells which were then plated 
onto plates containing ampicillin. Transformed colonies were grown up and 
used to prepare plasmid DNA. The DNA was cut with EcoRI and the 
orientation of the insert determined from the pattern of fragments 
obtained. The clones were called pJR76A and PJR76S according to the 
orientation of the insert. 
EXAMPLE 19 
Transfer of vectors pJR76A and pJR76S to Bin19 
A. Preparation of expression cassettes from pJR76A and pJR76S 
5 .mu.g of each clone was cut with HindIII at 37.degree. C. for 2 hours 
under standard conditions. The enzyme was inactivated by heating the 
reaction mixture to 70.degree. C. for 15 minutes. EcoRI was then added in 
concentration necessary to give partial restriction. The reactions were 
stopped by the addition of phenol and chloroform. The required 2.71 Kb 
EcoRI - HindIII fragments were isolated after agarose gel electrophoresis 
by electroelution. The fragments were extracted with phenol and chloroform 
and precipitated with ethanol. The fragments were then resuspended in 10 
.mu.l TE. 
B. Ligation of the expression cassettes to Bin19 
The two fragments from vectors pJR76A and pJR76S prepared in (A) were 
ligated separately to Bin19 (prepared as described in Example 11 B). The 
ligation mixture was used to transform competent TG2 cells. The 
transformation mix was plated onto L plates containing kanamycin and Xgal. 
Recombinant plasmids were identified by their white color. DNA was 
prepared from a number of these and analyzed for the presence of the 
required EcoR1 - HindIII fragments. 
EXAMPLE 20 
Construction of PG vectors pJR86A and pJR86S 
A. Ligation of PG fragment (c) to pJR2 
The products of Example 18 (A) fragment (c) and Example 16 (A) (i.e. pJR2 
cut with HincII) were ligated at 16.degree. C. for 24 hours under standard 
conditions and the mixture used to transform competent TG2 cells which 
were then plated on plates containing ampicillin. Transformed colonies 
were grown up and used to prepare plasmid DNA. The orientation of the 
insert was deduced using the EcoR1 restriction pattern. These clones were 
called pJR86A and pJR86S, according to the orientation of the insert. 
EXAMPLE 21 
Transfer of vectors pJR86A and pJR86S into Bin19 
This Example was carried out essentially as described in Example 19, i.e. 
the vectors were cut with HindIII under conditions of partial restriction, 
which was then followed by restriction with EcoR1. The resulting 3.63 Kb 
fragment was isolated and cloned into Bin19. 
All constructs in Bin19 were intended for use in separate triparental 
mating experiments to allow transfer to Agrobacterium, and from there to 
tomato plants. 
INHIBITION OF PECTIN ESTERASE 
In addition to polygalacturonase, pectin esterase (PE) has been implicated 
in softening of the tomato fruit. A ripe tomato fruit cDNA library was 
screened with mixed oligonucleotide probes designed from the published 
amino acid sequence of PE. One clone, pPE1, (NCIB Accession No. 12568) has 
been isolated and characterized. FIG. 3 shows the complete sequence of 
this cDNA clone. The deduced amino acid sequence of PE is substantially 
different from the sequence published by Markovic and Jornvall. 40 amino 
acid differences are found in the sequence of the mature PE protein; major 
rearrangements in the continuity of the published amino acid sequence are 
evident and an additional 11 amino acids are found in the protein 
presented here. In addition to the sequences of the mature PE, both 
N-terminal and C-terminal amino acid extensions are detected in the 
polypeptide encoded by pPE1. 
We have used fragments of the cDNA to construct antisense and sense 
vectors. These are summarized in Table 2. 
TABLE 2 
______________________________________ 
Name of 
Vectors Name of anti- 
sense 
based on sense vector vector Fragment 
______________________________________ 
pJR1 pJR101A pJR101S 420 bp PstI 
pJR111A pJR111S 351 bp BbvI 
pJR2 pJR102A pJR102S 420 bp PstI 
pJR112A pJR112S 351 bp BbvI 
______________________________________ 
CONSTRUCTION OF ANTISENSE PE VECTORS 
EXAMPLE 30 
Preparation of pJR101A and pJR101S 
A. Isolation of a 420 bp fragment from pPE1 
Plasmid pPE1 was cut with PstI at 37.degree. C. for 2 hours under standard 
conditions. The 420 bp PstI fragment was isolated after agarose gel 
electrophoresis by electroelution, extracted with phenol and chloroform 
and precipitated with ethanol. The DNA was then resuspended in 10 .mu.l 
TE. 
B. Preparation of pJR1 
pJR1 (from Experiment 11) was cut with PstI at 37.degree. C. for 2 hours 
under standard conditions. The reaction was stopped by the addition of 
phenol, precipitated with ethanol and resuspended in 20 .mu.l TE. 
C. Ligation of PE fragment to pJR1 
The products of steps (A) and (B) above were ligated under standard 
conditions and the ligation mixture was used to transform competent TG2 
cells. The transformation mix was subsequently plated onto ampicillin 
containing plates and incubated at 37.degree. C. overnight. Transformed 
colonies were grown up and used to prepare plasmid DNA. Clones were 
identified which gave 420 bp fragment on digestion with PstI. The 650 bp 
BamHI - HindIII fragments from these clones were isolated after agarose 
gele electrophoresis by electroelution and cloned into M13mp8. The 
orientation of the PstI insert was determined by sequence analysis. Clones 
identified were named pJR101A and pJR101S according to the orientation of 
the insert. 
EXAMPLE 31 
A. Isolation of a 1.2 Kb EcoR1 - HindIII fragment 
Plasmids pJR101A and pJR101S were cut separately with EcoR1 and HindIII at 
37.degree. C. for 2 hours under standard conditions. The resulting 1.2 Kb 
fragments were isolated after gel electrophoresis from agarose gels by 
electroelution. The DNA was then extracted with phenol and chloroform, 
precipitated with ethanol, and resuspended in 20 .mu.l TE. 
B. Preparation of Bin19 
Bin19 was cut with EcoR1 and HindIII for 2 hours at 37.degree. C. under 
standard conditions. The enzymes were removed by phenol extraction and the 
vector precipitated with ethanol. The DNA was then resuspended in water. 
C. Ligation of the PE expression cassette to Bin19 
Aliquots of the products of reactions A and B were ligated for 16 hours at 
16.degree. C. under standard conditions. The ligation mix was used to 
transform competent TG2 cells. The transformation mix was plated onto 
plates containing kanamycin. DNA was picked from individual clones and 
analyzed for the presence of the 1.2 Kb EcoR1 - HindIII fragment. 
EXAMPLE 32 
Construction of vectors pJR102A and pJR102S 
The construction of these vectors followed the construction of pJR101A and 
pJR101S (Example 30), except that the 420 bp Pstl PE fragment was inserted 
into pJR2 (from Experiment 14). 
EXAMPLE 33 
Transfer of vectors pJR102A and pJR102S to Bin19 
Transfer of the PE expression cassettes Bin 19 was carried out as described 
in Example 31 for the transfer of pJR102A and pJR102S into Bin19. 
EXAMPLE 34 
Construction of vectors pJR111A and pJR111S 
A. Isolation of a 351 bp fragment from pPE1 
Plasmid pPE1 was cut with BbvI at 37.degree. C. for 2 hours under standard 
conditions and the cohesive ends filled using T4 polymerase. The 351 bp 
fragment was isolated after agarose gel electrophoresis by electroelution, 
extracted with phenol and chloroform and precipitated with ethanol. It was 
then resuspended in 10 .mu.l TE. 
B. Preparation of PJR1 
1 .mu.g pJR1 (from Experiment 11) was cut with SmaI for 2 hours at 
37.degree. C. under standard conditions. The reaction was terminated by 
the addition of phenol and chloroform. After extraction the DNA was 
precipitated with ethanol, and resuspended in 10 .mu.l TE. 
C. Ligation of the PE fragment to pJR1 
The products of (A) and (B) were ligated at 16.degree. C. for 24 hours 
under standard conditions. The ligation mix was used to transform 
competent E.coli TG2 cells. The transformation mix was plated onto 
ampicillin-containing Plates. Single colonies were grown up and analyzed 
for the presence of a 900 bp EcoR1 - PstI fragment. This fragment was 
isolated by electroelution after agarose gel electrophoresis and cloned 
into M13 mp8 (commercially available vector). The orientation of the 
fragment was determined by DNA sequence analysis. 
EXAMPLE 35 
Transfer of vectors pJR111A and pJR111S to Bin 19 
A. Isolation of the 1.1 Kb EcoR1 - HindIII fragment Plamids pJR111A and 
pJR111S were cut with EcoR1 and HindIII at 37.degree. C. for 2 hours under 
standard conditions. The 1.1 Kb fragment was isolated after agarose gel 
electrophoresis by electroelution. It was extracted with phenol and 
chloroform, precipitated with ethanol and resuspended in 20 .mu.l TE. 
B. Ligation of the PE expression cassettes into Bin19. 
Aliquots of the products of (A) and Example 31 (B) were ligated at 
16.degree. C. for 24 hours. The ligation mixtures were used to transform 
competent E.coli TG2 cells. The transformation mix was plated onto plates 
containing kanamycin. Single colonies were used for DNA extraction and 
clones identified by the presence of the 1.1 Kb EcoR1 - HindIII fragment. 
EXAMPLE 36 
Construction of vectors pJR112A and pJR112S 
Construction of these vectors followed the procedure in Example 34 for the 
construction of vectors pJR111A and pJR111S except that the 351 bp BbvI 
fragment was inserted into pJR2 from Experiment 14, rather than pJR1. 
EXAMPLE 37 
Transfer of vectors pJR112A and pJR112S to Bin19 
Transfer of the PE expression cassettes from pJR112A and pJR112S into Bin19 
followed the protocol described in example 35. 
EXAMPLE 40 
Transformation of Tomato Stem Explants 
A. Transfer of Bin19 vectors to Agrobacterium 
The recombinant vectors prepared in Example 11 were mobilized from E.coli 
(TG-2) to Agrobacterium tumefaciens (LBA4404) (Hoekma A, Hirsch 
PR,Hooykaas PJJ and Schilperoort RA, 1983, Nature 303 pp 179-180) in a 
triparental mating on L-plates with E.coli (HB101) harboring pRK2013 
(Ditta G. et al, 1980 PNAS, USA, Vol 77, pp7347-7351) Transconjugants were 
selected on minimal medium containing kanamycin (50.mu.g/cm.sup.3) and 
streptomycin (500 .mu.g/cm.sup.3). 
B. Preparation of Agrobacteria for transformation 
L-Broth (5cm.sup.3) containing kanamycin at 50 .mu.g/cm.sup.3 was 
inoculated with a single bacterial colony. The culture was grown overnight 
at 30.degree. C. with shaking at 150 r.p.m. This culture (500 .mu.) was 
inoculated into L-Broth containing kanamycin (50 .mu.g/cm.sup.3) and grown 
as before. Immediately before use the Agrobacteria were pelleted by 
spinning at 3000 r.p.m. for 5 minutes and resuspended in an equal volume 
of liquid Murashige and Skoog (MS) medium. 
C. Preparation of plant tissue for transformation 
Feeder plates were prepared in 9 cm diameter petri dishes as follows. Solid 
MS medium supplemented with 5 uM zeatin riboside and 3 uM IAA aspartic 
acid was overlaid with Nicotiana tabacum var Samsun suspension culture 
(1cm.sup.3). One 9 cm and one 7 cm filter paper discs were placed on the 
surface. Hypocotyls from 4 week old seedlings grown on MS medium were 
excised and placed on feeder plates. The plates were sealed with Nescofilm 
and incubated overnight in the plant growth room (26.degree. C. under 
bright fluorescent light). 
D. Transformation Protocol 
Hypocotyls from the feeder plates were placed in the Agrobacteria 
suspension in 12 cm diameter petri dishes and cut into approximately 1 cm 
lengths, removing all leaf and cotelydon axes. After 20 minutes the 
hypocotyl segments were returned to the feeder plates which were sealed 
and replaced in the growth room. After 48 hours incubation in the growth 
room the plant material was transferred to MS medium supplemented with 5 
.mu.M zeatin riboside, 3 .mu.M IAA aspartic acid, 500 .mu.g/cm.sup.3 
carbenicillin and 50 .mu.g/cm.sup.3 kanamycin in petri dishes. The petri 
dishes were sealed and returned to the growth room. 
From six weeks after inoculation with Agrobacterium, shoots were removed 
from the explants and placed on MS medium supplemented with carbenicillin 
(200 .mu.g/cm.sup.3) for rooting. 
Transformed plants rooted 1-2 weeks after transfer. These plants were then 
grown in tissue culture for a number of weeks before being transferred to 
pots. These plants were then grown in growth rooms or greenhouses as 
appropriate. 
EXPERIMENT 40 
Analysis of Transformed Plants Produced in Example 40 
A. Analysis of leaves for the production of antisense RNA 
RNA was extracted from leaves following published procedures. RNA was 
probed for the presence of antisense RNA by dot hybridization using either 
sense or antisense specific probes. The results were considered to 
demonstrate the presence of antisense RNA in leaves from plants containing 
antisense constructs. 
B. Southern analysis Of transformed plants 
DNA was extracted from leaves of transformed plants. DNA was cut with 
various restriction enzymes, separated on agarose gels and transferred to 
nylon membranes. DNA was probed with appropriate labelled DNA fragments 
for the presence of the antisense and sense constructs. At the time of 
writing results are not to hand but a Southern blot has been obtained 
indicating that DNA from the construct JR16A has been incorporated into 
the genome of a tomato plant. 
C. Analysis of antisense RNA production in tomato fruit 
It is currently intended to extract RNA at different stages of ripening 
from transformed tomato fruit following published procedures. RNA will be 
probed with specific DNA probes for the production of antisense RNA. 
D. Effect of antisense RNA production of fruit softening 
Experiments are also planned to demonstrate how the presence of antisense 
RNA affects the process of fruit ripening.