Note: Descriptions are shown in the official language in which they were submitted.
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Y Delayed Ripenin~ Tomato Plants
BACKGROUND OF THE INVENTION
The present invention relates generally to the breeding of tomato plants.
More specifically, the invention relates to the introduction of a transgene that confers a
delayed ripening phenotype on tomato plants.
The plant hormone ethylene has a profound influence on plant physiology.
Active in trace amounts, it affects a number of processes such as fruit ripening, seed
germination, plant growth, leaf and flower senescence, pathogen infection, and the
interaction of plants with their environment. In particular, ethylene induces a number of
physio]ogical changes associated with fruit ripening such as accumulation of carotenoid
pigments, conversion of cholorplasts to chromoplasts, the increased expression of genes
encoding cell wall degradation enzymes, fruit softening and susceptibility to pathogens.
Control of the effects of ethylene is a particularly useful approach to
controlling fruit ripening in tomato. More than 80% of tomatoes (by volume) currently
sold in the United States are picked while green. Growers harvest green tomatoes for
several reasons: (1) green tomatoes are firmer, enabling them to withstand shipping and
handling witll less injury; (2) a green tomato harvest is less labor-intensive and less
costly than a vine-ripe harvest and (3) green fruit stays in the field for a shorter period of
time reducing the risk of loss from weather or pests.
After harvest, either the packer or the repacker exposes the green tomatoes
to an external source of ethy]ene gas to cause the tomatoes to develop red color. This
practice redclells the fruit, but these tomatoes will not always develop full flavor when
picked at the green stage. Fruit that have begun to ripen need to be shipped at a low
temperature to delay ripening, however these low temperat~lres decrease fruit quality.
Freq~lently, a portion of the green fmit is harvested at the imlllatllre green stage, whicll
means that it will never achieve full ripeness even with the application of ethylene. Such
tomatoes, together with other clistributioll-damaged tomatoes, do not achieve full flavor
potential and, we believe, are a major factor contributillg to cons~ er dissatisfactioll with
current fresh market lomatoes.
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In tomato (and other so called climacteric fruit), fruit ripening is associateclwith a burst of respiration and a concomitant increase in ethylene production. Once
ripening is initiated, the endogenous ethylene production rises autocatalytically.
If ethylene production could be controlled, tomato fruit could be left on
the vine longer to develop the fruit components which contribute to flavor. The present
invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides tomato plants comprising a genetic locus
having a sequence substantially identical to SEQ. ID. No. 1. The plants bear fruit that
display significant ripening impairment once the breaker stage is reached. In particular,
the fruit reach pink stage about 2 to about 3 weeks after breaker stage, when the fruit are
picked at breaker stage and stored at 15~C. Preferred plants are germinated from seed
deposited with the American Type Culture Collection under Accession No.
The invention further provides tomato fruit from the plants of the
invention. The fruit of the invention have ethylene levels less than about 1.0 nl/g/hr,
preferably less than about 0.5 nl/g/hr.
The invention also provides methods of tomato plants with decreased
ethylene production. The methods comprise crossing a parent tomato plant with a tomato
plant comprising a genetic locus of the invention. The tomato plant comprising the
genetic locus can be a plant germinated from seed deposited with American Type Culture
Collection under Accession No. . The method may furtller comprise the step
of selecting progeny bearing fmit that reach pink stage about 2 to about 3 weeks after
breaker stage, when the fruit are picked at breaker stage and stored at 15~C.
Definitions
The phrase "nucleic acid sequence" refers to a single or double-stranded
polymer of cleoxyribonllcleotide or ribonucleotide bases read from the 5' to the 3' end.
It includes both self-replicating plasmids, infectious polymers of DNA or RNA and non-
functional DNA or RNA.
The term " tomato plant" includes whole tomato plant<" tomato plant
organs (e.g., leaves, stems, roots, etc.), seeds and tolllato plant cells and progeny of
same.
A tomato plant is "derived from" seed or another plant, if it is germillaled
directly from the seed or is progeny of the plant or seecl (~.g., F" F2 (~t~.) as a result of
CA 02226088 1997-12-30
WO 971~1952 PC~/~JS96~ 96
standard sexual reproduction.
A "primary transformant" is a plant regenerated from one or more plant
cells transformed in vitro with a recombinant DNA construct.
A "heterologous sequence" is one that originates from a foreign species,
S or, if from the same species, is substantially modified from its original form. For
example, a heterologous promoter operably linked to structural gene is from a species
different from that from which the structural gene was derived, or, if from the same
species, is different from the promoter normally present with the gene, or is substantially
modified from its original form.
Two nucleic acid sequences or polypeptides are said to be "identical" if the
sequence of nucleotides or amino acid residues, respectively, in the two sequences is the
same when aligned for maximum correspondence as described below. The term
"complementary to" is used herein to mean that the sequence is complementary to all or
a portion of a reference polynucleotide sequence.
Sequence comparisons between two (or more) polynucleotides or
polypeptides are typically performed by comparing sequences of the two sequences over
a "comparison window" to identify and compare local regions of sequence similarity. A
"comparison window", as used herein, refers to a segment of at least about 20
contiguous pOSitiOllS, usually about 50 to about 200, more usually about 100 to abo~lt 150
in which a sequence may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted by the
local homology algorithm of Smith and Waterman Aclv. Appl. Math. 2: 4~2 (1981), by
the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad.
Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
These references are incorporated herein by reference.
"Percentage of seq~rence identity" is determined by comparing two
optimally aligned sequellces over a comparison window, wllereill the portion of tlle
polynucleotide sequellce in the comparison willdow may comprise additions or delctiolls
(i.e., gaps) as compared to the reference sequellce (whicll does not comprisc adc3itions or
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deletions) for optimal ~lignment of the two sequences. The percentage is calculated by
determining the number of positions at which the identical nucleic acid base or amino
acid residue occurs in both sequences to yield the number of matched positions, dividing
the number of matched positions by the total number of positions in the window of
5 comparison and multiplying the result by 100 to yield the percentage of sequence
identity.
The term "substantial identity" of polynucleotide sequences means that a
polynucleotide comprises a sequence that has at least 60% sequence identity, preferably
at least 80%, more preferably at least 90~o and most preferably at least 95%, compared
10 to a reference sequence using the programs described above (preferably BESTFIT) using
standard parameters. One of skill will recognize that these values can be appropriately
adjusted to determine corresponding identity of proteins encoded by two nucleotide
sequences by taking into account codon degeneracy, amino acid similarity, reading frame
positioning and the like. Substantial identity of amino acid sequences for these purposes
15 normally means sequence identity of at least 40%, preferably at least 60%, more
preferably at least 90%, and most preferably at least 95%.
Another indication that nucleotide sequences are substantially identical is if
two molecules hybridize to each other under stringent conditions. Stringent conditions
are sequence dependent and will be different in different circumstances. Generally,
20 stringent conditions are selected to be about 5~ C lower than the thermal melting point
(Tm) for the specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in
which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least
25 about 60~C.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l is a schematic diagram of the T-DNA region of
pWTT2 1 44/AccS .
Figure 2 is a restriction map of the T-DNA region of pWTT2144/AccS
30 - showing the location of the hybridization probes ~lsed in the genomic mapping.
Figllre 3 is a schelllatic diagram showing the Acc2 geno1ilic region and
A~c2 cDNA and the fiagments used as probes to determil1e transgene structure in the
1~45-4 transforlllallt.
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s
Figure 4 is schematic diagram showing the structure of the T-DNA insert
of the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides tomato plants (Lycopersicon esculentum)
comprising a stably incorporated locus that confers a delayed fruit ripening phenotype on
the plant. In particular, the tomato plants of the invention contain a T-DNA insert
comprising a secluence (the AccS transgene) derived from a tomato fruit-specificaminocyclopropane carboxylate (ACC) synthase gene, but that does not encode a
functional ACC synthase enzyme.
ACC synthase is the rate limiting enzyme that converts s-
adenosylmethionine to 1-aminocyclo~lo,oane-1-carboxylic acid, the immediate precursor
to ethylene. As explained in detail below, incorporation of the AccS transgene in a
tomato plant genome inhibits expression of the ACC synthase gene. Inhibition of ACC
synthase biosynthesis results in reduced levels of ethylene biosynthesis. Thus, fruit of
tomato plants comprising the transgene exhibit a delayed ripening phenotype, but ripen
normally when external ethylene is applied.
As used herein an "AccS transgene" is a truncated coding region from the
tomato Acc2 gene which is f~lsed to a 35S promoter from cauliflower mosaic virus and a
nos3' termination sequence from Agrobacterium tumef~Jciens. In particular, the sequence
from the Acc2 gene is a sequence consisting of nucleotide 149 to nucleotide 1237.
Rottman et al. J. Mol. Biol. 222:937-961 (1991).
The plants of the invention are derived, directly or indirectly, from
transformation of a tomato plant cell with a construct comprising a T-DNA having the
following regions: (1) LB, the left border region of A,~,~robc~cte~iun? T-DNA; (2) 2xpnos,
a tandem duplicate untranslated promoter region of the nopaline synthase gene from
Agr~b~let~ium, (3) nptII, the neomycin phosphotransl'erase gene from TnS; (4) ocs3',
the 3' untranslated genion of the octopine synthase gene from Agrobclclerium; (5) LacZ',
an ~Intranslated LacZ polylinker sequence; (6) p35S, the 35S promoter form cauliflower
mosaic virus (7) Cab22L, the leader sequence corresponding to the 5' untranslated region
of the Cab22R gene from petunia fused to the 35S promoter; (8) AccS, the truncated
coding regioll from base 149 to base 1237 of the tomato A~c2 gene; (9) nos 3', the
untranslated 3' region of the nopaline synthase gene from A~rob~lcteril~7?; and (10)
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RB, a right border region of Agrobacter7um T-DNA.
The "T-DNA insert" of the invention comprises three copies of the T-
DNA described above. The three copies are arranged in inverted repeats at the LB and
RB. At the LB-LB and RB-RB junction, one border is deleted such that there is only
5 one complete border at each junction. The LB and RB at either end of the T-DNA structure are also deleted.
An exemplary plasmid comprising the T-DNA described above,
pW'rr2144/AccS, is described in detail below. This plasmid is used to generate
transgenic tomato plants using Agrobacterium tumefaciens-mediated transformation10 techniques. Stable insertion of the AccS transgene into the tomato genome in the sense
orientation can result in down-regulation of expression of the corresponding endogenous
Acc2 gene and a reduction in ethylene biosynthesis in the ripening fruit. The technique
is an example of TranswitchTM suppression (U.S. Patent No. 5,283,184). One feature of
TranswitchTM gene suppression is the reduced accumulation of the targeted gene mRNA
15 (reduced steady state RNA). The trait (level of suppression), once selected through the
first sexual generation, behaves in a simple Mendelian fashion.
The methods of the present invention can be used to delay fruit ripening of
any tomato (large fruited or cherry) cultivar for fresh market or processing tomato
production. Exemplary cultivars that can be used include essentially all commercial
20 cultivars. For listings of suitable tomatoes s~e, Rick, in Evoluti~n of Crnp Plant.~ N.W.
Simmonds, ed. pp268-273 (Longman, London, 1976) and Taylor in Th~ Tomato Crop
ppl-35 (Chapman and Hall, London, 1986).
A tomato plant of the present invention can be obtained by crossing a p]ant
comprising the T-DNA insert of the invention with any tomato cultivar lacking the insert.
25 Any standard method used for crossing tomato plants can be used to introduce the
transgene into the genome of the desired plant. Generally, the methocls involve
emasculation of one parent, followed by application of pollen from the other parent to
the stigma of the first parent. The crosses can be perfontled using either parent as the
pollen parent. Embryo rescue can also be performed if the flowers abort after
30 pollination.
The plant containing the T-DNA insert can be a plant derived from
primary transforlnants or can be a plant in which thc f~ctor was intro(llIced througll a
sexual cross. Preferred plants of the invention are those derived from seed dcposited
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with the American Type Culture Collection (ATCC) Accession No.
A number of methods can be usecl to determine if a tomato plant exhibiting
a delaye~i ripening phenotype comprises the T-DNA insert of the invention in its genome.
The terms used herein to describe ripeness of tomato fruit are according to standard
S ripeness classes as described, for instance, in 77te Tomato Crop Atherton and Rudich eds.
(Chapman Hall, 1986). The ripeness classes for a given given fruit are set forth in Table
1.
Table 1
Score Class Description
1 Green Entirely light to dark-green, but mature.
2 Breaker First appearance of external pink, recl or tannish-
yellow color, not more than 10%.
3 Turning Over 10 ~, but not more than 30 % red, pink or
tannish-yellow.
4 Pink Over 305'o, but not more than 60% pinkish or
red.
Light red Over 60%, but not more than 90% red.
6 Red Over 90% red, desirable table ripeness.
Fruit of the plants of the invention reach breaker stage between about 40
and about 70 days after anthesis, usually between about 45 and about 60 days after
anthesis. The fruit, however, differ significantly from wild type fruit in terms of
20 ripening inhibition. On the vine, the fruit of plants of the invelltion typically remain at
the breaker stage 5 to 7 days longer than wild type. The transition from breaker stage to
light red stage in the ripening impaired fruit is further delayed witll respect to the
transition for wild type fruit. Under field conditions the fmit of the invention typically
require approximately 21 days to proceed from the breaker stage to light red stage,
25 whereas wild type fmit typically require 4 to 5 days to turn from the breaker stage to the
red stage. The ripening impaired fruit under field conditions can remain indefinitely at
the light red stage without ever reaching the red stage.
There are analogous differences in ripening for off the vine fruit picked at
the breaker stage anc'i storecl at 15~C. Ripening impaired fniit of thc> invention typically
30 require at least about 2 weeks (typically about 3 weeks) to reach the pink stage, and
while some may reach tile light red stage, the fruit can remain indefinitely without
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reaching the red stage. Fruit will, however, reach the red stage upon the application of
ethylene. This is to be contrasted with wild type fruit which typically require 5 to 7 days
to re~ch red ripe stage from the breaker stage under comparable conditions.
Fruit color development, as measured by the a/b ratio measures about 5 to
S about 10 fold less in the fruit of the invention as compared to control fruit at the breaker
or equivalent stage. Standard methods for determining tomato fruit color are described,
for instance, in Gull et al. J. Amer. Soc. Hort. Sci. 114:950-954 (1989) and Kader et al.
Hort. Sci. 13:577-578 (1978). At the time the control fruit are red ripe, the levels of
color in the fruit of the invention are 4 to 5 times lower.
In addition, the fruit do not synthesize normal levels of ethylene during
ripening. Typically, the level which is detected at the breaker or equivalent stage is less
than about 0.5 nl/g/hr, usually about 0.1 nl/g/hr as measured using a standard assay as
described in Grierson and Tucker, P~anta 157:174-179 (1983) and Sawamura et al. Plant
Cell Physiol. 19:1061-1069 (1978). At the pink stage which follows the breaker stage
the fruit continue to have ethylene levels which are reduced by about 50 to about 100
fold compared to control fruit.
Since the T-DNA inserts comprise NPTII genes, kanamycin resistance can
be detennined using selective media or by spraying 10-14 day old tomato seedlings with
a solution of kanamycin (Ig/L) on three consecutive days. The levels of Acc2 mRNA
can be measured using standard techniques such as Northern blots, RNAse protection
assays and the like. To further characterize the plants nucleic hybridization techniques
can be used to detem~ e structure of the T-DNA insert in the plant. The example
section, below, provides a detailed description of the molecular characterization of the T-
DNA insert of the invention.
The precise locus in which the T-DNA insert of the invention is integrated
can be determined using standard genetic and molecular mapping techniques well known
to those of skill in the art. Obviously, for plants derived either directly or indirectly
from a particular plant o~ seed containing the T-DNA insert (~ ., those deposited with
the ATCC under Accession No. ), the locus will be the same as the parent plant.
The following example is provided to illustrate, but not limit the claimed
inventiom
Example I
1~ . A~rol)acteri~ mediated tran.sfol mat ion svstem
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Introduction of DNA into plant tissue by Agrobactenum-mediated
trarlsformation as described in U.S. Patent No. 5,283,184. The vector system used to
transfer the AccS transgene into tomato is based on the Ti plasmid from Agrobact;erzum
tumefaciens,
S The T-DNA plasmid, pWTI'2144/AccS, used in these transformations is
composed of: (1) the replication of origin from pACYC184 that ensures replication in
Escherichia coli; (2) the pVS1 replicon (derived from Pseudomonas aeruginosa DNA)
that ensures replication in A. tumefaciens, (3) the tetracycline resistance marker from
plasmid RP1 that allows for selection of the binary plasmid in A. tumefaciens and E.
0 coli, and (4) the left and right border regions of T-DNA from an octopine strain of A,
n~mefaciens which surround the DNA insertion in the plant genome.
Within the T-DNA are the nptll gene from transposon TnS that encodes
enzyme neomycin phosphotransferase II and serves as a selectable marker for
transformed plant cells, fused to a nopaline synthase ~nos) promoter sequence and
octopine synthase (ocs3') termination sequence from A. tumc,~faciens, and the LacZ'
polylinker region with multiple restriction sites for cloning of genes to be transferred.
The T-DNA has an insertion of a truncated Acc2 gene coding region fused to the 35S
promoter from cauliflower mosaic virus and the nos3' termination sequence in the LacZ'
polylinker region of pWTT2144.
Plasmid pWTT2144 was transferred from E. coli to A. t,um~faciens
LBA4404, which carries the pAL4404 vir plasmid, by a triparental mating procedure as
described by Figurski et al., Proc. Natl. Acad . Sci. USA 76: l 648- 1652 (1979) .
The plasmid pWTT2144/AccS was used to transform the parental line
91103-114 to generate line 1345-4, described in detail below. The T-DNA region of this
plasmid consists of the following sequences (see, Figure 1):
CaMV35S. The 35S promoter region is derived from cauliflower mosaic
virus and controls expression of tlle AccS gene. Tlle 35S promoter directs high level
t constitutive expression and is widely used as a promoter for higll expression of
transgenes.
Cab22L leader. The Cab22L leader sequence (C'ab22L) is a 69 bp
fragment of P~t~ ia ~7~b~i~1a genomic DNA whicll was derived fronl the Cab22L gene
and correspon(ls to the 5' untrallslated region for that gene.
AccS. The AccS gene is a truncated codillg region derived from an ACC
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synthase gene (Acc2) isolated from tomato (L. esculentum). The AccS gene corresponds
to a 1088 bp region of the Acc2 gene from base 149 to base 1237. The AccS gene does
not encode a functional ACC synthase enzyme.
Termination sequences. The nopaline synthase (nos3') and octopine
S synthase focs3') gene termination sequences from A. tumefaciens function in the
expression of the AccS and nptII genes, respectively.
2Xpnos Promoter. The nos promoter is present in line 1345-4 as a
duplicate tandem repeat of the untranslated S' region of the nopaline synthase gene from
A. tum~aciens. It functions in line 1345-4 in the expression of the nptll selectable
marker gene. This sequence, as used in line 1345-4, no longer functions as a regulated
article since it is not associated with the nopaline synthase coding region which functions
in A. tum-~facien~.
NptII. The nptll gene is a coding region originally isolated from
transposon TnS. It encodes a protein, neomycin phosphotransferase II, which catalyzes
the phosphorylation of certain aminoglycoside antibiotics, rendering transformed cells
resistant to kanamycin. It functions in line 1345-4 as a selectable marker.
LacZ' polylinker sequence. The untranslated LacZ' polylinker sequence
functions in line 1345-4 as a site for cloning the AccS transgene into the binary vector
pWrr2144. Borders. The left and right border regions of T-DNA from
A. tum~f~Jci~n.~ function in the transfer of gene sequences into the tomato genome. The
border regions are the only necessary cis-acting elements in T-DNA for T-DNA
insertion. Tl1e use of a binary vector system allows for other necessary transfer elemel1ts
to act in trcl~7.S SO that only the border regions are required to be integrated into the plant
host genome. The T-DNA borders are only partially transferred to the tomato genome;
d~lring the transfomlation process, the left border is cut between nucleotides 293 and 294
(left border nick) wllile the right border is cut between nllcleotides 7603 and 7604 (right
border nick). Tllis cleavage reduces the length of the rigl1t border fragment ofpWTT2144/AccS from 1900 bp to 303 bp in tlle T-DNA and the left border fragment of
pWTT2144/AccS from 880 bp to 589 bp in tlle T-DNA (Figure l).
B. Description of Non-tr~ .sfol med Tom~to Cultiv~l 91103-114
DNAP tomato lille 91103-114 is a somaclone derived from the breeding
line FL7181 clevek~ped by Dr. Jay Scott at the University of Florida. Line FL7181 is
characterized as a deterl1lillate large-fruited variety witll an average fnlit weight of 8 oz.
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11
Fruit are globate to slightly ellipticai in shape and are substantially firmer than fruit of
comparable varieties (i.e., Floradade). Fruit shoulders are smooth and exhibit a darker
green shade than the rest of the fruit surface before ripening. Fruit ripen to a deep
climson red interior color due to the presence of the og' allele. The fruit stem (pedicel)
lacks a joint. This line is known to be resistant to Verticillium wilt race 1 and Fusarium
wilt races 1 and 2.
The DNAP line 91103-114 exhibits all of the traits described above, but
differs from FL7181 principally by reduced blossom end scar size, strong main stem and
increase~i foliage cover for fruit. Line 91103-114 also differs from FL7181 in its
adaptation to diverse growth environments; while FL7181 is specifically adapted to
Florida growing regions, 91103-114 has proven to grow well in other regions (e.g.,
California).
C. Description, History and Mendelian Inheritance of Delaved-Ripenin ~ Tomato
Line 1345-4
The line 1345-4 is a homozygous T2 selection from an original To
transformant 1345, obtained after Agrobac~erium ~um~faciens transformation of the
DNAP line 91103-114 with binary vector pWTT2144/AccS.
The primary transformant 1345 was selected in a greenhouse screen of
several hundred primary transformants to have fruit which did not ripen when left on the
vine. Subsequently, 10 of the 1345 T, seed (derived from self fertilization) which were
prescreened for kanamycin resistance were sceened in the greenhouse and observations
on the plant and fruit phenotype were made. Plant 1345-4 was selected as having fruit
which did not ripen on the vine. The 1345-4 plant was shown to be homozygous for the
T-DNA locus. Seed from the self-fertilization of 1345-4 have been subsequently
analyzed in multiple field trials over multiple growing seasons.
No instability in the delayed-ripening pl~enotype has been observed in any
field t~ials. Two indepelldellt ways have been used to monitor the stability of line
1345-4. First, it is possible to establish that the T-DNA insertion is stable and intact by
measuring kanamycin resistance in large populations of seedlings in the greenhouse. It
was establishcd that the 1345 plant was carrying a single T-DNA insertioll by evaluating
the segregation of kanalllycin resistallce in the primary transformant. Subsequcntly,
multiple ka-lalllycin-resistant T2 plants were selected and segregatioll of the
kanamycin-resistant phenotype in progeny plants arising trom sel~:fertilization of the
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12
selected T2 plants was evaluated. The T3 plants from 1345-4 were all kanamycin
resistant, hence it was deduced that the 1345-4 plant was homozygous.
The second approach to evaluating the stability of line 1345-4 is through
observation of the delayed-ripening phenotype in the field. A number of separate field
5 trials for the evaluation of the homozygous 1345-4 line and progeny derived from it have
been carried out. These trials have involved the evaluation of fruit on at least 2000
separate plants of 1345-4. During these evaluations, which involved observations of the
individual plants at multiple times during the ripening process and the harvest of fruit
from all of the plants, no exceptional plants in which all the fruit ripen at the normal rate
10 have been observed.
D. DNA Analysis of Delaved-Ripenin~ Tomato Line 1345-4
To determine the nature and number of insertions which have occurred in
line 1345, Southern hybridizations were used to characterize the structure of the T-DNA
inserts in the genomic DNA, in conjunction with the nptll segregation data described
15 above which indicates the T-DNA locus number. The T-DNA is defined as the region
between the left and right borders of the binary vector pWI-r2144/AccS that is
transferred into the plant (see Figure 1). This region includes the nptll selectable marker
and the truncated ACC synthase gene (AccS), together with the left and right border
sequences. Figure 2 shows the restriction enzymes used to cleave the DNA and the20 location of the four probes used to determine the struct-lre of T-DNA insert, the left
border (LB), nptll, Acc2, and right border (RB) probes.
1. Copv nulllber
The number of additional AccS genes in 1345 was determined by
digesting genolllic DNA from transgenic plants with HindllI and EcoRI, then after
25 electrophoresis and transfer to nylon membranes, hybridizing to a 32P-labeled Acc2
probe. This probe hybridizes to a 0.9 kb EcoRI-HindIII fragment from the transgene
(AccS) ancl a 1.2 kb HindIII fragment from tl'le endogenous Acc2 gene which includes an
additional 300 bp of intron sequence. By comparing the intensity of the transgene and
endogenous bands, a determinatioll of the nulllber of copies of the transgene can be made
30 in either the hemizygous primary transformant or holllozygous Sl progeny. Thehybridization patterns of gellolnic DNA from an untransformecl tomato plant, Baxter's
Early Busll Cherry (BEB) and 1345-4 digested with Hin(lIII and EcoRI and hybridized to
theAcc2 probe were analyzed. In 1345-4, the homozygous Sl progeny of 1345, the
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13
endogenous gene to transgene ratio is greater than 2 suggesting that there are at least 2
copies of AccS in the transgenic DNA. Since the nptll gene segregates as a single locus,
it is most probable that the 2-3 copies of the T-DNA are present at a single locus.
2. T-DNA Structure
It is known that a single intact copy or multiple T-DNA copies can be
inserted at a single locus as direct or inverted repeats around either the left or right
border, arld it is known that deletions of the T-DNA or insertions of genomic DNA may
be present between the T-DNA copies (Jorgenson et al., Mol. Gen. Genet. 207:471-477
(1987)). To determine the organization of the T-DNAs in the 1345 genome, we
hybridized several different probes to the 1345-4 genome digested with several restriction
enzymes. ~igure 2 shows the relative map position of the probes in the T-DNA.
Le.ft an~ Right B<~rders: Eco-RI digestions were done to determine the
number of intact left and right border fragments. EcoRI sites in the T-DNA are located
approximately 500 bp in from the LB and 2.1 kb in from the RB (within the AccS
transgene). The fragments hybridizing to the specific border probe will be at least this
size. If there is an inverted repeat at the LB and the borders are intact and flush, we
would expect to see a 1 kb EcoRI fragment hybridizing only to the LB. For the RB, an
intact inverted repeat would give a 4.2 kb fragment that hybridizes to the RB fragment as
well as to the AccS probe. A direct LB-RB repeat would result in a fragment of 2.6 kb
that hybridizes to both border probes and AccS.
In 1345-4 DNA cut with EcoRI, a 2.9 kb fragment hybridizes to the LB
probe only and a 3.5 kb fragment hybridizes to the RB probe only, indicating that there
is one intact left and right border. Likewise, the Ncol digestions show single hybridizing
bands with both probes and the fragments are of appropriate sizes (LB fragment, greater
than 1.6 kb and RB fragment greater than 2.3 kb). The HinciIII and XbaT digests also
show single bands of appropriate sizes which suggests that there is a single complete
T-DNA insertion. However, we know from the copy number blots (see above) that there
are at least two copies of the AccS transgelle. Together these results suggest that a
deleted form of T-DNA, containing an intact AccS gene but missing one or both of the
borders, is also present. There are no ciirect repeat stn~ctures since the LB and RB
probes do not hybriclize to the same fragment. There could, however, be invertecl or
indirect repeats around either border. Hybridizatiolls with the nptll and AccS proi~es
were done to furtller characterize the T-DNA insertions.
CA 02226088 1997-12-30
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14
n~tll: If the T-DNA is intact, the nptll probe will hybridize to a single
2.4 kb EcoRI fragment containing the entire 2Xpnos-nptll-ocs3' fusion (see Figure 2).
In 1345-4 genomic DNA cut with EcoRI, the expected 2.4 kb fragment is present inaddition to two other hybridizing fragments of 5.2 kb and 2.9 kb. The 2.9 kb fragment
S also hybridizes to the left border probe which indicates that there is probably one
complete internal copy and 2 inco m plete copies of T-DNA with deletions occurring at the
LB. Since the 2.9 kb EcoRI fragment hybridizes to both nptll and LB probes, thisjunction is likely an inverted repeat with a deletion of one of the borders that includes the
EcoRI site. Hybridization of an EcoRI fragment to both the L,B probe and the nptll
probe can only occur if a deletion eliminates one of the EcoRI sites.
Since Ncol cleaves within the nplll coding sequence, digestion of the
T-DNA with this enzyme will give two nptll fragments, one of 2.1 kb which spans the
nptll 3' coding region up to the NcoI site located at the ATG of the AccS transgene, and
one of 1.2 kb in length which includes the 5' nptll coding region and LB to the next
Ncol site in either genomic DNA or adjacent T-DNA insertion. If there is a perfect
inverted repeat at the LB, we expect a 3.3 kb Ncol fragment that hybridizes to both the
nptll and LB probes. Based on tlle results from the EcoRI digestion, we expect the 2.1
kb fragment to be present, as well as two fragments greater than 1.2 kb. One of these
will also hybridize to, the LB. As predicted, the 2.1 kb fragment is present as well as a
6.6 kb fragment and a 2.9 kb fragment which also hybridizes to the LB probe. This is
consistent with the presence of three T-DNA copies, one which is complete and intact,
one which is an inverted repeat with a deletion (approximately 600 bp) extending to the
LB, and a second inverted repeat with a deletion (approximately 50 bp) extending to the
right border.
Acc~S: Hybridization with the AccS probe will give fragments greater than
2.3 kb and 2.1 kb f~r Ncol and EcoRI digests respectively. These fragments will also
hybridize to the RB probe if the border is intact. The results sllow two hybridizing
fragments for NcoI, 15 kb and 3.8 kb, and two for EcoRI, 4 kb and 3.5 kb. The 3.8 kb
Ncol fragment and the 3.5 kb EcoRI fragment also hybridize to the RB. These results
confirm that there are at least 2 copies of AccS, and that one copy is present on a
T-DNA with a deleted RB. To delllollstratç that AccS itself has not been deleted or
rearranged, NcoI/Xl aI double digests were done to dl-(>p out tlle intact 1.1 kb fragment
conlainillg the complete truncated gene.
CA 02226088 1997-12-30
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Linlcage of AccS an~ nptll: XbaI and HindIII cleave the T-DNA once
approximately 1 kb in from the right border. Digestion with either of these enzymes will
generate fragments containing both the nptll and AccS genes. After XbaI digestion, a
9.2 kb and a 6.2 kb fragment hybridized with both probes. After HindIII digestion, a
5 9.2 kb and 6.4 W fragment hybridized to both probes confirming that each copy of AccS
is linked to a copy of nptll. The 9.2 kb fragment also hybridizes to the LB. The size of
the fragments are consistent with the presence of inverted T-DNA repeats at the LB and
RB.
The structure for 1345-4 T-DNA insertion is shown in Figure 4. It consists
10 of three T-DNAs assembled in inverted repeats at the LB and RB. At the LB-LB and
RB-RB junction, one border is deleted such that there is likely only one complete border
at each junction. The LB and RB at either end of the T-DNA structure are also deleted.
The endpoint rightward is internal to the nptll gene and deletes the internal NcoI site,
and the endpoint leftward lies between the Acc2 gene and the right border.
Finally, the nucleotide sequence of the T-DNA insert of 1345-4 has been
complied from the deduced restriction map and is present in SEQ. ID. No. 1.
The above examples are provided to illustrate the invention but not to limit
its scope. Other variants of the invention will be readily apparent to one of ordinary
skill in the art and are encompassed by the appended claims. All publications, patents,
20 and patent applications cited herein are hereby incorporated by reference.
CA 02226088 1997-12-30
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16
SEQ. ID. No. 1
1345-g T-DNA LOCU~
~cZ~ >no-3'
Ct~Cr,r~CCA~~ LAAG~ iLATGL~ iLAGGTCGATCTAGTAACATAG
ATGACACCGCGCGCG~TAATTTATCCTA~111~C~C1ATA11~
TCTATLGC~l-ATTAAATGTATAAll~ w ACTCTAATCATAAAAACCCA
TCTCATAAATAACGTCATGCATTACATGTTAATTATTACA1~C1_1AACGT
AATTCAACAGAAATTATATGATAATCATCGCAAGACCGGCAACAGGATTC
nos3'<-;
AATCTTAAGAAACTTTAl~I.i~LAAAl~ ~AACGAl--l'tjGCLLll~ACT
->ACCS
CTAGAAACAAACT-G~AACCAACL.LCLi_ LACATTCAAACGAAGATC
CAAGCGAGACGTT~AGCT~AACA1LG11lATAATAAC~L1LLATAACGAC
ATTTCGCTATCG~AAGTCGA1~L~1-~AAAAGTGGACGCAAATCCATCCA
ACAAAAAA~LLCGCATTA1111-1~AAGCATTTAA'~1CCCACTACTTCAA
GTCCATTAGTAAAATc,lll~ L~l1-~lACCTAACCTCAlCG~,~l~-l~l
CTTAGAAAATTATCGACGAAl1l-~ ~ATGGCAi1~L~_~iC'lAAAAA
ATA11~C~111~1~1AGATACTAAACCGAAACTCGACA1~ 1~1AGCAC
AATTAACGACATL~-GlLAAAAGAATATATGATTCCGACTCTAAATCCT
GGTAACCCCAT~1L11llGAAAGACTGTAGACGATGTGAACTAAATCTTT
GTTGCAGTAAGTCATTTCL1G11CATCGAGGATTTCAGCTATACTGACGA
ATTGAGGCGTGTCAAAGACAGTGGCTGCGTAGA11-~-~1CACAAACAAGG
TGGAl~l1~ lGcjl1GG;l~iAAACTCAAGACA~;l__lLA~ .il~1L'_''_''l'
GTCCAAAGTGGTGCCCAATGGATTTGATGGATTGGTCAAAATCAAACCTT
TTACTTTGAl~ GA~111l~ilGCAl11lCATATGCllLl11lACTGCT
TT~GAAGTAATTTTGAAATTATTGGAGL1~;1CACAGTGAATTGGAATAAG
TTGTACTCCAGTTCTCCATCTTAAATCTCTGTTAAATGCTGGGTAGTATG
GTGAAGGTACTAAAAATGCATCGCCAGGATCAGCCAAACAAAATATAATT
GTCTCATTAGCTCCAGTGGCACCACCAGCCATAACAACTCTTTCTGGATC
AAATCTAACTCTTCCTCCTL1l~il111CTCCATAAATTTCGCAATCGCTT
TTCTGAATTCAGGCAAGCCATGATAATCTTGAAAGTTGGCAATGGCCTTG
AATGATTTGATTCCTTCAGAACAAATTGAACCTTTTGGGTTTCTCTTAAT
ACC~
CCAATCTTCTATCAAGTCTAAAC.~AAGCTGATTTTCTGCTAAACCCATGG
->3S8Cab22~
TTTAATAAGAAGAGA~AAGAGTTCTTTTGTTATGGCTGAP.GT~ATAGAGA
CA 02226088 l997-l2-30
PCT~US96/IIO96
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17
AATGAGcTcGAG(~l~ lc~AAATGAAATG~A~ lATATAGA
GGAAGGGTCTTGCGAAGGATAGTGGGAll~l~GGTCAl~ llAcGTcAG
TGGAGATGTCACATCAATCCACTTGCTTTGAAGAC~'l'~'l'l'~AACGTCT
~l-~-l-~l~l~CACGATGCTC~l~ l~&GGTCCAil-''l'l'GG~ACCACT
GTCGGCAGAGGCATCTTGAATGATAGCC~ ATCGCAATGATGGC
A~ AGGAGccA~ c~ A~l-~l-l-l-~ATGAAGTGAcAG
ATAGCTGGGCAATGGAATCCGAGGA~ ~AAATTATCCTTTGTTG
AAAAGTCTCAATAGC~ GAGACTGTA'~ ~ACATTTT
TG&AGTAGACCAGAGTGTCGi-l-~ ACCATGTTGACGAAGAl1-1-1'~''1'1~
T'rGTCATTGAGTCGTAAAAGAC~ ;-lATGAA~ GCCAG;lL-l-l~AC
GGcGAGill'~i~illAGATCCTCGATTTGAATCTTAGACTCCATGCATGGCC
TTAGATTCAGTAGGAACTACG.~lllAGAGACTCC~ATCTCTATTACTTG
CCTTGGTTTATGAAGCAAGCCmTGAATCGTCCATACTGGAATAGTACTTC
TGATCTmGAGAAATAT~T~ lGATGCAATTAGl-~lG
AAl~~ GACTGCATCTTTAACCTTCTTGGGAAGGTATTTGATCTCCTG
GAGATTGTTACTCGGGTAGATCGTCTTGATGAGACCTG~l~--~lAGGCCT
CTCTAACCATCTGTGGGTCAGCAll~lllCTGAAATTGAAGAGGCTAACC
TTCTCATTATCA~l~lGAACATAGTGTCGTCACCTTCACCTTCGAACTT
CCTTCCTAGATCGTAAAGATAGAGGAAATCGTCCATTGTAATCTCCGGGG
3!58C~Iba2~- ¦ ->r.~cZ
CAAAGGAGATCCCGGGTACCGAGCTCGAATTCGTAATCATGGTCATAGCT
GTTTCCTGTGTGAAAll~llATCCGCTCACAATTCCACACAACATACGAG
CCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTC
ACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTC
GTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGC
Lac~-¦->ocs3'
GTATTGGGCAGCGGCCGCTGTTACCCGGCCGCCGTGCTGGACCGGGTTGA
ATGGTGCCCGTAACTTTCGGTAGAGCGGACGGCCAATACTCAACTTCAAG
GAATCTCACCCATGCGCGCCGGCGGGGAACCGGAGTTCCCTTCAGTGAAC
GTTATTAGTTCGCCGCTCGGTGTGTCGTAGATACTAGCCCCTGGGGCCTT
TTGAAATTTGAATAAGATTTATGTAATCAGTCTTTTAGGTTTGACCGGTT
CTGCCGClllllllAAAATTGGATTTGTAATAATAAAACGCAATTGTTTG
TTATTGTGGCGCTCTATCATAGATGTCGCTATAAACCTATTCAGCACAAT
ATATTGTTTTCATTTTAATATTGTACATATAAGTAGTAGGGTACAATCAG
TAAATTGAACGGAGAATATTATTCATAAAAATACG~TAGTAACGGGTGAT
ATATTCATTAGAATGAACCGAAACCGGCGGTAAGGATCTGAGCTACACAT
GCTCAGGTTTTTTACAACGTGCACAACAGA~TTGAAAGC~ATATCATGC
CA 02226088 l997-l2-30
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18
ocs3'<-~->NPTIT
GATcATAGGcGTcTcGcATATcTcATTAAAGcAGGGGGTGGGcGAAGAAc
TCCAGCATGAGATCCCCGC~-lG~AGGATCATCCAGCCGGC~ ~AA ~'
AACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCG~'l'~AATCGAAAT
~l~lGATGGCAGGTTGG~lCGCll~lCGGTCATTTCGAACCCCAGA
GTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGC
GAATCGGGAGCGGCGATACCGTAAAGCACGAGGAA~CG~lCAGCCCATTC
GCC~CCAAG~l~-llCAGCAATATCACGGGTAGCCAACGCTAl~l~AT
AGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGG
CCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGAC
GAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGllC~G
CTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGA
CCGGCllCCATCCGAGTACGTGCTCGCTCGATGCGATGlllCGCTTGGTG
GTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCAT
CAGCCATGATGGATA~'l'l"l~''l'CGGCAGGAGCAAGGTGAGATGACAGGAGA
TCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGT
GACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACG
ATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCG
GTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGC
GGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCC
TCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATC
ATGCGAAACGATCCTCATCCTGTCTCTTGATC
CA 02226088 1997-12-30
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19
j=r t~
GC~rr IGACA~UUA ~A~ ~C-~AG C$r~ Tr~ CCTrA~r~ ~-'L'~-n~AAoG C
pt'
AP~l~-~tX~ TCATGATCCA GA$1~T~TGG A5~GAÇaGTG AA~AIG~GAS 5c5~u~rn=;~ 5~Yt~3U3GG~
A~55~7~n~a~ fiCX~ GSaG ~ A~ ~ AL~AG~AATA '~ AoCr GA~S~:ADC ~lcAr~;r~ T
pnoS ~ L3 / L3 d~
ICfi3~UU~Gr3 AC~C~1LA~ 'G~-l~ ATGAI~
CA 02226088 l997-l2-30
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AAGGCAGGATATATTCAA
TTGTAA ~TGGCTTCA1~j-1CC~jG~AAATCTACATGGATCAGCAATGAGTAT
GATGGTCAATATGGAGAAAAAGAAAGAGTAATTACC~A1-11_1-1-1--1-1~AAT
TCAAAAATGTAGA1~-1~C~CAGCGTTATTATAAAATGAAAGTACATTTTG
ATAAAACGACAAATTACGA1~-~-1C~1ATTTATAGGCGAAAGCAATAAAC
AAATTATTCTAATTCGGAAA1~111A111CGA~L~L~1ACATTCACGTC
CAAATGGGGGCTTAGATGAGAAACTTCACGATCGAL~iC---1-1GA~ 1CGCC
AL~AGATACCCATTTCA1~1~AGA~ -L~-~AGATTATGCGAAAA
TATACACTCATATACATAAATACTGACAGTTTGAGCTACCAATTCAGTGT
AGCCCATTACCTCACATAATTCACTCAAATGCTAG~CAG-1~-1-~1CAACTC
G~CGTCAA111~1~GGCCACTATACGATAGTTGCGCAAATTTTCAAAGTC
CTGGCCTAACATCACAC~L~1~1~GGC~GTCCCA1-1--1~-1GATAAAT
CCACCCATCGGATCTGAATTCTCACTCATTAGGCNCCCCAGGCTTTACAC
TTTATGCTTCCGGCTCGTATAA1G1~1~AATTGTGAGCGGATAACAATT
CA 02226088 1997-12-30
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21
I,B~I.B dol~tod<--~-->yD,08
TCACACAGG~AACAGGATCATGAGCGGAGAATTAAGGGAGTCACGTTATG
ACCCCCGCCGATGACGCGGGACAAGC~-1111A~11GGAACTGACAGA
ACCGCAACG~TTGAAGGAGCCACTCAGCCGCGG~11..1~GAGTTTAATG
AGCTAAGCACATACGTCAGAAACC;F~TTATTGCGC~ AAAA~L~ A
AGGTCACTATCAGCTAGCAAATA~11~11~1~AAAAATGCTCCACTGACG
~TCCATAAATTCC~ ~1ATCCAATTAGAGTCTCATATTCAL1~1~AA
I - >~no~
TCCAAATAATCTGGATCATGAGCGGAGAATTAAGGGAGTCACGTTATGAC
CCCCGCCGATG~CGCGGGACAAGCC~11-11AC~111 WAACTGACAGAAC
CGCAACG~TTGAAGGAGCCACTCAGCCGCGGG11'~l~GAGTTTAATGAG
CTAAGCACATACGmCAGAAACG.TTATTGCGCGTTCAAAAGTCGCCTAAG
GTCACTATG~GCTAGCAAATAll1~-1l~lCAAAAATGCTCCACTGACGTT
CCATAAATTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATC
~non < 1 - >NPTTI
CAAATAATCTG'GATCTGATCAAGAGACAGGATGAGGATC~111CGCATGA
T~GAACAAGATGGATTGCACGCAG~1~1C~-~CCGCTTGGGTGGAGAGG
CTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGC
C~1~11~CGGCTGTCAGCGCAGGGGCGC~11~-1~111~L~-AAGACCG
ACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCG
TGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCAC
TGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATC
TCCTGTCA.CTCACCT.GCTCCTGCCGAGAAAGTATCCATCATGGCTGAT
GCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCA
CCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTC
TTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCC
GAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGT
CGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCC
GCL11-1~TGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTAT
CAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGA
ATGGGCTGACCGCTTCCT~L~iCLlLACGGTATCGCCGCTCCCGATTCGC
AGCGCATCGCCTTCTATCGC~11~11GACGAGTTCTTCTGAGCGGGACTC
I TGGGGTTCGAAATGACCGACC-AAGCGACGCCCAACCTGCCATCACGAGAT
TTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGG~ATCGTTTT
CCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGT
NPTII<- ¦-->ocs3 '
TCTTCGCCC,,CCCCCTGCTTTAATGAGATATGCGAGACGCCTATGATCGC
CA 02226088 1997-12-30
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22
ATGATATTTGCTTTCAAll~l~ll~l~CA~l~ lAAAAAACCTGAGCAT
GTGTAGCTCAGAl~llACCGCCGiil11~ ATTCTAATGAATATAT
CACCCGTTACTATCGTAl 1 1 1 lATGAATAATAl 1 L 1~ 1 1 ~AATTTACT
GATTGTACCCTACTACTTATATGTACAATATTAAAATGAAAACAATATAT
TGTGCTGAATAGGTTTATAGCGACATCTATGATAGAGCGCCACAATAACA
AACAATTGC~llllATTATTACAAATCCAATTTTaAAAAAAGCGGCAGAA
CCGGTCAAACCTAAAAGACTGATTACATAAATCTTATTCAAATTTCAAAA
GGCCCCAGGGGCTAGTATCTACGACACACCGAGCGGCGAACTAATAACGT
TCACTGAAGGGAACl~ il1CCLC ~;w CGCGCATGGGTGAGATTCCT
TGAAGTTGAGTAllG~C~l~CG~l~l-ACCGAAAGTTACGGGCACCATTC
oc~3'<-~->lacZ
AACCCGGTCCAGCACGG~ w CCGGGTAACAGCGGCC~lGC~CAATACGC
AAACCGCCTCTCCCCGCGC~11~ CGATTCATTAATGCAGCTGGCACGA
CAGGTTTCCCGACTGG~AAGCGGGCAGTGAGCGCAACGCAATTAATGTGA
GTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCT
CGTATGTTGl~lGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG
lacZ<-¦->35SCab22
CTATGACCATGATTACGAATTCGAG~l~ iLACCCGGGA1~1C~111GCC
CCGGAGATTACAATGGACGAlllC~l~lATCTTTACGATCTAGGAAGGAA
GTTCGAAGGTGAAGGTGACGACACTATGTTCACCACTGATAATGAGAAGG
TTAGCCTCTTCAATTTCAGAAAGAATGCTGACCCACAGATGGTTAGAGAG
GCCTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGTAACAATCTCCA
GGAGATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCA
GGACTAATTGCATCAAGAACACAGAGAAAGACATATTTCTCAAGATCAGA
AGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCATAAACCAAGGCA
AGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCTACTGAATCTAAGG
CCATGCATGGAGTCTAAGATTCAAATCGAGGATCTAACAGAACTCGCCGT
GAAGACTGGCGAACAGTTCATACAGA~l~llllACGACTCAATGACAAGA
AGAAAATCTTCGTCAACATGGTGGAGCACGACACTCTGGTCTACTCCAAA
AATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCA
ACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCT
GTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGC
CATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAG
TGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAG
ACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACT
GACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTC
TATATAAGG~AGTTCATTTCATTTGGAGAGGACACGCTCGAGCTCATTTC
=
CA 02226088 l997-l2-30
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23
35SCa~22 <-~
TCTATTACTTCAGCC~Tr~ACAAAAGAACTCTTTT~'1Ll _ lATTAAACC
-~ACCS
ATGGGTTTAGCAGAAAATCAG~~ AGACT~GATAGAAGATTGGAT
TAAGAGAAACCCAAAAGGTTCAA~ ~AAGGAATCAAATCATTCA
AGGCCAl1~iLLAACTTTCAAGATTATCA~ GCTG~ATTCAGAAAA
GCGATTGCGAAATTTATGGAGAAAACAAGAGGAGG~AGAGTTAGATTTGA
TCCAGAAAGA-~11~11ATGGL-1L~iW1~LLACTGGAGCTAATGAGACAA
TTATAllll~Llll~GCTGATCCTGGCGATGCA~ lAGTA~LllLACCA
TACTACCCAGCATTTAACAGAGATTTAAGATGGAGAACTGGAGTACAACT
TATTCCAATTCACTGTGAGAGCTCCAATAATTTCAAAATTACTTCAAAAG
CAGTAAAAGAAGCATATGAA~ATGCACAAAAATCAAACATCAAAGTAAAA
GGTTTGATT'"mGACCAATCCATC~AATCCATTGG&CACCACTTTGG~CAA
AGACACACTGAAAAGl~iLllGAGTTTCACCAACC~ACACAACATCCACC
ll~lll~ACG~AATC~ACGCAGCCACTGTCTTTGACACGCCTCAATTC
GTCAGTATAGCTGAAAlC~'L~iATGAACAGGAAATGACTTACTGCAACAA
AGATTTAGTTCACA1LL1~_lACAci1'~ll1CAAAAGAC;ATGGGGTTACCAG
GATTTAGAGTCGGAATCATATAllLl~--llAACGACGA-l~l~llAATTGT
GCTAGAAAAATGTCGA~i1lnL~iLlnlAGTATCTACACAAACGCAATATTT
TTTAGCGGCAATGCCATCGGACGAAAAA~ ~l~ATAATTTTCTAAGAG
AAAGCGCGATGAGGTTAGGTAAAAGGCACAAACATTTTACTAATGGACTT
GAAGTAGTGGGAATTAAATGCTTGAAAAATAATGCGGGG~llllll~GTTG
GATGGATTTGCGTCCA~l_llAAGGGAATCGACTTTCGATAGCGAAATGT
CGTTATGGAGAGTTATTATAAACGATGTTAAGCTTAACGTCTCGCTTGGA
ACCS~-~->~os3'
TCTTCGTTTGAATGTCAAGAGCCAGLllG~lllCGAGTTTGTTTCTAGAG
TCAAGGGGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGA
ATCCTGTTGCCGGTCTTGCGATGATTATCATATAAlllCl~llGAATTAC
GTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATG
GGlllllATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAA
AC~AAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCT
nos3'1-~lacZ
ATGTTACTAGATCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTT
; TTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCT
TGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCA
nos3~
CCGATCGCCCTTCCC~ACAGTTGCGCAGCCTGAATGGCGAATGGCGCCAG
CA 02226088 1997-12-30
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24
->RBJdeleted RB
Al~:CL'1~iAAAGCGAC~li~i~ATGTTAACATCTACAAATTGCCTTTTCTTA
TCGACCATGTACGTAAGCGCTTA~ ll~lGGA~ll~AG&AAACT
GGTAG~ GGGC~l~l~~ AAGATGGATCATTAATTTCCACCT
TCACCTACGATGGGGGGCATCGCAC~-l-~AGTAATATTGTACGGCTAAG
AGCGAATTTGGCCTGTAGACCTCAATTGCGA~l-l~lAATTTCAAACTA
TTCGGGCCTAA~llll~GTGTGATGATGCTGACTGGCAGGATATATACCG
TTGTAATCGCTCTTAGC~-~lACAATATTACTCACCGGTGCGATGCCCCCC
ATCGTAGGTGAAGGTG&AAATTAATGATCCATCTTGAGACCACAGGCCCA
CAACAGCTACCAGTTTCCTCAAGG~l~-CACCAAAAACGTAAGCGCTTACG
TACAl~l~GATAAGAAAAGGCAAlll~lAGATGTTAACATCCAACGTCG
RB~del~ted RB<-~->l~cZ
CTTTCAGGGATCTGGCGCCATTCGCCATTCAGGCTGCGCAA~l~ll~GGA
AGGGCGATCGGTGCGG&CC-l~llCGCTATTACGCCAGCTGGCGAAAGGGG
GATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGG~llllCCCAGTCA
l~cZ<- I
CGAC~ll~lAAAACGACGGCCAGTGCCAAGCTTGCATGCCTGCAGGTCGA
->nos3~
TCTAGTAACATAGATGACACCGCGCGCGATAATTTATCCTAGTTTGCGCG
CTATAllllGllllCTATCGCGTATTAAATGTATAATTGCGGGACTCTAA
TCATAAAAACCCATCTCATAAATAACGTCATGCATTACATGTTAATTATT
ACATGCTTAACGTAATTCAACAGAAATTATATGATAATCATCGCAAGACC
GGCAACAGGATTCAATCTTAAGAAACTTTATTGCCAAATGTTTGAACGAT
nos3~<-¦->ACCS
CTGCCCCTTGACTCTAGAAACAAACTCGAAACCAACCTGGCTCTTGACAT
TCAAACGAAGATCCAAGCGAGACGTTAAGCTTAACATCGTTTATAATAAC
TCTCCATAACGACATTTCGCTATCGAAAGTCGATTCCCTTAAAAGTGGAC
GCAAATCCATCCAACAAAAAAGCCCCGCATTATTTTTCAAGCATTTAATT
CCCACTACTTCAAGTCCATTAGTAAAAl~lll~lGCCTTTTACCTAACCT
CATCGCGCTTTCTCTTAGAAAATTATCGACGAA,TTTTTCGTCCGATGGCA
TTGCCGCTAAAAAATATTGC~lll~l~lAGATACTAAACCGAAACTCGAC
ATTTTTCTAGCACAATTAACGACATCGTCGTTAAAAGAATATATGATTCC
GACTCTAAATCCTGGTAACCCCATGT~~ GAAAGACTGTAGACGATGT
GAACTAAATCTTTGTTGCAGTAAGTCATTTCCTGTTCATCGAGGATTTCA
GCTATACTGACGAATTGAGGCGTGTCAAAGACAGTGGCTGCGTAGATTTC
GTCACAAACAAGGTGGATGTTGTGTTGGTTGGTGAAACTCAAGACACTTT
TCAGTGTGTCTTTGTCCAAAGTGGTGCCCAATGGATTTGATGGATTGGTC
CA 02226088 1997-12-30
W O 97/OlgS2 PCT~US96/II096
AAAATcAAAccTmmmAcTTTGA~ l~Alll~l~l~L'All~l~ATATGc
ACT~l 1 ~AAGTAA~lrll~AAATTATTGGAG~l~l~ACAGT
GAATTGG~ATAAGTTGTACTCCA~ ~ATCTTAAAl~l~ lAAAT
GCTGGGTi~GTATGGTGAAGGTACTAAAAATGCA-lLG;~;~AGGATCAGCCAA
ACAAAATATAA'~ --l~ATTAGCTCCAGTGGCACCACCAGCCATAACAA
~'L~'l"l''l ~' ~ATCAAATCTAA~l~ll~-~l~-~l~-ll-~ll-l-l~-l~-CATAAAT
TTCGCAATCG~ l~AATTCAGGCAAGCCATGATAATCTTGAAAGTT
GGCAATGGCCTTGAATGATTTGAl~ AGAACAAATTGAA~;~1l-~.L'~3
GGTTT~-l~ AATCCAAl~ -l~TCAAGTCTAAACAAAGCTGAl1ll~;
ACCS<~ 35SCab22~
GCTAAACCCATGGTTTAATAAGAAGAGAAAAGAGTTL-llll~llATGGCT
GAAGTAATAGAGA~ATGAGCT-GAG~-~i~l~ClCTCCAAATGAAATGAAC
TTCCTTATATAGAGG~AGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCA
TCCC,TACGTCAGTGGAGATGTCACATCAATCCALll~-lllGAAGACGT
GGTTGGAACGTCTTClllll~CACGATGCTCCTCGTGGGTGGGGGTCCAT
CTTTGGGACCACTGTCGGCAGAGGCATCTTGAATGATAG~lll~CTTTA
TCGCAATGATGGCAll-l~lAGGAGccAcLlLC~ -lACTGTCClll~:
GATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAG~lll~CCGAAA
TTATC~-lll~ll~AAAAGTCTCAATAGCL~lllGGTL-llLlGAGACTGTA
TCTTTGACAl 111 1 GGAGTAGACCAGAGTGTCti Lt~: 1 LCACCATGTTGAC
GAAGATTTT~~ ~lCATTGAGTCGTAAAAGACTCTGTATGAACTGTT
CGCCAGTCTTCACGGCGAGll-lGllAGATCCTCGATTTGAATCTTAGAC
TCCATGCATGGCCTTAGATTCAGTAGGAACTACClllllAGAGACTCCAA
TCTCTATTACTTGCCTTGGTTTATGAAGCAAGCCTTGAATCGTCCATACT
GGAATAGTACTTCTGATCTTGAGAAATATGTCTTTCTCTGTGTTCTTGAT
GC~ATTAGTCCTGAATCTTTTGACTGCATCTTTAACCTTCTTGGGAAGGT
ATTTGATCTCCTGGAGAll~llACTCGG~TAGATCGTCTTGATGAGACCT
GCTGCGTAGGCCTCTCTAACCATCTGTGGGTCAGCATTCTTTCTGAAATT
GAAGAGGCTAACCTTCTCATTATCAGTGGTGAACATAGTGTCGTCACCTT
CACCTTCGAACTTCCTTCCTAGATCGTAAAGATAGAGGAAATCGTCCATT
359Cnb22~ ->l~cZ
GTAATCTCCGGGGCAAAGGAGATCCCGGGTACCGAGCTCGAATTCGTAAT
CATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCA
CACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATG
AGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT
CGG&AAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGG
CA 02226088 1997-12-30
W O 97/01952 PCT~US96/11096
26
lacZ<-I->oc 8 3'
AGAGG~lll~CGTATTGGGCAGCGGCCGCTGTTA~C~C~C~l~C
TGGACCGGGTTGAATGGTGCCCGTAA~ll-l~lAGAGCGGACC~-~AAT
ACTCAACTTCAAGGAATCTCACCCATGCGCGCCGGCGGGGAACCGGAGTT
C-~l-l~AGTGAACGTTATTA~llC~lC~l~l~l~-~lAGATACTAG
C;~C~ i~ G~LllllGAAATTTGAATAAGATTTATGTAATCA~l~ lA
w lllGACCG~ll~l-G~-~lll-ll-l-lAAAATTGGATTTGTAATAATAAA
ACGCAAl-l~illl~llAll~ iLl~ ATCATAGATGTCGCTATAAACC
TATTCAGCACAATATALl-~~ CATTTTAATATTGTACATATAAGTAGT
AGGGTACAATCAGTAAATTGAACGGAGAATATTATTCATAAAAATACGAT
AGTAACGGGTGATATATTCATTAGAATGAACCGAAACCGGCGGTAAGG~T
CTGAGCTACACATGCTCA~Gi-i.lllACAACGTGCACAACAGAATTGAAA
ocs3'<-~->NPTII
GCAAATATCATGCGATCATAGGCGTCTCGCATATCTCATTAAAGCAGGGG
GTGGGCGAAGAACTCCAGCATGAGATCC~-C~CGCTGGAGGATCATCCAGC
CGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCG
GTGGAATCGAAATCTCGTGATGGCA~G-l-lG~CGTCGCTTGGTCGGTCAT
TTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAA
GGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGC
GGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAAC
GCTATGTCCTGATAGCG~lCCGCCACACCCAGCCGGCCACAGTCGATGAA
TCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGC
CATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCT-
GCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATC
CTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGAT
GTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGC
CGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTG
AGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCC
TTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTC
GTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGC
ACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACA
GCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCA
TAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAG~CCTGCGTGCAATCC
~ TII~-
ATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATC
C
