U.S. patent application number 10/737164 was filed with the patent office on 2005-04-07 for nor gene compositions and methods for use thereof.
Invention is credited to Giovannoni, James, Noensie, Frederick, Tanksley, Steven, Vrebalov, Julia.
Application Number | 20050076410 10/737164 |
Document ID | / |
Family ID | 32684525 |
Filed Date | 2005-04-07 |
United States Patent
Application |
20050076410 |
Kind Code |
A1 |
Giovannoni, James ; et
al. |
April 7, 2005 |
NOR gene compositions and methods for use thereof
Abstract
The current invention provides nucleic acid sequences encoding
the NOR gene. Compositions comprising this sequence are described,
as are plants transformed with such compositions. Further provided
are methods for the expression of the NOR gene. The methods of the
invention include the direct creation of transgenic plants with the
NOR gene by genetic transformation, as well as by plant breeding
methods. The sequences of the invention represent a valuable new
tool for the creation of transgenic plants, preferably having one
or more added beneficial characteristics.
Inventors: |
Giovannoni, James; (Ithaca,
NY) ; Tanksley, Steven; (Ithaca, NY) ;
Vrebalov, Julia; (Ithaca, NY) ; Noensie,
Frederick; (New York, NY) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
32684525 |
Appl. No.: |
10/737164 |
Filed: |
December 16, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10737164 |
Dec 16, 2003 |
|
|
|
09614408 |
Jul 12, 2000 |
|
|
|
6762347 |
|
|
|
|
60143357 |
Jul 12, 1999 |
|
|
|
Current U.S.
Class: |
800/288 ;
435/419; 435/468; 536/23.2 |
Current CPC
Class: |
C12N 15/8249 20130101;
C12N 15/825 20130101; C07K 14/415 20130101; C12N 15/8291
20130101 |
Class at
Publication: |
800/288 ;
536/023.2; 435/419; 435/468 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C12N 005/04 |
Goverment Interests
[0002] The government may own rights in this invention subject to
grant numbers USDA-NRICGP 92-373000-7653, USDA-NRICGP
92-373000-1575, Texas Advanced Technology Program 999902037, and
USDA-NRICGP 91-373000-6418.
Claims
1-64. (canceled)
65. An isolated nucleic acid sequence comprising an antisense
oligonucleotide complementary to a NOR gene mRNA encoded by SEQ ID
NO:7.
66. The isolated nucleic acid of claim 65, further comprising a
promoter operably linked to said antisense oligonucleotide.
67. The isolated nucleic acid of claim 65, further comprising an
enhancer.
68. The isolated nucleic acid of claim 67, wherein said enhancer
comprises an intron.
69. The isolated nucleic acid of claim 65, comprising a
transcriptional terminator.
70. An expression vector comprising the isolated nucleic acid
sequence of claim 65.
71. The expression vector of claim 70, further defined as a linear
nucleic acid segment.
72. The expression vector of claim 70, further defined as a plasmid
vector.
73. A plant transformed with the isolated nucleic acid sequence of
claim 65.
74. The transgenic plant of claim 73, further defined as a fertile
R.sub.0 transgenic plant.
75. The transgenic plant of claim 73, further defined as a progeny
plant of any generation of a fertile R.sub.0 transgenic plant,
wherein the progeny plant comprises the isolated nucleic acid
sequence.
76. A seed of the plant of claim 73, wherein the seed comprises the
isolated nucleic acid sequence.
77. A cell of the plant of claim 73.
78. The plant of claim 73, further defined as strawberry.
79. A method of manipulating the fruit ripening of a plant
comprising introducing the expression vector of claim 70 into the
plant.
80. The method of claim 79, wherein the expression vector is
introduced by a method comprising the steps of: (a) obtaining the
expression vector of claim 70; (b) transforming a recipient plant
cell with said expression vector; and (c) regenerating a transgenic
plant from said recipient plant cell, wherein the fruit ripening
phenotype of said plant is altered based on the expression of the
antisense oligonucleotide.
81. The method of claim 79, wherein the expression vector is
introduced by a method comprising the steps of: (a) obtaining a
transgenic plant comprising the expression vector of claim 70; and
(b) crossing the transgenic plant with itself or a second plant to
introduce the expression vector into a progeny plant.
82. The method of claim 79, wherein the plant is strawberry.
Description
[0001] This application claims the priority of U.S. Provisional
Application Ser. No. 60/143,357, filed Jul. 12, 1999, the
disclosure of which is specifically incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the NOR gene.
More specifically, it relates to methods and compositions for the
modification of plant phenotypes with the NOR gene.
[0005] 2. Description of the Related Art
[0006] The ripe phenotype is the summation of biochemical and
physiological changes occurring at the terminal stage of fruit
development rendering the organ edible and desirable to seed
dispersing animals and valuable as an agricultural commodity. These
changes, although variable among species, generally include
modification of cell wall ultrastructure and texture, conversion of
starch to sugars, increased susceptibility to post-harvest
pathogens, alterations in pigment biosynthesis/accumulation, and
heightened levels of flavor and aromatic volatiles (Rhodes, 1980;
Hobson and Grierson, 1993). Several of theses ripening attributes
translate to decreased shelf-life and high input harvest, shipping
and storage practices, particularly via changes in firmness and the
overall decrease in resistance to microbial infection of ripe
fruit. Currently acceptable techniques for minimizing the
consequences of undesirable ripening characteristics include
premature harvest, controlled atmosphere storage, pesticide
application, and chemically induced ripening to synchronize the
timing of maturation. Unfortunately, added production, shipping and
processing expenses, in addition to reduced fruit quality, are
often the consequence of these practices, challenging both the
competitiveness and long term sustainability of current levels of
crop production.
[0007] Although most fruit display modifications in color, texture,
flavor, and pathogen susceptibility during maturation, two major
classifications of ripening fruit, climacteric and non-climacteric,
have been utilized to distinguish fruit on the basis of respiration
and ethylene biosynthesis rates. Climacteric fruit such as tomato,
cucurbits, avocado, banana, peaches, plums, and apples, are
distinguished from non-climacteric fruits such as strawberry, grape
and citrus, by their increased respiration and ethylene
biosynthesis rates during ripening (Grierson, 1986). Ethylene has
been shown to be necessary for the coordination and completion of
ripening in climacteric fruit via analysis of inhibitors of
ethylene biosynthesis and perception (Yang, 1985; Tucker and Brady,
1987), in transgenic plants blocked in ethylene biosynthesis (Klee
et al., 1991; Oeller et al., 1991; Picton et al., 1993 a), and
through examination of the Never-ripe (Nr) ethylene perception
mutant of tomato (Lanahan et al., 1994).
[0008] Considerable attention has been directed toward elucidating
the molecular basis of ripening in the model system of tomato
during recent years (reviewed in Spiers and Brady, 1991; Gray et
al., 1992 and 1994; Giovannoni, 1993; Theologis 1992 and Theologis
et al., 1993). The critical role of ethylene in coordinating
climacteric ripening at the molecular level was first observed via
analysis of ethylene inducible ripening-related gene expression
(Tucker and Laties, 1984; Lincoln et al., 1987; Maunders et al.,
1987; DellaPenna et al., 1989; Starrett and Laties; 1993). Several
ripening genes, including ACC synthase and ACC oxidase, have been
shown via antisense gene repression to have profound influences on
the onset and degree of ripening (Hamilton et al., 1990; Oeller et
al., 1991). Although the sum effect of this research has been a
wealth of information pertaining to the regulation of ethylene
biosynthesis and its role in ripening, the molecular basis of
developmental cues which initiate ripening-related ethylene
biosynthesis, and additional aspects of ripening not directly
influenced by ethylene, remain largely unknown (Theologis et al.,
1993).
[0009] Single locus mutations which attenuate or arrest the normal
ripening process, and do not ripen in response to exogenous
ethylene, have been identified in tomato and are likely to
represent lesions in regulatory components necessary for initiation
of the ripening cascade, including ethylene biosynthesis
(Tigchelaar et al., 1978; Grierson, 1987; Giovannoni, 1993; Hobson
and Grierson, 1993; Gray et al., 1994). One such mutation, the Nr
mutation, has been identified and represents a gene responsible for
ethylene perception and/or signal transduction and is a tomato
homologue of the Arabidopsis Ethylene response 1 (Etr1) gene (Yen
et al., 1995; Wilkinson et al., 1995).
[0010] Tomato has served as a model for ripening of climacteric
fruit. Ripening-related genes have been isolated via differential
gene expression patterns (Slater et al., 1985, Lincoln et al.,
1987, Pear et al., 1989, Picton et al., 1993b) and biochemical
function (DellaPenna et al., 1986; Sheehy et al., 1987; Ray et al.,
1988; Biggs and Handa, 1989; Harriman and Handa, 1991; Oeller et
al., 1991; Yelle et al., 1991). Promoter analysis of ripening genes
has been performed via examination of promoter/reporter construct
activities in transient assay systems and transgenic plants. The
result has been the identification of cis-acting promoter elements
which are responsible for both ethylene and non-ethylene regulated
aspects of ripening (Deikman et al., 1992; Montgomery et al.,
1993). Trans-acting factors which interact with these promoters
also have been identified via gel-shift and footprint experiments,
although none have been isolated or cloned (Deikman and Fischer,
1988; Cordes et al., 1989; Montgomery et al., 1993).
[0011] The in vivo functions of several ripening-related genes
including polygalacturonase, pectinmethylesterase, ACC synthase,
ACC oxidase, and phytoene synthase have been tested via antisense
gene repression and/or mutant complementation in transgenic
tomatoes. For example, the cell wall pectinase, polygalacturonase,
was shown to be necessary for ripening-related pectin
depolymerization and pathogen susceptibility, however, the
inhibition of PG expression had minimal effects on fruit softening
(Smith et al., 1988, Giovannoni et al., 1989, Kramer et al., 1990).
Significant reduction in rates of ethylene evolution resulting in
inhibition of most ripening characteristics was observed in both
ACC synthase and ACC oxidase antisense mutants (Oeller et al.,
1991; Hamilton et al., 1990). Non-ripening antisense fruit were
subsequently restored to normal ripening phenotype with the
application of exogenous ethylene.
[0012] Further analysis of transgenic tomatoes inhibited in
ethylene biosynthesis demonstrates that climacteric ripening
represents a combination of both ethylene mediated and
developmental control (Theologis et al., 1993). Although antisense
ACC synthase tomatoes which failed to produce ethylene did not
ripen, gene expression analysis demonstrated that several
ripening-related genes, including polygalacturonase and E8 are
expressed in the absence of ethylene. This observation confirms the
presence of a developmental (or non-ethylene regulated) component
of ripening. In fact, an ethylene requirement was observed for
translation but not transcription of polygalacturonase mRNA,
suggesting interaction between ethylene and non-ethylene components
of ripening for expression of at least a subset of ripening genes
(Theologis et al., 1993).
[0013] While the above studies have provided some insight into the
ripening process in plants, there is still a great need in the art
for novel methods and compositions for the creation of plants
having enhanced phenotypes. In particular, there is a need in the
art for the isolation the RIN and NOR genes. The isolation of these
genes would allow the creation of novel transgenic plants altered
in their fruit characteristics and/or ethylene responsiveness, and
having one or more added beneficial properties.
SUMMARY OF THE INVENTION
[0014] In one aspect, the current invention provides an isolated
nucleic acid sequence comprising the NOR gene. In one embodiment of
the invention, the NOR gene may be further defined as isolatable
from the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:6 or SEQ
ID NO:7. In particular embodiments of the invention, the invention
provides an isolated nucleic acid corresponding to an open reading
frame of the NOR cDNA, for example, which may be denoted by the
nucleotides as indicated by bold letters in FIG. 6.
[0015] In another aspect, the invention provides an isolated
nucleic acid sequence having from about 17 to about 1209, about 25
to about 1209, about 30 to about 1209, about 40 to about 1209,
about 60 to about 1209, about 100 to about 1209, about 200 to about
1209, about 400 to about 1209, about 600 to about 1209, about 800
to about 1209, or about 1000 to about 1209 contiguous nucleotides
of the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7.
Similarly, the invention provides such nucleic acid segments from
SEQ ID NO:1. In particular embodiments of the invention, the
nucleic acid sequences of SEQ ID NO:6 and SEQ ID NO:7 are provided.
In particular embodiments of the invention, a nucleic acid sequence
of the invention may further comprising an enhancer, such as an
intron. A nucleic acid sequence of the invention may also include a
transcriptional terminator. Such sequences may be native to the NOR
gene or heterologous from potentially any species.
[0016] In yet another aspect, the invention provides an expression
vector comprising a NOR gene. Such a NOR gene may be in accordance
with any of the NOR-containing sequences described herein. The
expression vector may comprise the NOR gene operably linked to a
native or heterologous promoter, either in sense or antisense
orientation relative to the promoter. Potentially any heterologous
promoter may be used, for example, a promoter is selected from the
group consisting of CaMV .sup.35S, CaMV 19S, nos, Adh, actin,
histone, ribulose bisphosphate carboxylase, R-allele, root cell
promoter, .alpha.-tubulin, ABA-inducible promoter, turgor-inducible
promoter, rbcS, corn sucrose synthetase 1, corn alcohol
dehydrogenase 1, corn light harvesting complex, corn heat shock
protein, pea small subunit RuBP carboxylase, Ti plasmid mannopine
synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase,
bean glycine rich protein 1, CaMV 35s transcript, Potato patatin,
actin, cab, PEPCase and S-E9 small subunit RuBP carboxylase
promoter. In still further embodiments of the invention, the
expression vector may comprise any selectable marker, for example,
a selectable marker selected from the group consisting of
phosphinothricin acetyltransferase, glyphosate resistant EPSPS,
aminoglycoside phosphotransferase, hygromycin phosphotransferase,
neomycin phosphotransferase, dalapon dehalogenase, bromoxynil
resistant nitrilase, anthranilate synthase and glyphosate
oxidoreductase.
[0017] The expression vector may be either circular, for example,
as in the case of a plasmid vector, or could be a linear nucleic
acid segment, such as an expression cassette isolated from a
plasmid. In particular embodiments of the invention, the vector is
a plasmid vector. The expression vector may further comprise other
elements, such as a nucleic acid sequence encoding a transit
peptide, or potentially any terminator, for example, a heterologous
terminator such as the nos terminator.
[0018] In still yet another aspect, the invention provides a
transgenic plant comprising a stably transformed expression vector,
such as those described above. The transgenic plant may be any type
of plant, and in particular embodiments of the invention is a
tomato plant. In further embodiments of the invention, the
transgenic plant may be a fertile R.sub.0 transgenic plant. Also
included in the invention is a seed of such a fertile R.sub.0
transgenic plant, wherein said seed comprises said expression
vector. The transgenic plant may be a progeny plant of any
generation of a fertile R.sub.0 transgenic plant, wherein said
R.sub.0 transgenic plant comprises said expression vector. The
invention also includes a seed of such a progeny plant, wherein
said seed comprises said expression vector.
[0019] In still yet another aspect, the invention provides a
crossed fertile transgenic plant prepared according to the method
comprising the steps of: (i) obtaining a fertile transgenic plant
comprising a selected DNA comprising a NOR gene; (ii) crossing said
fertile transgenic plant with itself or with a second plant lacking
said selected DNA to prepare the seed of a crossed fertile
transgenic plant, wherein said seed comprises said selected DNA;
and (iii) planting said seed to obtain a crossed fertile transgenic
plant. In one embodiment of the invention, a seed is provided of
such a crossed fertile transgenic plant, wherein said seed
comprises said selected DNA. The crossed fertile transgenic plant
may be of any species, for example, a tomato plant. The plant may
also be inbred or hybrid.
[0020] In still yet another aspect, the invention provides a method
of manipulating the phenotype of a plant comprising the steps of:
(i) obtaining an expression vector comprising a NOR gene in sense
or antisense orientation; (ii) transforming a recipient plant cell
with said expression vector; and (iii) regenerating a transgenic
plant from said recipient plant cell, wherein the phenotype of said
plant is altered based on the expression of said NOR gene in sense
or antisense orientation. Any method of transforming a plant may be
used in accordance with the invention, including, microprojectile
bombardment, PEG mediated transformation of protoplasts,
electroporation, silicon carbide fiber mediated transformation, or
Agrobacterium-mediated transformation. In particular embodiments of
the invention, Agrobacterium-mediated transformation is used and
the plant is a tomato plant.
[0021] In still yet another aspect, the invention provides a method
of plant breeding comprising the steps of: (i) obtaining a
transgenic plant comprising a selected DNA comprising a NOR gene;
and (ii) crossing said transgenic plant with itself or a second
plant. The plant may be of any species and may be inbred or hybrid.
In particular embodiments of the invention, this method further
comprises the steps of: (iii) collecting seeds resulting from said
crossing; (iv) growing said seeds to produce progeny plants; (v)
identifying a progeny plant comprising said selected DNA; and (vi)
crossing said progeny plant with itself or a third plant. In one
embodiment of the invention, the second plant and third plant are
of the same genotype. The second and third plants may also be
inbred plants.
[0022] In still yet another aspect, the invention provides a
transgenic plant cell stably transformed with a selected DNA
comprising a NOR gene. The cell may be from any plant species, for
example, a cell from a tomato plant. The selected may comprise any
of the NOR gene comprising nucleic acid compositions disclosed
herein, for example, the expression vector compositions described
herein above. Such compositions include the open reading frame of
the NOR gene, as provided in SEQ ID NO:6 or SEQ ID NO:7 and
demarcated in FIG. 6.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein. The file of this patent contains at least one
drawing executed in color. Copies of this patent with color
drawing(s) will be provided by the Patent and Trademark Office upon
request and payment of the necessary fee.
[0024] FIG. 1. T-DNA constructs for delivery of sense or antisense
NOR gene cDNA (CD-11) sequences into plant genomes. The base
plasmid, termed NOR-pBI121 Sense/Antisense (Kanamicin
resistant--NPTII), had an approximate size of 13.0 kb.
Abbreviations are as follows: FB, LB: right and left T-DNA borders,
respectively; Nos-pro: nopaline synthase promoter driving
expression of the NPTII gene; NPTII: neomycin phosphotransferase
(kanamycin resistance) gene; nos-ter: transaction termination
sequence from the nopaline synthase gene; HindIII, SphI, PstI,
Xbal, BamHI, SmaI, EcoRI, SpeI, SacI: DNA restriction endonucleases
(enzymes); and Sma/EcoRV: the resulting chimeric sequence is
recognized by neither enzyme.
[0025] FIG. 2. Manipulation of fruit ripening and carotenoid
accumulation with the tomato NOR gene. Shown are representative
control and transformed fruit from tomato a line of the genotype
nor/nor in the cultivar MH1 and transformed with NOR-pBI121 Sense
(FIG. 1). Primary transformants (T0) were confirmed for transgene
integration via DNA gel-blot analysis and subsequently
self-pollinated. Resulting seed were harvested and grown (T1
generation) and analyzed for transgene segregation. Representative
fully mature fruit from T1 nor/nor individuals that either harbor
the sense NOR transgene (+) or have segregated it out (-) are
shown. In summary, transgene expression in the mutant background
partially recovers the non-ripening phenotype and confers ripening.
In this particular line, relatively low expression of the transgene
was observed as compared to expression of NOR in normally ripening
(Nor/Nor) fruit. Representative normal (Nor/Nor) and nearly
isogenic mutant (nor/nor) cultivar MH1 tomato fruit are shown as
controls. The partial recovery of ripening in the nor/nor fruit
harboring the NOR-pBI121 (+) transgene verified the isolation of
the NOR gene. Furthermore, the partial ripening phenotype observed
in this line demonstrated that regulated expression of the NOR gene
can be used to create a range of degrees of ripening and
ripening-associated characteristics (e.g., carotenoid accumulation,
ripe flavor, nutrient composition, softness, pathogen
susceptibility).
[0026] FIG. 3. DNA agarose gel showing the Nor versus nor alleles
as PCR amplification products. Genomic DNA was isolated from normal
(N/N) and homozygous nor mutant (n/n) nearly isogenic control lines
(cultivar MH1), in addition to individuals from a Nor/Nor X Nor/nor
back-cross (BC) population. DNA was amplified with one PCR primer
common to the coding region of both the Nor and nor alleles and
separately with either one primer specific to the Nor or nor
alleles, respectively. The allele-specific primers were based on
the 2 bp deletion which distinguished the normal (Nor) versus
mutant (nor) allele (see FIG. 5). PCR reactions with the normal
(Nor) allele primer were loaded on the top portion of the gel, and
those employing the mutant (nor) allele primer were loaded on the
bottom portion of the gel. PCR reactions from the same individual
plant but amplified separately with each allele-specific primer
were loaded directly above and below each other to facilitate
scoring. The normal (N) and mutant (n) alleles are indicated above
each lane and represent the corresponding genotype as determined by
analysis of band amplification.
[0027] FIG. 4. Expression of the NOR gene through plant development
and in normal and mutant fruit. RNA gel-blot analysis of expression
using the NOR full-length cDNA as probe. Expression was induced in
the transition from mature green to breaker fruit but was not
detected in any additional tissues examined including combined
cotyledons and hypocotyls (C/H), leaves, senescing leaves (S/Leaf),
stems or roots. It was noted that expression was also reduced in
identically aged nor (nor/nor) mutant fruit.
[0028] FIG. 5. DNA sequence of the region of tomato chromosome 10
harboring the NOR gene (SEQ ID NO:7). The sense genomic DNA
sequence of the complete transcribed region is shown in the 5'-3'
orientation. The coding sequence is in upper case while
non-translated sequences including the two NOR gene introns are in
lower case.
[0029] FIG. 6. Corrected DNA sequence of the NOR full-length cDNA
(CD-11) (SEQ ID NO:6). The full cDNA sequence is shown in 5'-3'
orientation. Lower case letters refer to non-translated portions of
the transcript while the upper case letters refer to the translated
(coding) sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The ripening of fleshy fruits represents a system of
eukaryotic development unique to plants as well as an important
component of agricultural quality and productivity. Greater
understanding of the genetic and molecular basis of the ripening
process will promote both our collective understanding of plant
development and yield tools useful for sustaining and improving
agricultural productivity and quality, while minimizing impact on
the resources necessary for production. The current invention
provides such understanding by providing the nucleic acids encoding
the NOR gene. By providing these sequences, the invention provides,
for the first time, the ability to use genetic transformation
techniques to manipulate a variety of plant characteristics which
are associated with these genes in ways that cannot be accomplished
via traditional breeding strategies including direct DNA transfer
to species other than tomato.
[0031] In tomato, ripening occurs over a period of several days,
depending on variety, and is characterized by softening, pectin
solubilization, increased respiration and ethylene biosynthesis,
enhanced pathogen susceptibility, heightened palatability, and
accumulation of the characteristic red and orange carotenoid
pigments lycopene and beta-carotene, respectively. NOR and also the
fruit ripening gene RIN segregate as single traits, result in
nearly complete inhibition of normal ripening as defined above, and
their effects on ripening cannot be restored via application of
exogenous ethylene (Tigchelaar et al., 1978). The ripening
phenotypes displayed by RIN and NOR demonstrate that the gene
products encoded by the normal alleles at these loci are involved
in the primary regulation of ripening (Hobson and Grierson, 1993;
Giovannoni 1993; Gray et al., 1994). Because virtually nothing is
known of the expression patterns or biochemical nature of the
normal NOR gene product, the inventors initiated a genetic
map-based cloning strategy for isolation of the corresponding
normal allele. All of the prerequisite tools for implementation of
this strategy are available in tomato including 1) the mutations
themselves, 2) DNA markers tightly linked to both rin and nor, 3)
large populations (>300 F2 progeny) segregating for target loci,
4) a library of high molecular weight tomato genomic DNA, and 5)
gene transfer technology for verification of cloned target genes
via complementation of the recessive phenotype with the dominant
allele.
[0032] Tomato has served for decades as a model system for both
plant genetics and fruit ripening, in part resulting in the
availability of the tools for ripening gene isolation mentioned
above. Numerous mutations regulating various aspects of tomato
fruit ripening have been identified over the years, most of which
result in alteration of pigment biosynthesis and/or accumulation
without significant effects on additional ripening characteristics
(Rick, 1980; Grierson, 1986; Gray et al., 1994). Examples include
the greenflesh (gf; Ramirez and Tomes, 1964) and yellowflesh (r;
Darby, 1978) mutants which inhibit ripening-related chlorophyll
degradation and lycopene accumulation, respectively. Tomato
mutations exerting complete or nearly complete inhibition of
overall ripening (i.e. blocking changes not just in color but also
texture, ethylene biosynthesis, pathogen susceptibility, flavor and
aroma) are few, the most extreme being rin and nor.
[0033] Neither mutation exerts any observable influence on aspects
of plant development or morphology other than ripening, suggesting
regulatory roles limited primarily to fruit development (the rin
mutation is associated with the mc or macrocalyx phenotype;
however, genetic evidence indicates that the rin mutant is actually
a double mutant at the linked RIN and MC loci Robinson and Tomes,
1968)). Fruit homozygous for either rin or nor are similar in
phenotype in that they attain full size, produce viable seed, yet
remain firm and green for weeks after normal fruit ripen. In
addition, homozygous rin and nor mutant fruit fail to display
climacteric respiration and ethylene biosynthesis characteristic of
normally ripening tomatoes (Tigchelaar et al., 1978), are highly
resistant to microbial infection (Grierson, 1986), and are
inhibited in their expression of ripening-related genes (DellaPenna
et al., 1989; Picton et al., 1993). Although often referred to as
recessive mutations, both rin and nor heterozygotes show
significant effects on some ripening parameters, including reduced
pathogen susceptibility and softening, resulting in extended
shelf-life (Tigchelaar et al., 1978; Biggs and Handa, 1989). For
this reason, heterozygosity at the RIN locus in particular has seen
increased commercial application in fresh market hybrids. Isolation
of the RIN and NOR genes by the current inventors permits
optimization of controlled ripening via controlled expression in
tomato and potentially other fruit crop species. From a broader
standpoint, the cloned RIN and NOR genes serve as cornerstones from
which to build a model system for analysis of the developmental
regulation of fruit ripening control.
[0034] I. Rationale and Significance of the Invention
[0035] Ripening is a unique and important plant process whose
understanding has great significance in the agricultural arts.
Isolation of genes regulating both the ethylene and non-ethylene
mediated components of fruit ripening represents an important step
in understanding the genetic basis of this complex developmental
pathway. Although most research emphasis in recent years has been
focused on elucidating the biosynthesis and function of ethylene
during climacteric fruit ripening, the genetic and molecular basis
of the developmental regulators which initiate ripening ethylene
biosynthesis, and control the non-ethylene mediated ripening
pathway, had remained a mystery. The mutant phenotypes of the
targeted nor locus demonstrates that this gene is essential for
normal ripening to occur and is a developmental regulator both of
ethylene biosynthesis and non-ethylene regulated aspects of
ripening. In addition, this gene may be related to those involved
in the regulation of other developmental programs. Insights gained
into the ripening process as a result of the current invention will
not only aid our understanding of overall plant development, but
may enhance understanding of developmental processes in other
eukaryotes as well.
[0036] From the standpoint of agriculture, ripening confers both
positive and negative attributes to the resulting commodity. While
ripening imparts desirable flavor, color, and texture, considerable
expense and crop loss result as a consequence of negative ripening
characteristics. For example, ripening related increases in fruit
pathogen susceptibility is a major contributor to fruit loss both
before and after harvest. This genetically regulated change in
fruit physiology currently necessitates the use of pesticides,
post-harvest fumigants, and controlled atmosphere storage and
shipping mechanisms in attempts to minimize loss. In addition to
being wasteful of energy and potentially harmful to the
environment, such practices represent major expenses in fruit
production.
[0037] The current inventors, however, have isolated the ripening
regulatory gene NOR, which allows for the first time the genetic
enhancement through manipulation of genes of positive ripening
attributes and reduction of undesirable qualities in tomato and
additional species. The ability to improve fruit quality while
reducing energy use, production costs, and environmental impact
will promote the long term productivity and sustainability of
commercial agriculture.
[0038] The current inventors employed a map-based cloning approach
for the isolation of the normal NOR locus in tomato. Tomato is the
best available system for the map-based cloning of ripening genes
because of the availability of: 1) single locus mutations
inhibiting normal fruit ripening, 2) a high density RFLP map, 3)
large populations segregating for targeted ripening loci, 4) a YAC
library, and 5) established procedures for transformation and
regeneration. Also, gene products have not been identified for
either of the target genes, thus precluding immunological cloning
strategies. In addition, previous to the invention one could only
speculate concerning patterns of normal NOR gene expression, thus
exacerbating the already difficult task of identifying appropriate
stages for differential screening strategies. Therefore, the
current invention represents a major advance over the prior art,
potentially allowing for the first time the creation of transgenic
plants having greatly enhanced agronomic characteristics.
[0039] II. Alteration of Plant Phenotypes with NOR Nucleic Acid
Compositions
[0040] (i) NOR Gene Function
[0041] The effects and thus potential uses of the NOR
(non-ripening) gene can be deduced from analysis of the well
characterized mutation at the nor locus (see Tigchellar et al.,
1978 and Giovannoni, 1993 for review). Further, the inventors have
shown that the NOR gene mutation (nor) greatly inhibits the
ripening process with minimal effects on other plant tissues or
even fruit prior to the onset of ripening. Consequently, the use of
the NOR gene may be indicated for manipulation of fruit ripening.
It is also apparent not only from the mutant phenotypes but also
from the transgenic expression of the NOR gene (i.e., expression is
primarily restricted to the tissues in which effects are observed,
fruits) that normal effects are centered on the developing flower,
specifically, the carpels (fruit). Nevertheless, manipulation of
NOR gene in non-fruit tissues via the tools of biotechnology could
be expected to yield various potentially useful effects in
non-fruit tissues as well (see examples below). It should be noted
that subsequent reference to "normal" and "mutant" refers to the
genotypes Nor/Nor and nor/nor, respectively.
[0042] Fruit ripening is a complex process ultimately rendering the
fruit palatable and/or susceptible to biotic or abiotic process
which result in seed liberation and dispersal. While specific
ripening attributes vary among species, the following general
process are common to many fruits (see Seymour et al., 1993 for
review), including tomato, and are all have been shown to be
influenced by the NOR gene via characterization of the
corresponding mutant:
[0043] A) Degradation of the photosynthetic pigment chlorophyll and
accumulation of various pigment compounds (often carotenoids and
flavonoids) resulting in changes of both color and nutritional
composition (Tigchellar et al., 1978; Yen et al., 1997).
[0044] B) Changes in cell wall metabolism and architecture
resulting in effects on texture and susceptibility to pathogen
infection with additional impacts on specific aspects of processing
qualities including viscosity and texture of whole and
chopped/pureed products (Tigchellar et al., 1978).
[0045] C) Changes in carbohydrate metabolism including the
conversion of starch to simple sugars (Seymour et al., 1993).
[0046] D) Changes in aroma and production of associated volatile
compounds.
[0047] E) Changes in ethylene hormone biosynthesis and perception
(DellaPenna et al., 1989) which directly influence many of the
specific ripening attributes mentioned here but may also impact
these and other areas via mechanisms not described above. Such
processes include effects on pathogen susceptibility, senescence,
abscission, seed germination, flowering, sex determination in
cucurbits and general stress responses (temperature, drought,
mechanical damage) See Ables et al., 1992 for review.
[0048] Previous observations, some of which are referenced above,
confirm the function of the NOR gene in most aspects of fruit
ripening and suggest that additional aspects of plant growth,
development and response to the environment could be altered via
expression of this gene in other plant tissues via alternate
promoters. As such, alteration of any of the forgoing phenotypes,
as well as other phenotypes conferred by the NOR gene, as well as
plants altered in such ways, specifically form a part of the
instant invention.
[0049] (ii) Examples of NOR Gene Use.
[0050] The NOR gene compositions provided by the inventors may find
numerous uses in manipulation of plant phenotypes. Exemplary uses
for the NOR gene are described herein below, although those of
skill in the art will recognize that the examples are in no way
limiting.
[0051] 1. Control of Fruit Ripening and Quality
[0052] Though currently less widely used than the tomato rin
(ripening-inhibitor) mutation, the nor mutation is currently used
in tomato breeding for development of hybrid lines with
slow-ripening/long-shelf-life characteristics. The NOR gene could
similarly be used for manipulation and control of ripening with
potential for accelerated ripening of important early season crops,
controlled or delayed ripening of crops permitting longer shipping
handling, storage and post-retail shelf-life. The fact that the
inventors have provided the cloned NOR gene will permit its
utilization in species other than tomato. Specific examples of use
would be in accelerated ripening of early season melons for
favorable market position and pricing, and ripening control of
bananas and strawberry--fruits which typically have short
shelf-lives making shipping and handling more costly.
[0053] Next, modified expression of the NOR gene in ripening fruits
may find use in elevating levels of important processing and
nutritional compounds such as antioxidant flavonoids and
carotenoids in fruits and non-fruit tissues. An example would be
potential over-expression in maize seeds to enhance accumulation of
antioxidant compounds for nutritional enhancement of the crop or
for extraction.
[0054] Finally, expression of NOR gene orthologues (functional
equivalents) in other species may regulate maturation of seed pods
(which are also carpels or "fruits", for example in soybean, pea,
common bean) and/or cereal grains (e.g., rice, maize, wheat,
sorghum). Thus over-expression or repression of the NOR gene may be
useful in controlling maturity and maturation time of various
cereals. Protracted or accelerated maturation via manipulation of
the NOR gene may additionally impact quality characteristics such
as total protein content, carbohydrate loading, nutritional
composition (e.g., via altered levels of carotenoids such as
beta-carotene and lycopene) and total yield.
[0055] In support of this example, the inventors have developed
T-DNA constructs (FIG. 1) for altering expression of the NOR gene
and have transformed such constructs into normal and mutant tomato
genotypes. FIG. 2 shows that delivery of the normal Nor allele into
the genome of mutant plants results in conversion of fruit from
unripe to ripe and results in a range of degrees of ripening and
pigment accumulation.
[0056] 2. Control of Senescence
[0057] The NOR gene controls fruit senescence as demonstrated by
the lack of senescence in tomato fruits harboring the nor mutation.
Senescence or tissue death is thus clearly regulated by NOR in
fruit and may be manipulated in non-fruit tissues via regulated
expression of the NOR gene. Examples of use may include late
fruit-ripening repression in banana or other tropical or
sub-tropical fruits subject to rapid decay to permit desirable
ripening but not advanced tissue damage reducing fruit quality and
desirability. Over-expression in anthers may result in senescence
yielding male-sterility, while if this gene is normally expressed
in other senescing tissues, gene repression may be useful to
inhibit senescence for example in vegetables (spinach, lettuce,
cabbage, broccoli).
[0058] Studies of the mutant nor phenotype have shown that the nor
mutation effects fruit senescence. The inventor's studies
comprising the cloning of the NOR gene and development and
observation of transgenic tomatoes confirm that the NOR gene
confers regulation of fruit ripening and senescence (FIG. 2), and
suggest the use of NOR gene nucleic acid compositions for
modification of fruit senescence.
[0059] 3. Control of Pathogen Infection.
[0060] Fruit tissue from nor mutant tomato plants are highly
resistant to infection by opportunistic microbial pathogens
(Tigchellar et al., 1978). Post-ripening repression (antisense or
co-suppression) of the gene in tomato, or other species (apple,
pear, peach, strawberry, citrus. etc), could thus be useful in
inhibiting subsequent over-ripening and pathogen susceptibility of
fruit. Along these same lines, activity of the NOR gene may
participate in non-fruit pathogen resistance for example via
repression of low-level of tissue or cell specific expression in
response to pathogen attack. Consequently NOR gene repression may
thus be used to provide a positive impact on pathogen resistance in
fruit and non-fruit tissues.
[0061] 4. Control of Ethylene Response
[0062] Again, phenotypic studies of fruit ripening effects of the
nor mutation and the transgenic complementation studies of the
inventor's (FIG. 2) demonstrate that the NOR gene influences both
ethylene biosynthesis and response in fruits. As such, NOR can be
utilized to manipulate ripening and quality as described above.
Nevertheless, ethylene impacts numerous aspects of plant growth and
development in addition to ripening, as mentioned and referenced
herein above. It is important to note here that inducible
over-expression or repression of the NOR gene may be useful in
controlling ethylene responses including abscission, senescence,
pathogen resistance, germination, and general stress responses
(drought, temperature, water, mechanical damage) leading to
increased yield and crop performance. Specific examples of use
might include 1) synchronized and controlled maturation of cereal
grains via high level NOR expression late in seed development, 2)
high level expression of NOR later in the growing season to induce
senescence and defoliation of cotton via over-expression in leaves
prior to boll harvest, and 3) synchronized and accelerated or
protracted maturation of seed pods via over-expression or
repression, respectively of NOR in soybean. Finally, as stress
responses such as responses to pathogen infection, and abiotic
stress (temperature, water, mechanical damage) are mediated in part
by ethylene, over-expression of the NOR gene may positively impact
the ability of plants to withstand biotic and abiotic insults, thus
resulting in enhanced crop performance and yield.
[0063] 5. DNA Markers for Assisted Breeding
[0064] The naturally-occurring nor mutation as stated above is
already used in breeding of fresh market and processing tomatoes.
Current phenotypic selection methods require confirmation of
genotype at the nor locus through analysis of fruit development
(i.e., the latest stage of plant development) with confirmation
requiring analysis of subsequent progeny. Such phenotypic screening
requires considerable growth space and 2-3 months per plant
generation cycle.
[0065] Isolation of the DNA sequences corresponding to the nor
mutation has permitted development of DNA markers based on sequence
variation between the normal versus mutant tomato genotypes. Use of
such markers allows for definitive genotyping of seedlings in a
matter of 1-5 days. An example of a DNA marker system based on the
nor mutation is shown in FIG. 3. In this example, the 2 bp deletion
resulting in the mutation (see below and FIG. 5) is exploited to
develop a set of PCR primers which distinguishes the normal versus
mutant allele. The sequence variation between the normal versus
mutant alleles would be the basis for development of virtually all
types of DNA-based markers for determining nor locus genotype
through the use of sequences located precisely at (thus 100%
accurate) the nor locus.
[0066] (iii) Summary
[0067] The NOR gene cDNA was identified by the inventors and termed
CD-11. The gene shows similar transcript size in mutant versus
normal fruit (FIG. 4) though it does show reduced accumulation in
the mutant. The inventors have also shown that the nor mutation
results from a 2 bp deletion in the coding sequence which results
in introduction of a premature stop codon through comparative
sequencing of the normal versus mutant alleles of the nor locus
(FIG. 5). The NOR gene is related to a family of plant
transcription factors associated with multiple aspects of plant
development including meristem and cotyledon development and leaf
senescence (Sour et al., 1996; Aida et al., 1997; John et al.,
1997). FIG. 6 depicts the DNA sequence NOR cDNA sequence.
[0068] The effects of manipulation of the NOR gene in tomato and
other plant species can be readily anticipated via phenotypic
observations of the effects of the nor mutation on fruit
development. In short, it is likely that most ripening related
parameters can be accelerated or inhibited in fruit via
over-expression or suppression, respectively of NOR. In addition it
is likely that at least a subset of these effects can also be
manifested in non-fruit tissues. It would seem particularly likely
that ectopic expression of the NOR gene could bring about effects
associated with ripening in non-fruit tissues (e.g., senescence,
abscission, cell wall alterations and starch conversion, in
addition to antioxidant pigment accumulation and associated
nutritional enhancement) either directly or in association with
other genetic modifications. If processes such as enhanced disease
resistance in non-fruit tissues are influenced for example by
repression of low level expression of NOR gene, then repression of
said gene may have a positive impact on enhancing disease
resistance as well.
[0069] (iv) Conclusion
[0070] An important advance of the instant invention is that it
provides novel methods for the modification of plant phenotypes. In
particular, by providing the NOR sequence, the invention allows the
creation of plants with modified phenotypes. The inventors
specifically contemplate the use of the NOR sequence, as well as
all of the derivatives thereof which are provided by the invention,
to genetically transform plant species for the purpose of altering
plant phenotypes. Exemplary phenotypic effects are those which are
associated with fruit ripening or ethylene response. In particular,
the inventors contemplate increasing the expression of NOR in order
to increase fruit ripening and/or ethylene responsiveness, or
alternatively, decreasing the effective expression of the NOR gene
in order to delay, protract, and/or inhibited fruit ripening or
ethylene responses. The expression of NOR sequences in accordance
with the invention may be carried out using the native promoter, or
alternatively, promoters that are inducible, viral, synthetic,
constitutive as described (Poszkowski et al., 1989; Odell et al.,
1985), and temporally regulated, spatially regulated (e.g.,
tissue-specific), and spatio-temporally regulated (Chau et al.,
1989).
[0071] Types of effects which could be recognized on fruit ripening
include, as described in detail above, processes related to changes
in color, texture, flavor, aroma, shelf-life, ethylene responses,
nutrient composition, cell wall metabolism, and susceptibility to
pathogenesis associated with the ripening process. Similarly,
effects on ethylene responsiveness which could be effected with the
invention include either increased or decreased responsiveness to
ethylene. Changes in response to ethylene may effect fruit
ripening, organ abscission, seed or pollen dehiscence/shattering,
tissue senescence, disease resistance, and response to
environmental stresses including but not limited to drought,
flooding, heat, cold, nutrient deficiency, high or low light
intensity, mechanical damage and insect or pathogen infection.
Modification of any of the foregoing effects in a plant, as well as
any other effects associated with the NOR gene, is specifically
contemplated by the inventors and a part of the current
invention.
[0072] Potentially any method employing the sequences described by
the inventors may be used to realize the above-mentioned phenotypic
effects in potentially any plant species, although fruiting effects
can be expected to be realized only in species producing fruit. For
example, fruit ripening or ethylene responsiveness could be
decreased in a given plant by transformation of the plant with an
expression vector comprising an antisense NOR gene. Such a NOR gene
could comprise the sequences provided herein, or could represent
copies of homologous sequences from other plants isolated using the
sequences of the invention. Decreases in fruit ripening or ethylene
responsiveness could alternatively be realized by use of
co-suppression by way of introduction of additional NOR sequences
into a host genome. In this case, for example, by introducing
multiple exogenous copies of NOR sequences, preferably comprising a
functional expression unit, cosuppression of any functional native
NOR sequences could be realized, and thereby the phenotype of the
plant be modified with respect to traits effected by NOR
expression. The effect could be realized potentially by use of the
NOR promoter, coding sequences or terminators, or using
heterologous versions thereof.
[0073] In order to realize the phenotypic effects contemplated by
the inventors, it is not required that a particular plant be
directly transformed. In particular, once a transgene comprising a
sequence of the invention has been introduced into a host plant,
that transgene may be passed to any subsequent generation by
standard plant breeding protocols. Such breeding can allow the
transgene to be introduced into different lines, preferably of an
elite agronomic background, or even to different species which can
be made sexually compatible with the plant having the transgene.
Breeding protocols may be aided by the use of genetic markers which
are closely linked to the genes of interest. As such, the instant
invention extends to any plant which has been directly introduced
with a transgene prepared in accordance with the invention, or
which has received the transgene by way of crossing with a plant
having such a transgene. The invention may additionally be applied
to any plant species. Preferably, a plant prepared in accordance
with the invention will be a fruiting plant, for example, tomato,
berries such as strawberries and raspberries, banana, kiwi,
avocado, melon, mango, papaya, lychee, pear, stone fruits such as
peach, apricot, plum and cherry, in addition to true (anatomical)
fruits commonly referred to as "vegetables" including peppers,
eggplant, okra, and other non-melon curcubuts such as cucumber and
squash. Specific examples of other plant species which could be
used in accordance with the invention include, but are not limited
to, wheat, maize, rye, rice, turfgrass, oat, barley, sorghum,
millet, sugarcane, carrot, tobacco, tomato, potato, soybean,
canola, sunflower, alfalfa and cotton.
[0074] By way of example, one may utilize an expression vector
containing a sense or antisense NOR coding region and an
appropriate selectable marker to transform a plant cell of a
selected species. Any method capable of introducing the expression
vector into the cell may be used in accordance with the invention,
for example, use of Agrobacterium-mediated DNA transfer,
microprojectile bombardment, direct DNA transfer into pollen, by
injection of DNA into reproductive organs of a plant, or by direct
injection of DNA into the cells of immature embryos followed by the
rehydration of desiccated embryos, or by direct DNA uptake by
protoplasted cells. The development or regeneration of plants
containing the foreign, exogenous gene that encodes a polypeptide
of interest introduced by Agrobacterium from leaf explants can be
achieved by methods well known in the art such as described (Horsch
et al., 1985). In this procedure, transformants are cultured in the
presence of a selection agent and in a medium that induces the
regeneration of shoots in the plant strain being transformed as
described (Fraley et al., 1983). This procedure typically produces
shoots within two to four months and those shoots are then
transferred to an appropriate root-inducing medium containing the
selective agent and an antibiotic to prevent bacterial growth.
Shoots that rooted in the presence of the selective agent to form
plantlets are then transplanted to soil or other media to allow the
production of roots. These procedures vary depending upon the
particular plant strain employed, such variations being well known
in the art. By inclusion of a selectable or screenable marker with
an expression vector, those cells receiving the expression vector
may efficiently be isolated from those that have not received the
vector.
[0075] The ultimate goal in the production of transgenic plants
having altered phenotypes is to produce plants which are useful to
man. In this respect, transgenic plants created in accordance with
the current invention may be used for virtually any purpose deemed
of value to the grower or to the consumer. For example, the fruit
of tomato plants with enhanced fruit ripening characteristics may
be harvested and sold to consumers or used in the production of
various food products. Additionally, seed could be harvested from
the fruit of a plant prepared in accordance with the instant
invention, and the seed may be sold to farmers for planting in the
field or may be directly used as food, either for animals or
humans. Alternatively, products may be made from the seed itself,
for example, oil, starch, pharmaceuticals, and various industrial
products. Such products may be made from particular plant parts or
from the entire plant.
[0076] Means for preparing products from plants, such as those that
may be made with the current invention, have been well known since
the dawn of agriculture and will be known to those of skill in the
art. Specific methods for crop utilization may be found in, for
example, Sprague and Dudley (1988), and Watson and Ramstad
(1987).
[0077] III. Plant Transformation Constructs
[0078] The construction of vectors which may be employed in
conjunction with plant transformation techniques according to the
invention will be known to those of skill of the art in light of
the present disclosure (see, for example, Sambrook et al., 1989;
Gelvin et al., 1990). The techniques of the current invention are
thus not limited to any particular nucleic acid sequences in
conjunction with the NOR nucleic acid sequences provided herein.
Exemplary sequences for use with the invention include those
provided in SEQ ID NO:1, SEQ ID NO:6 and SEQ ID NO:7.
[0079] One important use of the sequences of the invention will be
in the alteration of plant phenotypes by genetic transformation of
plants with sense or antisense NOR genes. The NOR gene may be
provided with other sequences. Where an expressible coding region
that is not necessarily a marker coding region is employed in
combination with a marker coding region, one may employ the
separate coding regions on either the same or different DNA
segments for transformation. In the latter case, the different
vectors are delivered concurrently to recipient cells to maximize
cotransformation.
[0080] The choice of any additional elements used in conjunction
with the NOR sequences will often depend on the purpose of the
transformation. One of the major purposes of transformation of crop
plants is to add commercially desirable, agronomically important
traits to the plant. Such traits include, but are not limited to,
processes related to changes in fruit color, texture, flavor,
aroma, shelf-life, ethylene responses, nutrient composition, cell
wall metabolism, susceptibility to pathogenesis associated with the
ripening process, organ abscission, seed or pollen
dehiscence/shattering, tissue senescence, disease resistance, and
response to environmental stresses including but not limited to
drought, flooding, heat, cold, nutrient deficiency, high or low
light intensity, mechanical damage and insect or pathogen
infection. In certain embodiments, the present inventors
contemplate the transformation of a recipient cell with more than
transformation construct. Two or more transgenes can be created in
a single transformation event using either distinct selected-gene
encoding vectors, or using a single vector incorporating two or
more gene coding sequences.
[0081] In other embodiments of the invention, it is contemplated
that one may wish to employ replication-competent viral vectors for
plant transformation. Such vectors include, for example, wheat
dwarf virus (WDV) "shuttle" vectors, such as pW1-11 and PW1-GUS
(Ugaki et al., 1991). These vectors are capable of autonomous
replication in plant cells as well as E. coli, and as such may
provide increased sensitivity for detecting DNA delivered to
transgenic cells. A replicating vector also may be useful for
delivery of genes flanked by DNA sequences from transposable
elements such as Ac, Ds, or Mu. It also is contemplated that
transposable elements would be useful for introducing DNA fragments
lacking elements necessary for selection and maintenance of the
plasmid vector in bacteria, e.g., antibiotic resistance genes and
origins of DNA replication. It also is proposed that use of a
transposable element such as Ac, Ds, or Mu would actively promote
integration of the desired DNA and hence increase the frequency of
stably transformed cells.
[0082] It further is contemplated that one may wish to co-transform
plants or plant cells with 2 or more vectors. Co-transformation may
be achieved using a vector containing the marker and another gene
or genes of interest. Alternatively, different vectors, e.g.,
plasmids, may contain the different genes of interest, and the
plasmids may be concurrently delivered to the recipient cells.
Using this method, the assumption is made that a certain percentage
of cells in which the marker has been introduced, also have
received the other gene(s) of interest. Thus, not all cells
selected by means of the marker, will express the other genes of
interest which had been presented to the cells concurrently.
[0083] Vectors used for plant transformation may include, for
example, plasmids, cosmids, YACs (yeast artificial chromosomes),
BACs (bacterial artificial chromosomes) or any other suitable
cloning system. Thus when the term "vector" or "expression vector"
is used, all of the foregoing types of vectors, as well as nucleic
acid sequences isolated therefrom, are included. It is contemplated
that utilization of cloning systems with large insert capacities
will allow introduction of large DNA sequences comprising more than
one selected gene. Introduction of such sequences may be
facilitated by use of bacterial or yeast artificial chromosomes
(BACs or YACs, respectively), or even plant artificial chromosomes.
For example, the use of BACs for Agrobacterium-mediated
transformation was disclosed by Hamilton et al. (1996).
[0084] Particularly useful for transformation are expression
cassettes which have been isolated from such vectors. DNA segments
used for transforming plant cells will, of course, generally
comprise the cDNA, gene or genes which one desires to introduced
into and have expressed in the host cells. These DNA segments can
further include, in addition to a NOR coding sequence, structures
such as promoters, enhancers, polylinkers, or even regulatory genes
as desired. The DNA segment or gene chosen for cellular
introduction will often encode a protein which will be expressed in
the resultant recombinant cells resulting in a screenable or
selectable trait and/or which will impart an improved phenotype to
the resulting transgenic plant. However, this may not always be the
case, and the present invention also encompasses transgenic plants
incorporating non-expressed transgenes. Preferred components likely
to be included with vectors used in the current invention are as
follows.
[0085] (i) Regulatory Elements
[0086] The construction of vectors which may be employed in
conjunction with the present invention will be known to those of
skill of the art in light of the present disclosure (see e.g.,
Sambrook et al., 1989; Gelvin et al., 1990). Preferred constructs
will generally include a plant promoter such as the CaMV 35S
promoter (Odell et al., 1985), or others such as CaMV 19S (Lawton
et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987),
sucrose synthase (Yang & Russell, 1990), a-tubulin, actin (Wang
et al., 1992), cab (Sullivan et al., 1989), PEPCase (Hudspeth &
Grula, 1989) or those associated with the R gene complex (Chandler
et al., 1989). Tissue specific promoters such as root cell
promoters (Conkling et al., 1990) and tissue specific enhancers
(Fromm et al., 1989) are also contemplated to be particularly
useful, as are inducible promoters such as ABA- and
turgor-inducible promoters.
[0087] As the DNA sequence between the transcription initiation
site and the start of the coding sequence, i.e., the untranslated
leader sequence, can influence gene expression, one may also wish
to employ a particular leader sequence. Preferred leader sequences
are contemplated to include those which include sequences predicted
to direct optimum expression of the attached gene, i.e., to include
a preferred consensus leader sequence which may increase or
maintain mRNA stability and prevent inappropriate initiation of
translation. The choice of such sequences will be known to those of
skill in the art in light of the present disclosure. Sequences that
are derived from genes that are highly expressed in plants, and in
tomato in particular, will be most preferred.
[0088] It is contemplated that vectors for use in accordance with
the present invention may be constructed to include the ocs
enhancer element. This element was first identified as a 16 bp
palindromic enhancer from the octopine synthase (ocs) gene of
Agrobacterium (Ellis et al., 1987), and is present in at least 10
other promoters (Bouchez et al., 1989). It is proposed that the use
of an enhancer element, such as the ocs element and particularly
multiple copies of the element, will act to increase the level of
transcription from adjacent promoters when applied in the context
of plant transformation.
[0089] It is specifically envisioned that NOR coding sequences may
be introduced under the control of novel promoters or enhancers,
etc., or perhaps even homologous or tissue specific (e.g., root-,
collar/sheath-, whorl-, stalk-, earshank-, kernel- or
leaf-specific) promoters or control elements. Indeed, it is
envisioned that a particular use of the present invention will be
the targeting sense or antisense NOR expression in a
tissue-specific manner. For example, these sequences could be
targeted to the fruit.
[0090] Vectors for use in tissue-specific targeting of genes in
transgenic plants will typically include tissue-specific promoters
and may also include other tissue-specific control elements such as
enhancer sequences. Promoters which direct specific or enhanced
expression in certain plant tissues will be known to those of skill
in the art in light of the present disclosure. These include, for
example, the rbcS promoter, specific for green tissue; the ocs, nos
and mas promoters which have higher activity in roots or wounded
leaf tissue; a truncated (-90 to +8) 35S promoter which directs
enhanced expression in roots, and an a-tubulin gene that directs
expression in roots.
[0091] It also is contemplated that tissue specific expression may
be functionally accomplished by introducing a constitutively
expressed gene (all tissues) in combination with an antisense gene
that is expressed only in those tissues where the gene product is
not desired. For example, a gene coding for a NOR sequence may be
introduced such that it is expressed in all tissues using the 35S
promoter from Cauliflower Mosaic Virus. Expression of an antisense
transcript of the same NOR gene in the fruit of a plant would
prevent expression of the NOR gene only in the fruit.
[0092] Alternatively, one may wish to obtain novel tissue-specific
promoter sequences for use in accordance with the present
invention. To achieve this, one may first isolate cDNA clones from
the tissue concerned and identify those clones which are expressed
specifically in that tissue, for example, using Northern blotting.
Ideally, one would like to identify a gene that is not present in a
high copy number, but which gene product is relatively abundant in
specific tissues. The promoter and control elements of
corresponding genomic clones may then be localized using the
techniques of molecular biology known to those of skill in the
art.
[0093] It is contemplated that expression of sense or antisense NOR
genes in transgenic plants may in some cases be desired only under
specified conditions. It is contemplated that expression of such
sequences at high levels may have detrimental effects. It is known
that a large number of genes exist that respond to the environment.
For example, expression of some genes such as rbcS, encoding the
small subunit of ribulose bisphosphate carboxylase, is regulated by
light as mediated through phytochrome. Other genes are induced by
secondary stimuli. A number of genes have been shown to be induced
by ABA (Skriver and Mundy, 1990). Therefore, in particular
embodiments, inducible expression of the nucleic acid sequences of
the invention may be desired.
[0094] It also is contemplated by the inventors that in some
embodiments of the present invention expression of a NOR gene will
be desired only in a certain time period during the development of
the plant. Developmental timing is frequently correlated with
tissue specific gene expression. For example, expression of certain
genes associated with fruit ripening will only be expressed at
certain stages of fruit development.
[0095] Additionally, vectors may be constructed and employed in the
intracellular targeting of a specific gene product within the cells
of a transgenic plant or in directing a protein to the
extracellular environment. This will generally be achieved by
joining a DNA sequence encoding a transit or signal peptide
sequence to the coding sequence of a particular gene. The resultant
transit, or signal, peptide will transport the protein to a
particular intracellular, or extracellular destination,
respectively, and will then be post-translationally removed.
Transit or signal peptides act by facilitating the transport of
proteins through intracellular membranes, e.g., vacuole, vesicle,
plastid and mitochondrial membranes, whereas signal peptides direct
proteins through the extracellular membrane.
[0096] A particular example of such a use concerns the direction of
a herbicide resistance selectable marker gene, such as the EPSPS
gene, to a particular organelle such as the chloroplast rather than
to the cytoplasm. This is exemplified by the use of the rbcS
transit peptide which confers plastid-specific targeting of
proteins. In addition, it is proposed that it may be desirable to
target NOR genes to the extracellular spaces or to the vacuole.
[0097] It also is contemplated that it may be useful to target DNA
itself within a cell. For example, it may be useful to target
introduced DNA to the nucleus as this may increase the frequency of
transformation. Within the nucleus itself it would be useful to
target a gene in order to achieve site specific integration. For
example, it would be useful to have an gene introduced through
transformation replace an existing gene in the cell.
[0098] (ii) Terminators
[0099] Transformation constructs prepared in accordance with the
invention will typically include a 3' end DNA sequence that acts as
a signal to terminate transcription and allow for the
poly-adenylation of the mRNA produced by coding sequences operably
linked to a NOR gene. In one embodiment of the invention, the
native NOR gene is used. Alternatively, a heterologous 3' end may
enhance the expression of sense or antisense NOR sequences.
Terminators which are deemed to be particularly useful in this
context include those from the nopaline synthase gene of
Agrobacterium tumefaciens (nos 3' end) (Bevan et al., 1983), the
terminator for the T7 transcript from the octopine synthase gene of
Agrobacterium tumefaciens, and the 3' end of the protease inhibitor
I or II genes from potato or tomato. Regulatory elements such as
Adh intron (Callis et al., 1987), sucrose synthase intron (Vasil et
al., 1989) or TMV omega element (Gallie et at, 1989), may further
be included where desired.
[0100] (iii) Transit or Signal Peptides
[0101] Sequences that are joined to the coding sequence of an
expressed gene, which are removed post-translationally from the
initial translation product and which facilitate the transport of
the protein into or through intracellular or extracellular
membranes, are termed transit (usually into vacuoles, vesicles,
plastids and other intracellular organelles) and signal sequences
(usually to the endoplasmic reticulum, golgi apparatus and outside
of the cellular membrane). By facilitating the transport of the
protein into compartments inside and outside the cell, these
sequences may increase the accumulation of gene product protecting
them from proteolytic degradation. These sequences also allow for
additional mRNA sequences from highly expressed genes to be
attached to the coding sequence of the genes. Since mRNA being
translated by ribosomes is more stable than naked mRNA, the
presence of translatable mRNA in front of the gene may increase the
overall stability of the mRNA transcript from the gene and thereby
increase synthesis of the gene product. Since transit and signal
sequences are usually post-translationally removed from the initial
translation product, the use of these sequences allows for the
addition of extra translated sequences that may not appear on the
final polypeptide. It further is contemplated that targeting of
certain proteins may be desirable in order to enhance the stability
of the protein (U.S. Pat. No. 5,545,818, incorporated herein by
reference in its entirety).
[0102] Additionally, vectors may be constructed and employed in the
intracellular targeting of a specific gene product within the cells
of a transgenic plant or in directing a protein to the
extracellular environment. This generally will be achieved by
joining a DNA sequence encoding a transit or signal peptide
sequence to the coding sequence of a particular gene. The resultant
transit, or signal, peptide will transport the protein to a
particular intracellular, or extracellular destination,
respectively, and will then be post-translationally removed.
[0103] (iv) Marker Genes
[0104] By employing a selectable or screenable marker protein, one
can provide or enhance the ability to identify transformants.
"Marker genes" are genes that impart a distinct phenotype to cells
expressing the marker protein and thus allow such transformed cells
to be distinguished from cells that do not have the marker. Such
genes may encode either a selectable or screenable marker,
depending on whether the marker confers a trait which one can
"select" for by chemical means, i.e., through the use of a
selective agent (e.g., a herbicide, antibiotic, or the like), or
whether it is simply a trait that one can identify through
observation or testing, i.e., by "screening"' (e.g., the green
fluorescent protein). Of course, many examples of suitable marker
proteins are known to the art and can be employed in the practice
of the invention.
[0105] Included within the terms selectable or screenable markers
also are genes which encode a "secretable marker" whose secretion
can be detected as a means of identifying or selecting for
transformed cells. Examples include markers which are secretable
antigens that can be identified by antibody interaction, or even
secretable enzymes which can be detected by their catalytic
activity. Secretable proteins fall into a number of classes,
including small, diffusible proteins detectable, e.g., by ELISA;
small active enzymes detectable in extracellular solution (e.g.,
.alpha.-amylase, .beta.-lactamase, phosphinothricin
acetyltransferase); and proteins that are inserted or trapped in
the cell wall (e.g., proteins that include a leader sequence such
as that found in the expression unit of extensin or tobacco
PR-S).
[0106] With regard to selectable secretable markers, the use of a
gene that encodes a protein that becomes sequestered in the cell
wall, and which protein includes a unique epitope is considered to
be particularly advantageous. Such a secreted antigen marker would
ideally employ an epitope sequence that would provide low
background in plant tissue, a promoter-leader sequence that would
impart efficient expression and targeting across the plasma
membrane, and would produce protein that is bound in the cell wall
and yet accessible to antibodies. A normally secreted wall protein
modified to include a unique epitope would satisfy all such
requirements.
[0107] 1. Selectable Markers
[0108] Many selectable marker coding regions may be used in
connection with the NOR sequences of the present invention
including, but not limited to, neo (Potrykus et al., 1985), which
provides kanamycin resistance and can be selected for using
kanamycin, G418, paromomycin, etc.; bar, which confers bialaphos or
phosphinothricin resistance; a mutant EPSP synthase protein
(Hinchee et al., 1988) conferring glyphosate resistance; a
nitrilase such as bxn from Klebsiella ozaenae which confers
resistance to bromoxynil (Stalker et al., 1988); a mutant
acetolactate synthase (ALS) which confers resistance to
imidazolinone, sulfonylurea or other ALS inhibiting chemicals
(European Patent Application 154,204, 1985); a methotrexate
resistant DHFR (Thillet et al., 1988), a dalapon dehalogenase that
confers resistance to the herbicide dalapon; or a mutated
anthranilate synthase that confers resistance to 5-methyl
tryptophan. Where a mutant EPSP synthase is employed, additional
benefit may be realized through the incorporation of a suitable
chloroplast transit peptide, CTP (U.S. Pat. No. 5,188,642) or OTP
(U.S. Pat. No. 5,633,448) and use of a modified maize EPSPS (PCT
Application WO 97/04103).
[0109] An illustrative embodiment of selectable marker capable of
being used in systems to select transformants are those that encode
the enzyme phosphinothricin acetyltransferase, such as the bar gene
from Streptomyces hygroscopicus or the pat gene from Streptomyces
viridochromogenes. The enzyme phosphinothricin acetyl transferase
(PAT) inactivates the active ingredient in the herbicide bialaphos,
phosphinothricin (PPT). PPT inhibits glutamine synthetase,
(Murakami et al., 1986; Twell et al., 1989) causing rapid
accumulation of ammonia and cell death.
[0110] Where one desires to employ a bialaphos resistance gene in
the practice of the invention, the inventor has discovered that
particularly useful genes for this purpose are the bar or pat genes
obtainable from species of Streptomyces (e.g., ATCC No. 21,705).
The cloning of the bar gene has been described (Murakami et al.,
1986; Thompson et al., 1987) as has the use of the bar gene in the
context of plants (De Block et al., 1987; De Block et al., 1989;
U.S. Pat. No. 5,550,318).
[0111] 2. Screenable Markers
[0112] Screenable markers that may be employed include a
.beta.-glucuronidase (GUS) or uidA gene which encodes an enzyme for
which various chromogenic substrates are known; an R-locus gene,
which encodes a product that regulates the production of
anthocyanin pigments (red color) in plant tissues (Dellaporta et
al., 1988); a .beta.-lactamase gene (Sutcliffe, 1978), which
encodes an enzyme for which various chromogenic substrates are
known (e.g., PADAC, a chromogenic cephalosporin); a xyle gene
(Zukowsky et al., 1983) which encodes a catechol dioxygenase that
can convert chromogenic catechols; an .alpha.-amylase gene (Ikuta
et al., 1990); a tyrosinase gene (Katz et al., 1983) which encodes
an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone
which in turn condenses to form the easily-detectable compound
melanin; a .beta.-galactosidase gene, which encodes an enzyme for
which there are chromogenic substrates; a luciferase (lux) gene (Ow
et al., 1986), which allows for bioluminescence detection; an
aequorin gene (Prasher et al., 1985) which may be employed in
calcium-sensitive bioluminescence detection; or a gene encoding for
green fluorescent protein (Sheen et al., 1995; Haseloff et al.,
1997; Reichel et al., 1996; Tian et al., 1997; WO 97/41228).
[0113] Genes from the maize R gene complex can also be used as
screenable markers. The R gene complex in maize encodes a protein
that acts to regulate the production of anthocyanin pigments in
most seed and plant tissue. Maize strains can have one, or as many
as four, R alleles which combine to regulate pigmentation in a
developmental and tissue specific manner. Thus, an R gene
introduced into such cells will cause the expression of a red
pigment and, if stably incorporated, can be visually scored as a
red sector. If a maize line carries dominant alleles for genes
encoding for the enzymatic intermediates in the anthocyanin
biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a
recessive allele at the R locus, transformation of any cell from
that line with R will result in red pigment formation. Exemplary
lines include Wisconsin 22 which contains the rg-Stadler allele and
TR112, a K55 derivative which is r-g, b, Pl. Alternatively, any
genotype of maize can be utilized if the C1 and R alleles are
introduced together.
[0114] Another screenable marker contemplated for use in the
present invention is firefly luciferase, encoded by the lux gene.
The presence of the lux gene in transformed cells may be detected
using, for example, X-ray film, scintillation counting, fluorescent
spectrophotometry, low-light video cameras, photon counting cameras
or multiwell luminometry. It also is envisioned that this system
may be developed for populational screening for bioluminescence,
such as on tissue culture plates, or even for whole plant
screening. The gene which encodes green fluorescent protein (GFP)
is contemplated as a particularly useful reporter gene (Sheen et
al., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tian et
al., 1997; WO 97/41228). Expression of green fluorescent protein
may be visualized in a cell or plant as fluorescence following
illumination by particular wavelengths of light. Where use of a
screenable marker gene such as lux or GFP is desired, the inventors
contemplated that benefit may be realized by creating a gene fusion
between the screenable marker gene and a selectable marker gene,
for example, a GFP-NPTII gene fusion. This could allow, for
example, selection of transformed cells followed by screening of
transgenic plants or seeds.
[0115] 3. Negative Selectable Markers
[0116] Introduction of genes encoding traits that can be selected
against may be useful for eliminating undesirable linked genes. It
is contemplated that when two or more genes are introduced together
by cotransformation that the genes will be linked together on the
host chromosome. For example, a gene encoding Bt that confers
insect resistance on the plant may be introduced into a plant
together with a bar gene that is useful as a selectable marker and
confers resistance to the herbicide Liberty.RTM. on the plant.
However, it may not be desirable to have an insect resistant plant
that also is resistant to the herbicide Liberty.RTM.. It is
proposed that one also could introduce an antisense bar gene that
is expressed in those tissues where one does not want expression of
the bar gene, e.g., in whole plant parts. Hence, although the bar
gene is expressed and is useful as a selectable marker, it is not
useful to confer herbicide resistance on the whole plant. The bar
antisense gene is a negative selectable marker.
[0117] It also is contemplated that negative selection is necessary
in order to screen a population of transformants for rare
homologous recombinants generated through gene targeting. For
example, a homologous recombinant may be identified through the
inactivation of a gene that was previously expressed in that cell.
The antisense gene to neomycin phosphotransferase II (NPT II) has
been investigated as a negative selectable marker in tobacco
(Nicotiana tabacum) and Arabidopsis thaliana (Xiang. and Guerra,
1993). In this example, both sense and antisense NPT II genes are
introduced into a plant through transformation and the resultant
plants are sensitive to the antibiotic kanamycin. An introduced
gene that integrates into the host cell chromosome at the site of
the antisense NPT II gene, and inactivates the antisense gene, will
make the plant resistant to kanamycin and other aminoglycoside
antibiotics. Therefore, rare, site-specific recombinants may be
identified by screening for antibiotic resistance. Similarly, any
gene, native to the plant or introduced through transformation,
that when inactivated confers resistance to a compound, may be
useful as a negative selectable marker.
[0118] It is contemplated that negative selectable markers also may
be useful in other ways. One application is to construct transgenic
lines in which one could select for transposition to unlinked
sites. In the process of tagging it is most common for the
transposable element to move to a genetically linked site on the
same chromosome. A selectable marker for recovery of rare plants in
which transposition has occurred to an unlinked locus would be
useful. For example, the enzyme cytosine deaminase may be useful
for this purpose (Stouggard, 1993). In the presence of this enzyme
the compound 5-fluorocytosine is converted to 5-fluorouracil which
is toxic to plant and animal cells. If a transposable element is
linked to the gene for the enzyme cytosine deaminase, one may
select for transposition to unlinked sites by selecting for
transposition events in which the resultant plant is now resistant
to 5-fluorocytosine. The parental plants and plants containing
transpositions to linked sites will remain sensitive to
5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of
the cytosine deaminase gene through genetic segregation of the
transposable element and the cytosine deaminase gene. Other genes
that encode proteins that render the plant sensitive to a certain
compound will also be useful in this context. For example, T-DNA
gene 2 from Agrobacterium tumefaciens encodes a protein that
catalyzes the conversion of .alpha.-naphthalene acetamide (NAM) to
.alpha.-naphthalene acetic acid (NAA) renders plant cells sensitive
to high concentrations of NAM (Depicker et al., 1988).
[0119] It also is contemplated that negative selectable markers may
be useful in the construction of transposon tagging lines. For
example, by marking an autonomous transposable element such as Ac,
Master Mu, or En/Spn with a negative selectable marker, one could
select for transformants in which the autonomous element is not
stably integrated into the genome. It is proposed that this would
be desirable, for example, when transient expression of the
autonomous element is desired to activate in trans the
transposition of a defective transposable element, such as Ds, but
stable integration of the autonomous element is not desired. The
presence of the autonomous element may not be desired in order to
stabilize the defective element, i.e., prevent it from further
transposing. However, it is proposed that if stable integration of
an autonomous transposable element is desired in a plant the
presence of a negative selectable marker may make it possible to
eliminate the autonomous element during the breeding process.
[0120] (iv) Ribozymes
[0121] DNA may be introduced into plants for the purpose of
expressing RNA transcripts that function to affect plant phenotype
yet are not translated into protein. Two examples are antisense
RNA, which is discussed in detail below, and RNA with ribozyme
activity. Both may serve possible functions in reducing or
eliminating expression of native or introduced plant genes, for
example, a NOR gene. However, as detailed below, DNA need not be
expressed to effect the phenotype of a plant. Genes also may be
constructed or isolated, which when transcribed, produce RNA
enzymes (ribozymes) which can act as endoribonucleases and catalyze
the cleavage of RNA molecules with selected sequences. The cleavage
of selected messenger RNAs can result in the reduced production of
their encoded polypeptide products. These genes may be used to
prepare novel transgenic plants which possess them. The transgenic
plants may possess reduced levels of polypeptides including, but
not limited to, the polypeptides cited above.
[0122] Ribozymes are RNA-protein complexes that cleave nucleic
acids in a site-specific fashion. Ribozymes have specific catalytic
domains that possess endonuclease activity (Kim and Cech, 1987;
Gerlach et al., 1987; Forster and Symons, 1987). For example, a
large number of ribozymes accelerate phosphoester transfer
reactions with a high degree of specificity, often cleaving only
one of several phosphoesters in an oligonucleotide substrate (Cech
et al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub,
1992). This specificity has been attributed to the requirement that
the substrate bind via specific base-pairing interactions to the
internal guide sequence ("IGS") of the ribozyme prior to chemical
reaction.
[0123] Ribozyme catalysis has primarily been observed as part of
sequence-specific cleavage/ligation reactions involving nucleic
acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No.
5,354,855 reports that certain ribozymes can act as endonucleases
with a sequence specificity greater than that of known
ribonucleases and approaching that of the DNA restriction
enzymes.
[0124] Several different ribozyme motifs have been described with
RNA cleavage activity (Symons, 1992). Examples include sequences
from the Group I self splicing introns including Tobacco Ringspot
Virus (Prody et al., 1986), Avocado Sunblotch Viroid (Palukaitis et
al., 1979), and Lucerne Transient Streak Virus (Forster and Symons,
1987). Sequences from these and related viruses are referred to as
hammerhead ribozyme based on a predicted folded secondary
structure.
[0125] Other suitable ribozymes include sequences from RNase P with
RNA cleavage activity (Yuan et al., 1992, Yuan and Altman, 1994,
U.S. Pat. Nos. 5,168,053 and 5,624,824), hairpin ribozyme
structures (Berzal-Herranz et al., 1992; Chowrira et al., 1994) and
Hepatitis Delta virus based ribozymes (U.S. Pat. No. 5,625,047).
The general design and optimization of ribozyme directed RNA
cleavage activity has been discussed in detail (Haseloff and
Gerlach, 1988, Symons, 1992, Chowrira et al., 1994; Thompson et
al., 1995).
[0126] The other variable on ribozyme design is the selection of a
cleavage site on a given target RNA. Ribozymes are targeted to a
given sequence by virtue of annealing to a site by complimentary
base pair interactions. Two stretches of homology are required for
this targeting. These stretches of homologous sequences flank the
catalytic ribozyme structure defined above. Each stretch of
homologous sequence can vary in length from 7 to 15 nucleotides.
The only requirement for defining the homologous sequences is that,
on the target RNA, they are separated by a specific sequence which
is the cleavage site. For hammerhead ribozyme, the cleavage site is
a dinucleotide sequence on the target RNA is a uracil (U) followed
by either an adenine, cytosine or uracil (A, C or U) (Perriman et
al., 1992; Thompson et al., 1995). The frequency of this
dinucleotide occurring in any given RNA is statistically 3 out of
16. Therefore, for a given target messenger RNA of 1000 bases, 187
dinucleotide cleavage sites are statistically possible.
[0127] Designing and testing ribozymes for efficient cleavage of a
target RNA is a process well known to those skilled in the art.
Examples of scientific methods for designing and testing ribozymes
are described by Chowrira et al., (1994) and Lieber and Strauss
(1995), each incorporated by reference. The identification of
operative and preferred sequences for use in down regulating a
given gene is simply a matter of preparing and testing a given
sequence, and is a routinely practiced "screening" method known to
those of skill in the art.
[0128] (v) Induction of Gene Silencing
[0129] It also is possible that genes may be introduced to produce
novel transgenic plants which have reduced expression of a native
gene product by the mechanism of co-suppression, thus this
technique could be used in accordance with the invention. It has
been demonstrated in tobacco, tomato, and petunia (Goring et al.,
1991; Smith et al., 1990; Napoli et al., 1990; van der Krol et al.,
1990) that expression of the sense transcript of a native gene will
reduce or eliminate expression of the native gene in a manner
similar to that observed for antisense genes. The introduced gene
may encode all or part of the targeted native protein but its
translation may not be required for reduction of levels of that
native protein.
[0130] IV. Antisense Constructs
[0131] Antisense treatments are one way of altering fruit quality
and/or ethylene response and the characteristics associated
therewith in accordance with the invention. In particular,
constructs comprising the NOR gene in antisense orientation may be
used to decrease or effectively eliminate the expression of the
gene in a plant. As such, antisense technology may be used to
"knock-out" the function of a NOR gene or homologous sequences
thereof, thereby causing the delay, protraction or inhibition of
fruit ripening and/or decreased ethylene responsiveness, as well as
the effects associated therewith.
[0132] Antisense methodology takes advantage of the fact that
nucleic acids tend to pair with "complementary" sequences. By
complementary, it is meant that polynucleotides are those which are
capable of base-pairing according to the standard Watson-Crick
complementarity rules. That is, the larger purines will base pair
with the smaller pyrimidines to form combinations of guanine paired
with cytosine (G:C) and adenine paired with either thymine (A:T) in
the case of DNA, or adenine paired with uracil (A:U) in the case of
RNA. Inclusion of less common bases such as inosine,
5-methylcytosine, 6-methyladenine, hypoxanthine and others in
hybridizing sequences does not interfere with pairing.
[0133] Targeting double-stranded (ds) DNA with polynucleotides
leads to triple-helix formation; targeting RNA will lead to
double-helix formation. Antisense polynucleotides, when introduced
into a target cell, specifically bind to their target
polynucleotide and interfere with transcription, RNA processing,
transport, translation and/or stability. Antisense RNA constructs,
or DNA encoding such antisense RNA's, may be employed to inhibit
gene transcription or translation or both within a host cell,
either in vitro or in vivo, such as within a host animal, including
a human subject.
[0134] Antisense constructs may be designed to bind to the promoter
and other control regions, exons, introns or even exon-intron
boundaries of a gene. It is contemplated that the most effective
antisense constructs will include regions complementary to
intron/exon splice junctions. Thus, it is proposed that a preferred
embodiment includes an antisense construct with complementarity to
regions within 50-200 bases of an intron-exon splice junction. It
has been observed that some exon sequences can be included in the
construct without seriously affecting the target selectivity
thereof. The amount of exonic material included will vary depending
on the particular exon and intron sequences used. One can readily
test whether too much exon DNA is included simply by testing the
constructs in vitro to determine whether normal cellular function
is affected or whether the expression of related genes having
complementary sequences is affected.
[0135] As stated above, "complementary" or "antisense" means
polynucleotide sequences that are substantially complementary over
their entire length and have very few base mismatches. For example,
sequences of fifteen bases in length may be termed complementary
when they have complementary nucleotides at thirteen or fourteen
positions. Naturally, sequences which are completely complementary
will be sequences which are entirely complementary throughout their
entire length and have no base mismatches. Other sequences with
lower degrees of homology also are contemplated. For example, an
antisense construct which has limited regions of high homology, but
also contains a non-homologous region (e.g., ribozyme; see above)
could be designed. These molecules, though having less than 50%
homology, would bind to target sequences under appropriate
conditions.
[0136] It may be advantageous to combine portions of genomic DNA
with cDNA or synthetic sequences to generate specific constructs.
For example, where an intron is desired in the ultimate construct,
a genomic clone will need to be used. The cDNA or a synthesized
polynucleotide may provide more convenient restriction sites for
the remaining portion of the construct and, therefore, would be
used for the rest of the sequence.
[0137] V. Methods for Plant Transformation
[0138] Suitable methods for plant transformation for use with the
current invention are believed to include virtually any method by
which DNA can be introduced into a cell, such as by direct delivery
of DNA such as by PEG-mediated transformation of protoplasts
(Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA
uptake (Potrykus et al, 1985), by electroporation (U.S. Pat. No.
5,384,253, specifically incorporated herein by reference in its
entirety), by agitation with silicon carbide fibers (Kaeppler et
al., 1990; U.S. Pat. No. 5,302,523, specifically incorporated
herein by reference in its entirety; and U.S. Pat. No. 5,464,765,
specifically incorporated herein by reference in its entirety), by
Agrobacterium-mediated transformation (U.S. Pat. No. 5,591,616 and
U.S. Pat. No. 5,563,055; both specifically incorporated herein by
reference) and by acceleration of DNA coated particles (U.S. Pat.
No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.
5,538,880; each specifically incorporated herein by reference in
its entirety), etc. Through the application of techniques such as
these, the cells of virtually any plant species may be stably
transformed, and these cells developed into transgenic plants.
[0139] (i) Agrobacterium-Mediated Transformation
[0140] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example, the
methods described by Fraley et al., (1985), Rogers et al., (1987)
and U.S. Pat. No. 5,563,055, specifically incorporated herein by
reference in its entirety.
[0141] Agrobacterium-mediated transformation is most efficient in
dicotyledonous plants and is the preferable method for
transformation of dicots, including Arabidopsis, tobacco, tomato,
and potato. Indeed, while Agrobacterium-mediated transformation has
been routinely used with dicotyledonous plants for a number of
years, it has only recently become applicable to monocotyledonous
plants. Advances in Agrobacterium-mediated transformation
techniques have now made the technique applicable to nearly all
monocotyledonous plants. For example, Agrobacterium-mediated
transformation techniques have now been applied to rice (Hiei et
al., 1997; U.S. Pat. No. 5,591,616, specifically incorporated
herein by reference in its entirety), wheat (McCormac et al.,
1998), barley (Tingay et al., 1997; McCormac et al., 1998), and
maize (Ishidia et al., 1996).
[0142] Modern Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., 1985).
Moreover, recent technological advances in vectors for
Agrobacterium-mediated gene transfer have improved the arrangement
of genes and restriction sites in the vectors to facilitate the
construction of vectors capable of expressing various polypeptide
coding genes. The vectors described (Rogers et al., 1987) have
convenient multi-linker regions flanked by a promoter and a
polyadenylation site for direct expression of inserted polypeptide
coding genes and are suitable for present purposes. In addition,
Agrobacterium containing both armed and disarmed Ti genes can be
used for the transformations. In those plant strains where
Agrobacterium-mediated transformation is efficient, it is the
method of choice because of the facile and defined nature of the
gene transfer.
[0143] (ii) Electroporation
[0144] Where one wishes to introduce DNA by means of
electroporation, the method of Krzyzek et al. (U.S. Pat. No.
5,384,253, incorporated herein by reference in its entirety) may be
particularly advantageous. In this method, certain cell
wall-degrading enzymes, such as pectin-degrading enzymes, are
employed to render the target recipient cells more susceptible to
transformation by electroporation than untreated cells.
Alternatively, recipient cells are made more susceptible to
transformation by mechanical wounding.
[0145] To effect transformation by electroporation, one may employ
either friable tissues, such as a suspension culture of cells or
embryogenic callus or alternatively one may transform immature
embryos or other organized tissue directly. In this technique, one
would partially degrade the cell walls of the chosen cells by
exposing them to pectin-degrading enzymes (pectolyases) or
mechanically wounding in a controlled manner. Examples of some
species which have been transformed by electroporation of intact
cells include maize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995;
D'Halluin et al., 1992), wheat (Zhou et al., 1993), tomato (Hou and
Lin, 1996), soybean (Christou et al., 1987) and tobacco (Lee et
al., 1989).
[0146] One also may employ protoplasts for electroporation
transformation of plants (Bates, 1994; Lazzeri, 1995). For example,
the generation of transgenic soybean plants by electroporation of
cotyledon-derived protoplasts is described by Dhir and Widholm in
Intl. Patent Appl. Publ. No. WO 9217598 (specifically incorporated
herein by reference). Other examples of species for which
protoplast transformation has been described include barley
(Lazerri, 1995), sorghum (Battraw et al., 1991), maize
(Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato
(Tsukada, 1989).
[0147] (iii) Microprojectile Bombardment
[0148] Another method for delivering transforming DNA segments to
plant cells in accordance with the invention is microprojectile
bombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S.
Pat. No. 5,610,042; and PCT Application WO 94/09699; each of which
is specifically incorporated herein by reference in its entirety).
In this method, particles may be coated with nucleic acids and
delivered into cells by a propelling force. Exemplary particles
include those comprised of tungsten, platinum, and preferably,
gold. It is contemplated that in some instances DNA precipitation
onto metal particles would not be necessary for DNA delivery to a
recipient cell using microprojectile bombardment. However, it is
contemplated that particles may contain DNA rather than be coated
with DNA. Hence, it is proposed that DNA-coated particles may
increase the level of DNA delivery via particle bombardment but are
not, in and of themselves, necessary.
[0149] For the bombardment, cells in suspension are concentrated on
filters or solid culture medium. Alternatively, immature embryos or
other target cells may be arranged on solid culture medium. The
cells to be bombarded are positioned at an appropriate distance
below the macroprojectile stopping plate.
[0150] An illustrative embodiment of a method for delivering DNA
into plant cells by acceleration is the Biolistics Particle
Delivery System, which can be used to propel particles coated with
DNA or cells through a screen, such as a stainless steel or Nytex
screen, onto a filter surface covered with monocot plant cells
cultured in suspension. The screen disperses the particles so that
they are not delivered to the recipient cells in large aggregates.
It is believed that a screen intervening between the projectile
apparatus and the cells to be bombarded reduces the size of
projectiles aggregate and may contribute to a higher frequency of
transformation by reducing the damage inflicted on the recipient
cells by projectiles that are too large.
[0151] Microprojectile bombardment techniques are widely
applicable, and may be used to transform virtually any plant
species. Examples of species for which have been transformed by
microprojectile bombardment include monocot species such as maize
(PCT Application WO 95/06128), barley (Ritala et al., 1994;
Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055,
specifically incorporated herein by reference in its entirety),
rice (Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et
al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower et al.,
1992), and sorghum (Casa et al., 1993; Hagio et al., 1991); as well
as a number of dicots including tobacco (Tomes et al., 1990;
Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783,
specifically incorporated herein by reference in its entirety),
sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997),
cotton (McCabe and Martinell, 1993), tomato (VanEck et al. 1995),
and legumes in general (U.S. Pat. No. 5,563,055, specifically
incorporated herein by reference in its entirety).
[0152] (iv) Other Transformation Methods
[0153] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation, and combinations of these
treatments (see, e.g., Potrykus et al., 1985; Lorz et al., 1985;
Omirulleh et al., 1993; Fromm et al., 1986; Uchimiya et al., 1986;
Callis et al., 1987; Marcotte et al., 1988).
[0154] Application of these systems to different plant strains
depends upon the ability to regenerate that particular plant strain
from protoplasts. Illustrative methods for the regeneration of
cereals from protoplasts have been described (Fujimara et al.,
1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al.,
1986; Omirulleh et al, 1993 and U.S. Pat. No. 5,508,184; each
specifically incorporated herein by reference in its entirety).
Examples of the use of direct uptake transformation of cereal
protoplasts include transformation of rice (Ghosh-Biswas et al.,
1994), sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995),
oat (Zheng and Edwards, 1990) and maize (Omirulleh et al.,
1993).
[0155] To transform plant strains that cannot be successfully
regenerated from protoplasts, other ways to introduce DNA into
intact cells or tissues can be utilized. For example, regeneration
of cereals from immature embryos or explants can be effected as
described (Vasil, 1989). Also, silicon carbide fiber-mediated
transformation may be used with or without protoplasting (Kaeppler,
1990; Kaeppler et al., 1992; U.S. Pat. No. 5,563,055, specifically
incorporated herein by reference in its entirety). Transformation
with this technique is accomplished by agitating silicon carbide
fibers together with cells in a DNA solution. DNA passively enters
as the cell are punctured. This technique has been used
successfully with, for example, the monocot cereals maize (PCT
Application WO 95/06128, specifically incorporated herein by
reference in its entirety; Thompson, 1995) and rice (Nagatani,
1997).
[0156] VI. Site Specific Integration or Excision of Transgenes
[0157] It is specifically contemplated by the inventors that one
could employ techniques for the site-specific integration or
excision of transformation constructs prepared in accordance with
the instant invention. Alternatively, site-specific integration
techniques could be used to insertionally mutagenize or replace a
native NOR gene sequence. An advantage of site-specific integration
or excision is that it can be used to overcome problems associated
with conventional transformation techniques, in which
transformation constructs typically randomly integrate into a host
genome in multiple copies. This random insertion of introduced DNA
into the genome of host cells can be lethal if the foreign DNA
inserts into an essential gene. In addition, the expression of a
transgene may be influenced by "position effects" caused by the
surrounding genomic DNA. Further, because of difficulties
associated with plants possessing multiple transgene copies,
including gene silencing, recombination and unpredictable
inheritance, it is typically desirable to control the copy number
of the inserted DNA, often only desiring the insertion of a single
copy of the DNA sequence.
[0158] Site-specific integration or excision of transgenes or parts
of transgenes can be achieved in plants by means of homologous
recombination (see, for example, U.S. Pat. No. 5,527,695,
specifically incorporated herein by reference in its entirety).
Homologous recombination is a reaction between any pair of DNA
sequences having a similar sequence of nucleotides, where the two
sequences interact (recombine) to form a new recombinant DNA
species. The frequency of homologous recombination increases as the
length of the shared nucleotide DNA sequences increases, and is
higher with linearized plasmid molecules than with circularized
plasmid molecules. Homologous recombination can occur between two
DNA sequences that are less than identical, but the recombination
frequency declines as the divergence between the two sequences
increases.
[0159] Introduced DNA sequences can be targeted via homologous
recombination by linking a DNA molecule of interest to sequences
sharing homology with endogenous sequences of the host cell. For
example, conserved NOR sequences could be used to replace a native
NOR sequence with one of the NOR sequences provided herein. Once
the DNA enters the cell, the two homologous sequences can interact
to insert the introduced DNA at the site where the homologous
genomic DNA sequences were located. Therefore, the choice of
homologous sequences contained on the introduced DNA will determine
the site where the introduced DNA is integrated via homologous
recombination. For example, if the DNA sequence of interest is
linked to DNA sequences sharing homology to a single copy gene of a
host plant cell, the DNA sequence of interest will be inserted via
homologous recombination at only that single specific site.
However, if the DNA sequence of interest is linked to DNA sequences
sharing homology to a multicopy gene of the host eukaryotic cell,
then the DNA sequence of interest can be inserted via homologous
recombination at each of the specific sites where a copy of the
gene is located.
[0160] DNA can be inserted into the host genome by a homologous
recombination reaction involving either a single reciprocal
recombination (resulting in the insertion of the entire length of
the introduced DNA) or through a double reciprocal recombination
(resulting in the insertion of only the DNA located between the two
recombination events). For example, if one wishes to insert a
foreign gene into the genomic site where a selected gene is
located, the introduced DNA should contain sequences homologous to
the selected gene. A single homologous recombination event would
then result in the entire introduced DNA sequence being inserted
into the selected gene. Alternatively, a double recombination event
can be achieved by flanking each end of the DNA sequence of
interest (the sequence intended to be inserted into the genome)
with DNA sequences homologous to the selected gene. A homologous
recombination event involving each of the homologous flanking
regions will result in the insertion of the foreign DNA. Thus only
those DNA sequences located between the two regions sharing genomic
homology become integrated into the genome.
[0161] Although introduced sequences can be targeted for insertion
into a specific genomic site via homologous recombination, in
higher eukaryotes homologous recombination is a relatively rare
event compared to random insertion events. In plant cells, foreign
DNA molecules find homologous sequences in the cell's genome and
recombine at a frequency of approximately 0.5-4.2.times.10.sup.-4.
Thus any transformed cell that contains an introduced DNA sequence
integrated via homologous recombination will also likely contain
numerous copies of randomly integrated introduced DNA sequences.
Therefore, to maintain control over the copy number and the
location of the inserted DNA, these randomly inserted DNA sequences
can be removed. One manner of removing these random insertions is
to utilize a site-specific recombinase system. In general, a site
specific recombinase system consists of three elements: two pairs
of DNA sequence (the site-specific recombination sequences) and a
specific enzyme (the site-specific recombinase). The site-specific
recombinase will catalyze a recombination reaction only between two
site-specific recombination sequences.
[0162] A number of different site specific recombinase systems
could be employed in accordance with the instant invention,
including, but not limited to, the Cre/lox system of bacteriophage
P1 (U.S. Pat. No. 5,658,772, specifically incorporated herein by
reference in its entirety), the FLP/FRT system of yeast (Golic and
Lindquist, 1989), the Gin recombinase of phage Mu (Maeser et al.,
1991), the Pin recombinase of E. coli (Enomoto et al., 1983), and
the R/RS system of the pSR1 plasmid (Araki et al., 1992). The
bacteriophage P1 Cre/lox and the yeast FLP/FRT systems constitute
two particularly useful systems for site specific integration or
excision of transgenes. In these systems a recombinase (Cre or FLP)
will interact specifically with its respective site-specific
recombination sequence (lox or FRT, respectively) to invert or
excise the intervening sequences. The sequence for each of these
two systems is relatively short (34 bp for lox and 47 bp for FRT)
and therefore, convenient for use with transformation vectors.
[0163] Experiments on the performance of the FLP/FRT system in both
maize and rice protoplasts indicate that FRT site structure, and
amount of the FLP protein present, affects excision activity. In
general, short incomplete FRT sites leads to higher accumulation of
excision products than the complete full-length FRT sites. The
systems can catalyze both intra- and intermolecular reactions in
maize protoplasts, indicating its utility for DNA excision as well
as integration reactions. The recombination reaction is reversible
and this reversibility can compromise the efficiency of the
reaction in each direction. Altering the structure of the
site-specific recombination sequences is one approach to remedying
this situation. The site-specific recombination sequence can be
mutated in a manner that the product of the recombination reaction
is no longer recognized as a substrate for the reverse reaction,
thereby stabilizing the integration or excision event.
[0164] In the Cre-lox system, discovered in bacteriophage P1,
recombination between loxP sites occurs in the presence of the Cre
recombinase (see, e.g., U.S. Pat. No. 5,658,772, specifically
incorporated herein by reference in its entirety). This system has
been utilized to excise a gene located between two lox sites which
had been introduced into a yeast genome (Sauer, 1987). Cre was
expressed from an inducible yeast GAL1 promoter and this Cre gene
was located on an autonomously replicating yeast vector.
[0165] Since the lox site is an asymmetrical nucleotide sequence,
lox sites on the same DNA molecule can have the same or opposite
orientation with respect to each other. Recombination between lox
sites in the same orientation results in a deletion of the DNA
segment located between the two lox sites and a connection between
the resulting ends of the original DNA molecule. The deleted DNA
segment forms a circular molecule of DNA. The original DNA molecule
and the resulting circular molecule each contain a single lox site.
Recombination between lox sites in opposite orientations on the
same DNA molecule results in an inversion of the nucleotide
sequence of the DNA segment located between the two lox sites. In
addition, reciprocal exchange of DNA segments proximate to lox
sites located on two different DNA molecules can occur. All of
these recombination events are catalyzed by the product of the Cre
coding region.
[0166] VI. Biological Functional Equivalents
[0167] Modification and changes may be made in the nucleic acids
provided by the present invention and accordingly the structure of
the polypeptides encoded thereby, and still obtain functional
molecules that encode a NOR polypeptide. The following is a
discussion based upon alerting nucleic acids in the NOR sequences
to result in a changing of the amino acids of a NOR polypeptide to
create an equivalent, or even an improved, second-generation
molecule. In particular embodiments of the invention, mutated NOR
proteins are contemplated to be useful for increasing the activity
of the protein. The amino acid changes may be achieved by changing
the codons of the DNA sequence, according to the codons given in
Table 1.
1TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K
AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0168] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like
properties. It is thus contemplated by the inventors that various
changes may be made in the peptide sequences of the disclosed
compositions, or corresponding DNA sequences which encode said
peptides without appreciable loss of their biological utility or
activity.
[0169] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte et al., 1982, incorporate
herein by reference). It is accepted that the relative hydropathic
character of the amino acid contributes to the secondary structure
of the resultant protein, which in turn defines the interaction of
the protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like.
[0170] Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte et
al., 1982), these are: isoleucine (+4.5); valine (+4.2); leucine
(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine
(+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine
(-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6);
histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate
(-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[0171] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e., still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0172] It also is understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein.
[0173] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0174] It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still obtain a
biologically equivalent, and in particular, an immunologically
equivalent protein. In such changes, the substitution of amino
acids whose hydrophilicity values are within .+-.2 is preferred,
those which are within .+-.1 are particularly preferred, and those
within .+-.0.5 are even more particularly preferred.
[0175] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0176] Another embodiment for the preparation of polypeptides
according to the invention is the use of peptide mimetics. Mimetics
are peptide-containing molecules that mimic elements of protein
secondary structure. See, for example, Johnson et al., "Peptide
Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds.,
Chapman and Hall, New York (1993). The underlying rationale behind
the use of peptide mimetics is that the peptide backbone of
proteins exists chiefly to orient amino acid side chains in such a
way as to facilitate molecular interactions, such as those of
antibody and antigen. A peptide mimetic is expected to permit
molecular interactions similar to the natural molecule. These
principles may be used, in conjunction with the principles outline
above, to engineer second generation molecules having many of the
natural properties of the starting gene product, but with altered
and even improved characteristics.
[0177] VIII. Production and Characterization of Stably Transformed
Plants
[0178] After effecting delivery of exogenous DNA to recipient
cells, the next steps generally concern identifying the transformed
cells for further culturing and plant regeneration. As mentioned
herein, in order to improve the ability to identify transformants,
one may desire to employ a selectable or screenable marker gene
with a transformation vector prepared in accordance with the
invention. In this case, one would then generally assay the
potentially transformed cell population by exposing the cells to a
selective agent or agents, or one would screen the cells for the
desired marker gene trait.
[0179] (i) Selection
[0180] It is believed that DNA is introduced into only a small
percentage of target cells in any one experiment. In order to
provide an efficient system for identification of those cells
receiving DNA and integrating it into their genomes one may employ
a means for selecting those cells that are stably transformed. One
exemplary embodiment of such a method is to introduce into the host
cell, a marker gene which confers resistance to some normally
inhibitory agent, such as an antibiotic or herbicide. Examples of
antibiotics which may be used include the aminoglycoside
antibiotics neomycin, kanamycin and paromomycin, or the antibiotic
hygromycin. Resistance to the aminoglycoside antibiotics is
conferred by aminoglycoside phosphostransferase enzymes such as
neomycin phosphotransferase II (NPT II) or NPT I, whereas
resistance to hygromycin is conferred by hygromycin
phosphotransferase.
[0181] Potentially transformed cells then are exposed to the
selective agent. In the population of surviving cells will be those
cells where, generally, the resistance-conferring gene has been
integrated and expressed at sufficient levels to permit cell
survival. Cells may be tested further to confirm stable integration
of the exogenous DNA.
[0182] One herbicide which constitutes a desirable selection agent
is the broad spectrum herbicide bialaphos. Bialaphos is a
tripeptide antibiotic produced by Streptomyces hygroscopicus and is
composed of phosphinothricin (PPT), an analogue of L-glutamic acid,
and two L-alanine residues. Upon removal of the L-alanine residues
by intracellular peptidases, the PPT is released and is a potent
inhibitor of glutamine synthetase (GS), a pivotal enzyme involved
in ammonia assimilation and nitrogen metabolism (Ogawa et al.,
1973). Synthetic PPT, the active ingredient in the herbicide
Liberty.TM. also is effective as a selection agent. Inhibition of
GS in plants by PPT causes the rapid accumulation of ammonia and
death of the plant cells.
[0183] The organism producing bialaphos and other species of the
genus Streptomyces also synthesizes an enzyme phosphinothricin
acetyl transferase (PAT) which is encoded by the bar gene in
Streptomyces hygroscopicus and the pat gene in Streptomyces
viridochromogenes. The use of the herbicide resistance gene
encoding phosphinothricin acetyl transferase (PAT) is referred to
in DE 3642 829 A, wherein the gene is isolated from Streptomyces
viridochromogenes. In the bacterial source organism, this enzyme
acetylates the free amino group of PPT preventing auto-toxicity
(Thompson et al., 1987). The bar gene has been cloned (Murakami et
al., 1986; Thompson et al., 1987) and expressed in transgenic
tobacco, tomato, potato (De Block et al., 1987) Brassica (De Block
et al., 1989) and maize (U.S. Pat. No. 5,550,318). In previous
reports, some transgenic plants which expressed the resistance gene
were completely resistant to commercial formulations of PPT and
bialaphos in greenhouses.
[0184] Another example of a herbicide which is useful for selection
of transformed cell lines in the practice of the invention is the
broad spectrum herbicide glyphosate. Glyphosate inhibits the action
of the enzyme EPSPS which is active in the aromatic amino acid
biosynthetic pathway. Inhibition of this enzyme leads to starvation
for the amino acids phenylalanine, tyrosine, and tryptophan and
secondary metabolites derived thereof U.S. Pat. No. 4,535,060
describes the isolation of EPSPS mutations which confer glyphosate
resistance on the Salmonella typhimurium gene for EPSPS, aroA. The
EPSPS gene was cloned from Zea mays and mutations similar to those
found in a glyphosate resistant aroA gene were introduced in vitro.
Mutant genes encoding glyphosate resistant EPSPS enzymes are
described in, for example, International Patent WO 97/4103. The
best characterized mutant EPSPS gene conferring glyphosate
resistance comprises amino acid changes at residues 102 and 106,
although it is anticipated that other mutations will also be useful
(PCT/WO97/4103).
[0185] To use the bar-bialaphos or the EPSPS-glyphosate selective
system, bombarded tissue is cultured for 0-28 days on nonselective
medium and subsequently transferred to medium containing from 1-3
mg/l bialaphos or 1-3 mM glyphosate as appropriate. While ranges of
1-3 mg/l bialaphos or 1-3 mM glyphosate will typically be
preferred, it is proposed that ranges of 0.1-50 mg/l bialaphos or
0.1-50 mM glyphosate will find utility in the practice of the
invention. Tissue can be placed on any porous, inert, solid or
semi-solid support for bombardment, including but not limited to
filters and solid culture medium. Bialaphos and glyphosate are
provided as examples of agents suitable for selection of
transformants, but the technique of this invention is not limited
to them.
[0186] It further is contemplated that the herbicide DALAPON,
2,2-dichloropropionic acid, may be useful for identification of
transformed cells. The enzyme 2,2-dichloropropionic acid
dehalogenase (deh) inactivates the herbicidal activity of
2,2-dichloropropionic acid and therefore confers herbicidal
resistance on cells or plants expressing a gene encoding the
dehalogenase enzyme (Buchanan-Wollaston et al., 1992; U.S. patent
application Ser. No. 08/113,561, filed Aug. 25, 1993; U.S. Pat. No.
5,508,468; and U.S. Pat. No. 5,508,468; each of the disclosures of
which is specifically incorporated herein by reference in its
entirety).
[0187] Alternatively, a gene encoding anthranilate synthase, which
confers resistance to certain amino acid analogs, e.g.,
5-methyltryptophan or 6-methyl anthranilate, may be useful as a
selectable marker gene. The use of an anthranilate synthase gene as
a selectable marker was described in U.S. Pat. No. 5,508,468; and
U.S. patent application Ser. No. 08/604,789.
[0188] An example of a screenable marker trait is the red pigment
produced under the control of the R-locus in maize. This pigment
may be detected by culturing cells on a solid support containing
nutrient media capable of supporting growth at this stage and
selecting cells from colonies (visible aggregates of cells) that
are pigmented. These cells may be cultured further, either in
suspension or on solid media. The R-locus is useful for selection
of transformants from bombarded immature embryos. In a similar
fashion, the introduction of the C1 and B genes will result in
pigmented cells and/or tissues.
[0189] The enzyme luciferase may be used as a screenable marker in
the context of the present invention. In the presence of the
substrate luciferin, cells expressing luciferase emit light which
can be detected on photographic or x-ray film, in a luminometer (or
liquid scintillation counter), by devices that enhance night
vision, or by a highly light sensitive video camera, such as a
photon counting camera. All of these assays are nondestructive and
transformed cells may be cultured further following identification.
The photon counting camera is especially valuable as it allows one
to identify specific cells or groups of cells which are expressing
luciferase and manipulate those in real time. Another screenable
marker which may be used in a similar fashion is the gene coding
for green fluorescent protein.
[0190] It further is contemplated that combinations of screenable
and selectable markers will be useful for identification of
transformed cells. In some cell or tissue types a selection agent,
such as bialaphos or glyphosate, may either not provide enough
killing activity to clearly recognize transformed cells or may
cause substantial nonselective inhibition of transformants and
nontransformants alike, thus causing the selection technique to not
be effective. It is proposed that selection with a growth
inhibiting compound, such as bialaphos or glyphosate at
concentrations below those that cause 100% inhibition followed by
screening of growing tissue for expression of a screenable marker
gene such as luciferase would allow one to recover transformants
from cell or tissue types that are not amenable to selection alone.
It is proposed that combinations of selection and screening may
enable one to identify transformants in a wider variety of cell and
tissue types. This may be efficiently achieved using a gene fusion
between a selectable marker gene and a screenable marker gene, for
example, between an NPTII gene and a GFP gene.
[0191] (ii) Regeneration and Seed Production
[0192] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In an
exemplary embodiment, MS and N6 media may be modified by including
further substances such as growth regulators. A preferred growth
regulator for such purposes is dicamba or 2,4-D. However, other
growth regulators may be employed, including NAA, NAA+2,4-D or
perhaps even picloram. Media improvement in these and like ways has
been found to facilitate the growth of cells at specific
developmental stages. Tissue may be maintained on a basic media
with growth regulators until sufficient tissue is available to
begin plant regeneration efforts, or following repeated rounds of
manual selection, until the morphology of the tissue is suitable
for regeneration, at least 2 wk, then transferred to media
conducive to maturation of embryoids. Cultures are transferred
every 2 wk on this medium. Shoot development will signal the time
to transfer to medium lacking growth regulators.
[0193] The transformed cells, identified by selection or screening
and cultured in an appropriate medium that supports regeneration,
will then be allowed to mature into plants. Developing plantlets
are transferred to soiless plant growth mix, and hardened, e.g., in
an environmentally controlled chamber at about 85% relative
humidity, 600 ppm CO.sub.2, and 25-250 microeinsteins m.sup.-2
s.sup.-1 of light. Plants are preferably matured either in a growth
chamber or greenhouse. Plants are regenerated from about 6 wk to 10
months after a transformant is identified, depending on the initial
tissue. During regeneration, cells are grown on solid media in
tissue culture vessels. Illustrative embodiments of such vessels
are petri dishes and Plant Cons. Regenerating plants are preferably
grown at about 19 to 28.degree. C. After the regenerating plants
have reached the stage of shoot and root development, they may be
transferred to a greenhouse for further growth and testing.
[0194] Note, however, that seeds on transformed plants may
occasionally require embryo rescue due to cessation of seed
development and premature senescence of plants. To rescue
developing embryos, they are excised from surface-disinfected seeds
10-20 days post-pollination and cultured. An embodiment of media
used for culture at this stage comprises MS salts, 2% sucrose, and
5.5 g/l agarose. In embryo rescue, large embryos (defined as
greater than 3 mm in length) are germinated directly on an
appropriate media. Embryos smaller than that may be cultured for 1
wk on media containing the above ingredients along with 10.sup.-5M
abscisic acid and then transferred to growth regulator-free medium
for germination.
[0195] Progeny may be recovered from transformed plants and tested
for expression of the exogenous expressible gene by localized
application of an appropriate substrate to plant parts such as
leaves. In the case of bar transformed plants, it was found that
transformed parental plants (R.sub.0) and their progeny of any
generation tested exhibited no bialaphos-related necrosis after
localized application of the herbicide Basta to leaves, if there
was functional PAT activity in the plants as assessed by an in
vitro enzymatic assay. All PAT positive progeny tested contained
bar, confirming that the presence of the enzyme and the resistance
to bialaphos were associated with the transmission through the
germline of the marker gene.
[0196] (iii) Characterization
[0197] To confirm the presence of the exogenous DNA or
"transgene(s)" in the regenerating plants, a variety of assays may
be performed. Such assays include, for example, "molecular
biological" assays, such as Southern and Northern blotting and
PCR.TM.; "biochemical" assays, such as detecting the presence of a
protein product, e.g., by immunological means (ELISAs and Western
blots) or by enzymatic function; plant part assays, such as leaf or
root assays; and also, by analyzing the phenotype of the whole
regenerated plant.
[0198] 1. DNA Integration, RNA Expression and Inheritance
[0199] Genomic DNA may be isolated from callus cell lines or any
plant parts to determine the presence of the exogenous gene through
the use of techniques well known to those skilled in the art. Note,
that intact sequences will not always be present, presumably due to
rearrangement or deletion of sequences in the cell.
[0200] The presence of DNA elements introduced through the methods
of this invention may be determined by polymerase chain reaction
(PCR.TM.). Using this technique discreet fragments of DNA are
amplified and detected by gel electrophoresis. This type of
analysis permits one to determine whether a gene is present in a
stable transformant, but does not prove integration of the
introduced gene into the host cell genome. It is typically the
case, however, that DNA has been integrated into the genome of all
transformants that demonstrate the presence of the gene through
PCR.TM. analysis. In addition, it is not possible using PCR.TM.
techniques to determine whether transformants have exogenous genes
introduced into different sites in the genome, i.e., whether
transformants are of independent origin. It is contemplated that
using PCR.TM. techniques it would be possible to clone fragments of
the host genomic DNA adjacent to an introduced gene.
[0201] Positive proof of DNA integration into the host genome and
the independent identities of transformants may be determined using
the technique of Southern hybridization. Using this technique
specific DNA sequences that were introduced into the host genome
and flanking host DNA sequences can be identified. Hence the
Southern hybridization pattern of a given transformant serves as an
identifying characteristic of that transformant. In addition it is
possible through Southern hybridization to demonstrate the presence
of introduced genes in high molecular weight DNA, i.e., confirm
that the introduced gene has been integrated into the host cell
genome. The technique of Southern hybridization provides
information that is obtained using PCR.TM., e.g., the presence of a
gene, but also demonstrates integration into the genome and
characterizes each individual transformant.
[0202] It is contemplated that using the techniques of dot or slot
blot hybridization which are modifications of Southern
hybridization techniques one could obtain the same information that
is derived from PCR.TM., e.g., the presence of a gene.
[0203] Both PCR.TM. and Southern hybridization techniques can be
used to demonstrate transmission of a transgene to progeny. In most
instances the characteristic Southern hybridization pattern for a
given transformant will segregate in progeny as one or more
Mendelian genes (Spencer et al., 1992) indicating stable
inheritance of the transgene.
[0204] Whereas DNA analysis techniques may be conducted using DNA
isolated from any part of a plant, RNA will only be expressed in
particular cells or tissue types and hence it will be necessary to
prepare RNA for analysis from these tissues. PCR.TM. techniques
also may be used for detection and quantitation of RNA produced
from introduced genes. In this application of PCR.TM. it is first
necessary to reverse transcribe RNA into DNA, using enzymes such as
reverse transcriptase, and then through the use of conventional
PCR.TM. techniques amplify the DNA. In most instances PCR.TM.
techniques, while useful, will not demonstrate integrity of the RNA
product. Further information about the nature of the RNA product
may be obtained by Northern blotting. This technique will
demonstrate the presence of an RNA species and give information
about the integrity of that RNA. The presence or absence of an RNA
species also can be determined using dot or slot blot Northern
hybridizations. These techniques are modifications of Northern
blotting and will only demonstrate the presence or absence of an
RNA species.
[0205] 2. Gene Expression
[0206] While Southern blotting and PCR.TM. may be used to detect
the gene(s) in question, they do not provide information as to
whether the corresponding protein is being expressed. Expression
may be evaluated by specifically identifying the protein products
of the introduced genes or evaluating the phenotypic changes
brought about by their expression.
[0207] Assays for the production and identification of specific
proteins may make use of physical-chemical, structural, functional,
or other properties of the proteins. Unique physical-chemical or
structural properties allow the proteins to be separated and
identified by electrophoretic procedures, such as native or
denaturing gel electrophoresis or isoelectric focusing, or by
chromatographic techniques such as ion exchange or gel exclusion
chromatography. The unique structures of individual proteins offer
opportunities for use of specific antibodies to detect their
presence in formats such as an ELISA assay. Combinations of
approaches may be employed with even greater specificity such as
western blotting in which antibodies are used to locate individual
gene products that have been separated by electrophoretic
techniques. Additional techniques may be employed to absolutely
confirm the identity of the product of interest such as evaluation
by amino acid sequencing following purification. Although these are
among the most commonly employed, other procedures may be
additionally used.
[0208] Assay procedures also may be used to identify the expression
of proteins by their functionality, especially the ability of
enzymes to catalyze specific chemical reactions involving specific
substrates and products. These reactions may be followed by
providing and quantifying the loss of substrates or the generation
of products of the reactions by physical or chemical procedures.
Examples are as varied as the enzyme to be analyzed and may include
assays for PAT enzymatic activity by following production of
radiolabeled acetylated phosphinothricin from phosphinothricin and
.sup.14C-acetyl CoA or for anthranilate synthase activity by
following loss of fluorescence of anthranilate, to name two.
[0209] Very frequently the expression of a gene product is
determined by evaluating the phenotypic results of its expression.
These assays also may take many forms including but not limited to
analyzing changes in the chemical composition, morphology, or
physiological properties of the plant. Chemical composition may be
altered by expression of genes encoding enzymes or storage proteins
which change amino acid composition and may be detected by amino
acid analysis, or by enzymes which change starch quantity which may
be analyzed by near infrared reflectance spectrometry.
Morphological changes may include greater stature or thicker
stalks. Most often changes in response of plants or plant parts to
imposed treatments are evaluated under carefully controlled
conditions termed bioassays.
[0210] IX. Assays of Transgene Expression
[0211] Assays may be employed with the instant invention for
determination of the relative efficiency of transgene expression.
Such methods would also be useful in evaluating, for example,
random or site-specific mutants of the NOR sequences provided
herein. Alternatively, assays could be used to determine the
efficacy of expression when various different enhancers,
terminators or other types of elements potentially used in the
preparation of transformation constructs.
[0212] For plants, expression assays may comprise a system
utilizing embryogenic or non-embryogenic cells, or alternatively,
whole plants. An advantage of using cellular assays is that
regeneration of large numbers of plants is not required. However,
the systems are limited in that promoter activity in the
non-regenerated cells may not directly correlate with expression in
a plant. Additionally, assays of tissue or developmental specific
promoters are generally not feasible.
[0213] The biological sample to be assayed may comprise nucleic
acids isolated from the cells of any plant material according to
standard methodologies (Sambrook et al, 1989). The nucleic acid may
be genomic DNA or fractionated or whole cell RNA. Where RNA is
used, it may be desired to convert the RNA to a complementary DNA.
In one embodiment of the invention, the RNA is whole cell RNA; in
another, it is poly-A RNA. Normally, the nucleic acid is
amplified.
[0214] Depending on the format, the specific nucleic acid of
interest is identified in the sample directly using amplification
or with a second, known nucleic acid following amplification. Next,
the identified product is detected. In certain applications, the
detection may be performed by visual means (e.g., ethidium bromide
staining of a gel). Alternatively, the detection may involve
indirect identification of the product via chemiluminescence,
radioactive scintigraphy of radiolabel or fluorescent label or even
via a system using electrical or thermal impulse signals (Affymax
Technology; Bellus, 1994).
[0215] Following detection, one may compare the results seen in a
given plant with a statistically significant reference group of
non-transformed control plants. Typically, the non-transformed
control plants will be of a genetic background similar to the
transformed plants. In this way, it is possible to detect
differences in the amount or kind of protein detected in various
transformed plants. Alternatively, clonal cultures of cells, for
example, callus or an immature embryo, may be compared to other
cells samples.
[0216] As indicated, a variety of different assays are contemplated
in the screening of cells or plants of the current invention and
associated promoters. These techniques may in cases be used to
detect for both the presence and expression of the particular genes
as well as rearrangements that may have occurred in the gene
construct. The techniques include but are not limited to,
fluorescent in situ hybridization (FISH), direct DNA sequencing,
pulsed field gel electrophoresis (PFGE) analysis, Southern or
Northern blotting, single-stranded conformation analysis (SSCA),
RNAse protection assay, allele-specific oligonucleotide (ASO), dot
blot analysis, denaturing gradient gel electrophoresis, RFLP and
PCR.TM.-SSCP.
[0217] (i) Quantitation of Gene Expression with Relative
Quantitative RT-PCR.TM.
[0218] Reverse transcription (RT) of RNA to cDNA followed by
relative quantitative PCR.TM. (RT-PCR.TM.) can be used to determine
the relative concentrations of specific mRNA species isolated from
plants. By determining that the concentration of a specific mRNA
species varies, it is shown that the gene encoding the specific
mRNA species is differentially expressed. In this way, a promoters
expression profile can be rapidly identified, as can the efficacy
with which the promoter directs transgene expression.
[0219] In PCR.TM., the number of molecules of the amplified target
DNA increase by a factor approaching two with every cycle of the
reaction until some reagent becomes limiting. Thereafter, the rate
of amplification becomes increasingly diminished until there is no
increase in the amplified target between cycles. If a graph is
plotted in which the cycle number is on the X axis and the log of
the concentration of the amplified target DNA is on the Y axis, a
curved line of characteristic shape is formed by connecting the
plotted points. Beginning with the first cycle, the slope of the
line is positive and constant. This is said to be the linear
portion of the curve. After a reagent becomes limiting, the slope
of the line begins to decrease and eventually becomes zero. At this
point the concentration of the amplified target DNA becomes
asymptotic to some fixed value. This is said to be the plateau
portion of the curve.
[0220] The concentration of the target DNA in the linear portion of
the PCR.TM. amplification is directly proportional to the starting
concentration of the target before the reaction began. By
determining the concentration of the amplified products of the
target DNA in PCR.TM. reactions that have completed the same number
of cycles and are in their linear ranges, it is possible to
determine the relative concentrations of the specific target
sequence in the original DNA mixture. If the DNA mixtures are cDNAs
synthesized from RNAs isolated from different tissues or cells, the
relative abundances of the specific mRNA from which the target
sequence was derived can be determined for the respective tissues
or cells. This direct proportionality between the concentration of
the PCR.TM. products and the relative mRNA abundances is only true
in the linear range of the PCR.TM. reaction.
[0221] The final concentration of the target DNA in the plateau
portion of the curve is determined by the availability of reagents
in the reaction mix and is independent of the original
concentration of target DNA. Therefore, the first condition that
must be met before the relative abundances of a mRNA species can be
determined by RT-PCR.TM. for a collection of RNA populations is
that the concentrations of the amplified PCR.TM. products must be
sampled when the PCR.TM. reactions are in the linear portion of
their curves.
[0222] The second condition that must be met for an RT-PCR.TM.
study to successfully determine the relative abundances of a
particular mRNA species is that relative concentrations of the
amplifiable cDNAs must be normalized to some independent standard.
The goal of an RT-PCR.TM. study is to determine the abundance of a
particular mRNA species relative to the average abundance of all
mRNA species in the sample.
[0223] Most protocols for competitive PCR.TM. utilize internal
PCR.TM. standards that are approximately as abundant as the target.
These strategies are effective if the products of the PCR.TM.
amplifications are sampled during their linear phases. If the
products are sampled when the reactions are approaching the plateau
phase, then the less abundant product becomes relatively over
represented. Comparisons of relative abundances made for many
different RNA samples, such as is the case when examining RNA
samples for differential expression, become distorted in such a way
as to make differences in relative abundances of RNAs appear less
than they actually are. This is not a significant problem if the
internal standard is much more abundant than the target. If the
internal standard is more abundant than the target, then direct
linear comparisons can be made between RNA samples.
[0224] The above discussion describes theoretical considerations
for an RT-PCR.TM. assay for plant tissue. The problems inherent in
plant tissue samples are that they are of variable quantity (making
normalization problematic), and that they are of variable quality
(necessitating the co-amplification of a reliable internal control,
preferably of larger size than the target). Both of these problems
are overcome if the RT-PCR.TM. is performed as a relative
quantitative RT-PCR.TM. with an internal standard in which the
internal standard is an amplifiable cDNA fragment that is larger
than the target cDNA fragment and in which the abundance of the
mRNA encoding the internal standard is roughly 5-100 fold higher
than the mRNA encoding the target. This assay measures relative
abundance, not absolute abundance of the respective mRNA
species.
[0225] Other studies may be performed using a more conventional
relative quantitative RT-PCR.TM. assay with an external standard
protocol. These assays sample the PCR.TM. products in the linear
portion of their amplification curves. The number of PCR.TM. cycles
that are optimal for sampling must be empirically determined for
each target cDNA fragment. In addition, the reverse transcriptase
products of each RNA population isolated from the various tissue
samples must be carefully normalized for equal concentrations of
amplifiable cDNAs. This consideration is very important since the
assay measures absolute mRNA abundance. Absolute mRNA abundance can
be used as a measure of differential gene expression only in
normalized samples. While empirical determination of the linear
range of the amplification curve and normalization of cDNA
preparations are tedious and time consuming processes, the
resulting RT-PCR.TM. assays can be superior to those derived from
the relative quantitative RT-PCR.TM. assay with an internal
standard.
[0226] One reason for this advantage is that without the internal
standard/competitor, all of the reagents can be converted into a
single PCR.TM. product in the linear range of the amplification
curve, thus increasing the sensitivity of the assay. Another reason
is that with only one PCR.TM. product, display of the product on an
electrophoretic gel or another display method becomes less complex,
has less background and is easier to interpret.
[0227] (ii) Marker Gene Expression
[0228] Markers represent an efficient means for assaying the
expression of transgenes. Using, for example, a selectable marker,
one could quantitatively determine the resistance conferred upon a
plant or plant cell by a construct comprising the selectable marker
coding region operably linked to the promoter to be assayed, e.g.,
an RS324 promoter. Alternatively, various plant parts could be
exposed to a selective agent and the relative resistance provided
in these parts quantified, thereby providing an estimate of the
tissue specific expression of the promoter.
[0229] Screenable markers constitute another efficient means for
quantifying the expression of a given transgene. Potentially any
screenable marker could be expressed and the marker gene product
quantified, thereby providing an estimate of the efficiency with
which the promoter directs expression of the transgene.
Quantification can readily be carried out using either visual
means, or, for example, a photon counting device.
[0230] A preferred screenable marker gene assay for use with the
current invention constitutes the use of the screenable marker gene
.beta.-glucuronidase (GUS). Detection of GUS activity can be
performed histochemically using 5-bromo-4-chloro-3-indolyl
glucuronide (X-gluc) as the substrate for the GUS enzyme, yielding
a blue precipitate inside of cells containing GUS activity. This
assay has been described in detail (Jefferson, 1987). The blue
coloration can then be visually scored, and estimates of expression
efficiency thereby provided. GUS activity also can be determined by
immunoblot analysis or a fluorometric GUS specific activity assay
(Jefferson, 1987).
[0231] (iii) Purification and Assays of Proteins
[0232] One means for determining the efficiency with which a
particular transgene is expressed is to purify and quantify a
polypeptide expressed by the transgene. Protein purification
techniques are well known to those of skill in the art. These
techniques involve, at one level, the crude fractionation of the
cellular milieu to polypeptide and non-polypeptide fractions.
Having separated the polypeptide from other proteins, the
polypeptide of interest may be further purified using
chromatographic and electrophoretic techniques to achieve partial
or complete purification (or purification to homogeneity).
Analytical methods particularly suited to the preparation of a pure
peptide are ion-exchange chromatography, exclusion chromatography;
polyacrylamide gel electrophoresis; and isoelectric focusing. A
particularly efficient method of purifying peptides is fast protein
liquid chromatography or even HPLC.
[0233] Various techniques suitable for use in protein purification
will be well known to those of skill in the art. These include, for
example, precipitation with ammonium sulphate, PEG, antibodies and
the like or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of such and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
[0234] There is no general requirement that the protein or peptide
being assayed always be provided in their most purified state.
Indeed, it is contemplated that less substantially purified
products will have utility in certain embodiments. Partial
purification may be accomplished by using fewer purification steps
in combination, or by utilizing different forms of the same general
purification scheme. For example, it is appreciated that a
cation-exchange column chromatography performed utilizing an HPLC
apparatus will generally result in a greater "-fold" purification
than the same technique utilizing a low pressure chromatography
system. Methods exhibiting a lower degree of relative purification
may have advantages in total recovery of protein product, or in
maintaining the activity of an expressed protein.
[0235] It is known that the migration of a polypeptide can vary,
sometimes significantly, with different conditions of SDS/PAGE
(Capaldi et al., 1977). It will therefore be appreciated that under
differing electrophoresis conditions, the apparent molecular
weights of purified or partially purified expression products may
vary.
[0236] High Performance Liquid Chromatography (HPLC) is
characterized by a very rapid separation with extraordinary
resolution of peaks. This is achieved by the use of very fine
particles and high pressure to maintain an adequate flow rate.
Separation can be accomplished in a matter of minutes, or at most
an hour. Moreover, only a very small volume of the sample is needed
because the particles are so small and close-packed that the void
volume is a very small fraction of the bed volume. Also, the
concentration of the sample need not be very great because the
bands are so narrow that there is very little dilution of the
sample.
[0237] Gel chromatography, or molecular sieve chromatography, is a
special type of partition chromatography that is based on molecular
size. The theory behind gel chromatography is that the column,
which is prepared with tiny particles of an inert substance that
contain small pores, separates larger molecules from smaller
molecules as they pass through or around the pores, depending on
their size. As long as the material of which the particles are made
does not adsorb the molecules, the sole factor determining rate of
flow is the size. Hence, molecules are eluted from the column in
decreasing size, so long as the shape is relatively constant. Gel
chromatography is unsurpassed for separating molecules of different
size because separation is independent of all other factors such as
pH, ionic strength, temperature, etc. There also is virtually no
adsorption, less zone spreading and the elution volume is related
in a simple matter to molecular weight.
[0238] Affinity Chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule that it can specifically bind to. This is a
receptor-ligand type interaction. The column material is
synthesized by covalently coupling one of the binding partners to
an insoluble matrix. The column material is then able to
specifically adsorb the substance from the solution. Elution occurs
by changing the conditions to those in which binding will not occur
(alter pH, ionic strength, temperature, etc.).
[0239] A particular type of affinity chromatography useful in the
purification of carbohydrate containing compounds is lectin
affinity chromatography. Lectins are a class of substances that
bind to a variety of polysaccharides and glycoproteins. Lectins are
usually coupled to agarose by cyanogen bromide. Conconavalin A
coupled to Sepharose was the first material of this sort to be used
and has been widely used in the isolation of polysaccharides and
glycoproteins other lectins that have been include lentil lectin,
wheat germ agglutinin which has been useful in the purification of
N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins
themselves are purified using affinity chromatography with
carbohydrate ligands. Lactose has been used to purify lectins from
castor bean and peanuts; maltose has been useful in extracting
lectins from lentils and jack bean; N-acetyl-D galactosamine is
used for purifying lectins from soybean; N-acetyl glucosaminyl
binds to lectins from wheat germ; D-galactosamine has been used in
obtaining lectins from clams and L-fucose will bind to lectins from
lotus.
[0240] The matrix should be a substance that itself does not adsorb
molecules to any significant extent and that has a broad range of
chemical, physical and thermal stability. The ligand should be
coupled in such a way as to not affect its binding properties. The
ligand should also provide relatively tight binding. And it should
be possible to elute the substance without destroying the sample or
the ligand. One of the most common forms of affinity chromatography
is immunoaffinity chromatography. The generation of antibodies that
would be suitable for use in accord with the present invention is
discussed below.
[0241] X. Oligonucleotide Probes and Primers
[0242] Naturally, the present invention also encompasses DNA
segments that are complementary, or essentially complementary, to
the sequences set forth in SEQ ID NO:1, SEQ ID NO:6, and SEQ ID
NO:7. Nucleic acid sequences that are "complementary" are those
that are capable of base-pairing according to the standard
Watson-Crick complementary rules. As used herein, the term
"complementary sequences" means nucleic acid sequences that are
substantially complementary, as may be assessed by the same
nucleotide comparison set forth above, or as defined as being
capable of hybridizing to the nucleic acid segment of SEQ ID NO:1,
SEQ ID NO:6, or SEQ ID NO:7 under relatively stringent conditions
such as those described herein. Such sequences may encode the
entire NOR protein or functional or non-functional fragments
thereof. Alternatively, the hybridizing segments may be shorter
oligonucleotides. Sequences of 17 bases long should occur only once
in the genome of most plant species and, therefore, suffice to
specify a unique target sequence. Although shorter oligomers are
easier to make, numerous other factors are involved in determining
the specificity of hybridization. Both binding affinity and
sequence specificity of an oligonucleotide to its complementary
target increases with increasing length. It is contemplated that
exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be
used, although others are contemplated. Longer polynucleotides
encoding 250, 500, 1000, 1212, 1500, 2000, 2500, or 3000 bases and
longer are contemplated as well. Such oligonucleotides will find
use, for example, as probes in Southern and Northern blots and as
primers in amplification reactions.
[0243] Suitable hybridization conditions will be well known to
those of skill in the art. In certain applications, for example,
substitution of amino acids by site-directed mutagenesis, it is
appreciated that lower stringency conditions are required. Under
these conditions, hybridization may occur even though the sequences
of probe and target strand are not perfectly complementary, but are
mismatched at one or more positions. Conditions may be rendered
less stringent by increasing salt concentration and decreasing
temperature. For example, a medium stringency condition could be
provided by about 0.1 to 0.25 M NaCl at temperatures of about
37.degree. C. to about 55.degree. C., while a low stringency
condition could be provided by about 0.15 M to about 0.9 M salt, at
temperatures ranging from about 20.degree. C. to about 55.degree.
C. Thus, hybridization conditions can be readily manipulated, and
thus will generally be a method of choice depending on the desired
results.
[0244] In other embodiments, hybridization may be achieved under
conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl.sub.2, 10 mM dithiothreitol, at temperatures between
approximately 20.degree. C. to about 37.degree. C. Other
hybridization conditions utilized could include approximately 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, at temperatures
ranging from approximately 40.degree. C. to about 72.degree. C.
Formamide and SDS also may be used to alter the hybridization
conditions.
[0245] One method of using probes and primers of the present
invention is in the search for genes related to NOR from other
species. Normally, the target DNA will be a genomic or cDNA
library, although screening may involve analysis of RNA molecules.
By varying the stringency of hybridization, and the region of the
probe, different degrees of homology may be discovered.
[0246] Another way of exploiting probes and primers of the present
invention is in site-directed, or site-specific mutagenesis.
Site-specific mutagenesis is a technique useful in the preparation
of individual peptides, or biologically functional equivalent
proteins or peptides, through specific mutagenesis of the
underlying DNA. The technique further provides a ready ability to
prepare and test sequence variants, incorporating one or more of
the foregoing considerations, by introducing one or more nucleotide
sequence changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent nucleotides, to provide a
primer sequence of sufficient size and sequence complexity to form
a stable duplex on both sides of the deletion junction being
traversed. Typically, a primer of about 17 to 25 nucleotides in
length is preferred, with about 5 to 10 residues on both sides of
the junction of the sequence being altered.
[0247] The technique typically employs a bacteriophage vector that
exists in both a single stranded and double stranded form. Typical
vectors useful in site-directed mutagenesis include vectors such as
the M13 phage. These phage vectors are commercially available and
their use is generally well known to those skilled in the art.
Double stranded plasmids are also routinely employed in site
directed mutagenesis, which eliminates the step of transferring the
gene of interest from a phage to a plasmid.
[0248] In general, site-directed mutagenesis is performed by first
obtaining a single-stranded vector, or melting of two strands of a
double stranded vector which includes within its sequence a DNA
sequence encoding the desired protein. An oligonucleotide primer
bearing the desired mutated sequence is synthetically prepared.
This primer is then annealed with the single-stranded DNA
preparation, taking into account the degree of mismatch when
selecting hybridization conditions, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are
selected that include recombinant vectors bearing the mutated
sequence arrangement.
[0249] The preparation of sequence variants of the selected gene
using site-directed mutagenesis is provided as a means of producing
potentially useful species and is not meant to be limiting, as
there are other ways in which sequence variants of genes may be
obtained. For example, recombinant vectors encoding the desired
gene may be treated with mutagenic agents, such as hydroxylamine,
to obtain sequence variants.
[0250] XI. Breeding Plants of the Invention
[0251] In addition to direct transformation of a particular
genotype with a construct prepared according to the current
invention, transgenic plants may be made by crossing a plant having
a construct of the invention to a second plant lacking the
construct. For example, a nucleic acid sequence encoding a NOR
coding sequence can be introduced into a particular plant variety
by crossing, without the need for ever directly transforming a
plant of that given variety. Therefore, the current invention not
only encompasses a plant directly created from cells which have
been transformed in accordance with the current invention, but also
the progeny of such plants. As used herein the term "progeny"
denotes the offspring of any generation of a parent plant prepared
in accordance with the instant invention, wherein the progeny
comprises a construct prepared in accordance with the invention.
"Crossing" a plant to provide a plant line having one or more added
transgenes relative to a starting plant line, as disclosed herein,
is defined as the techniques that result in a transgene of the
invention being introduced into a plant line by crossing a starting
line with a donor plant line that comprises a transgene of the
invention. To achieve this one could, for example, perform the
following steps:
[0252] (a) plant seeds of the first (starting line) and second
(donor plant line that comprises a transgene of the invention)
parent plants;
[0253] (b) grow the seeds of the first and second parent plants
into plants that bear flowers;
[0254] (c) pollinate a female flower of the first parent plant with
the pollen of the second parent plant; and
[0255] (d) harvest seeds produced on the parent plant bearing the
female flower.
[0256] Backcrossing is herein defined as the process including the
steps of:
[0257] (a) crossing a plant of a first genotype containing a
desired gene, DNA sequence or element to a plant of a second
genotype lacking said desired gene, DNA sequence or element;
[0258] (b) selecting one or more progeny plant containing the
desired gene, DNA sequence or element;
[0259] (c) crossing the progeny plant to a plant of the second
genotype; and
[0260] (d) repeating steps (b) and (c) for the purpose of
transferring said desired gene, DNA sequence or element from a
plant of a first genotype to a plant of a second genotype.
[0261] Introgression of a DNA element into a plant genotype is
defined as the result of the process of backcross conversion. A
plant genotype into which a DNA sequence has been introgressed may
be referred to as a backcross converted genotype, line, inbred, or
hybrid. Similarly a plant genotype lacking said desired DNA
sequence may be referred to as an unconverted genotype, line,
inbred, or hybrid.
[0262] XII. Definitions
[0263] Genetic Transformation: A process of introducing a DNA
sequence or construct (e.g., a vector or expression cassette) into
a cell or protoplast in which that exogenous DNA is incorporated
into a chromosome or is capable of autonomous replication.
[0264] Exogenous gene: A gene which is not normally present in a
given host genome in the exogenous gene's present form In this
respect, the gene itself may be native to the host genome, however,
the exogenous gene will comprise the native gene altered by the
addition or deletion of one or more different regulatory
elements.
[0265] Expression: The combination of intracellular processes,
including transcription and translation undergone by a coding DNA
molecule such as a structural gene to produce a polypeptide.
[0266] Expression vector: A nucleic acid comprising one or more
coding sequences which one desires to have expressed in a
transgenic organism.
[0267] Progeny: Any subsequent generation, including the seeds and
plants therefrom, which is derived from a particular parental plant
or set of parental plants.
[0268] Promoter: A recognition site on a DNA sequence or group of
DNA sequences that provide an expression control element for a
structural gene and to which RNA polymerase specifically binds and
initiates RNA synthesis (transcription) of that gene.
[0269] R.sub.0 Transgenic Plant: A plant which has been directly
transformed with a selected DNA or has been regenerated from a cell
or cell cluster which has been transformed with a selected DNA.
[0270] Regeneration: The process of growing a plant from a plant
cell (e.g., plant protoplast, callus or explant).
[0271] Selected DNA: A DNA which one desires to have expressed in a
transgenic plant, plant cell or plant part. A selected DNA may be
native or foreign to a host genome, but where the selected DNA is
present in the host genome, may include one or more regulatory or
functional elements which alter the expression profile of the
selected gene relative to native copies of the gene.
[0272] Selected Gene: A gene which one desires to have expressed in
a transgenic plant, plant cell or plant part. A selected gene may
be native or foreign to a host genome, but where the selected gene
is present in the host genome, will include one or more regulatory
or functional elements which differ from native copies of the
gene.
[0273] Transformation construct: A chimeric DNA molecule which is
designed for introduction into a host genome by genetic
transformation. Preferred transformation constructs will comprise
all of the genetic elements necessary to direct the expression of
one or more exogenous genes, for example, NOR genes. Included
within in this term are, for example, expression cassettes isolated
from a starting vector molecule.
[0274] Transformed cell: A cell the DNA complement of which has
been altered by the introduction of an exogenous DNA molecule into
that cell.
[0275] Transgene: A segment of DNA which has been incorporated into
a host genome or is capable of autonomous replication in a host
cell and is capable of causing the expression of one or more
cellular products. Exemplary transgenes will provide the host cell,
or plants regenerated therefrom, with a novel phenotype relative to
the corresponding non-transformed cell or plant. Transgenes may be
directly introduced into a plant by genetic transformation, or may
be inherited from a plant of any previous generation which was
transformed with the DNA segment.
[0276] Transgenic plant: A plant or progeny plant of any subsequent
generation derived therefrom, wherein the DNA of the plant or
progeny thereof contains an introduced exogenous DNA segment not
originally present in a non-transgenic plant of the same strain.
The transgenic plant may additionally contain sequences which are
native to the plant being transformed, but wherein the "exogenous"
gene has been altered in order to alter the level or pattern of
expression of the gene.
[0277] Transit peptide: A polypeptide sequence which is capable of
directing a polypeptide to a particular organelle or other location
within a cell.
XII. EXAMPLES
[0278] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the concept, spirit and scope
of the invention. More specifically, it will be apparent that
certain agents which are both chemically and physiologically
related may be substituted for the agents described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.
Example 1
Genetic Mapping of Fruit Ripening Loci
[0279] Three F2 populations, each segregating for one of three
ripening mutants, were generated from interspecific crosses between
L. esculentum cultivars homozygous for the respective mutant allele
and a normally ripening L. cheesmannii parent (accession LA483). As
a result RFLP markers less than one cM from both RIN (CT63-0.24 cM)
and NOR (CT16-0.9 cm) were identified.
[0280] A) RFLP Mapping of the rin Locus using Pooled DNA Samples:
1840 F2 individuals segregating for the mutant rin allele and RFLP
markers were generated from the interspecific cross between L.
esculentum homozygous for the mutant rin allele and L. cheesmannii
homozygous for the allele conferring the normal ripening phenotype.
45 DNA pools representing a total of 225 mutant individuals (5
plants per pool) were generated from this population. Lanes 1-7
represented a subset of these pools and 35 mutant individuals.
Using this subset of DNA pools, the rin locus was initially mapped
to a 10 cM region of chromosome 5 flanked by RFLP markers CT93 and
TG448. Sample results from gel blot hybridization of pooled DNA
samples with RFLP probes are shown for TG448 (tightly linked) and
TG185 (unlinked--greater than 50 cM away). "C" represents a DNA
pool derived from 5 individuals with ripening fruit. "e" and "c"
designate the L. esculentum (mutant) and L. cheesmannii (normal)
alleles of the RFLP probes employed, respectively. Hybridization to
the "c" allele of TG448 in pools 1, 3 and 6 represents individuals
within these pools which have undergone recombination between the
TG448 and rin loci. The entire set of 45 pools was used to generate
an RFLP map of the rin region of chromosome 5.
[0281] B) Generation of linked markers via RAPD analysis of nearly
isogenic DNA pools: Methodology for isolating molecular markers
linked to targeted regions of the genome is important in the event
that markers close enough for initiating a chromosome walk are not
available. Until recently, methods for the isolation of molecular
markers linked to targeted loci relied upon the availability of
nearly isogenic lines (NILs) in which the target locus resides in a
highly polymorphic introgressed region (Young et al 1998, Martin et
al 1991). Although nearly isogenic lines do exist for the rin, nor
and Nr loci, in all cases, both the donor and recurrent parents
used are L. esculentum cultivars. Unfortunately, extremely low
levels of RFLP polymorphism are detected among L. esculentum
cultivars (Miller and Tanksley, 1990). Consequently, these NILs are
not useful for the identification of new markers linked to ripening
loci.
[0282] A new strategy was employed that rapidly identifies markers
linked to previously mapped target genes for which NILs are not
available (Giovannoni et al 1991). This strategy is based on the
generation of nearly isogenic DNA pools from existing RFLP mapping
populations. In short, once a target locus has been mapped to a
genomic region between flanking RFLP markers, two DNA pools are
generated from an RFLP mapping population. Membership in either
pool depends on the individuals' parental origin for the target
region (as defined by scoring the flanking markers in the RFLP
mapping population employed). The result is two DNA pools
representing individual progeny selected to be homozygous for loci
derived from either one parent or the other across the target
region defined by the flanking markers. Since inclusion in a pool
is dependent only on the parental origin of the target region,
distantly linked and unlinked loci are equally likely to be derived
from either parent (i.e. they are not selected for). By combining
DNA from multiple individuals into each pool, the chances for
homozygosity at loci other than those within the target region
becomes minimal. The resulting DNA pools are nearly isogenic for
the targeted genomic region. These pools are subsequently utilized
as templates for RAPD reactions employing random primer PCR
amplification of genomic templates (Williams, et al 1990).
Amplification products which differ between the two nearly isogenic
DNA pools are likely to be derived from the target region
(Giovannoni et al, 1991).
[0283] In order to identify additional markers tightly linked to
targeted ripening loci, pairs of DNA pools nearly isogenic for all
three ripening locus regions were generated and screened for
polymorphic RAPD products. Random primers will be purchased from
Operon Technologies and the population used for the generation of
isogenic DNA pools is the L. esculentum X L. pennellli F2 mapping
population used to generate the tomato RFLP map. This population
will be used rather than the actual ripening gene mapping
populations because of the higher degree of polymorphism between
the parents (Miller and Tanksley, 1990). The purpose of this effort
will be to identify tightly linked molecular probes to be utilized
in chromosome walks.
[0284] In previous analysis employing tomato genomic DNA templates,
3-7 amplification products were detected, on average, per primer.
Consequently, 500 primers should result in approximately 2,500
amplified loci of the tomato genome. Given the estimated map size
of 1500 cM for the tomato genome, 500 primers should yield
approximately 1-2 markers within 1 cM on either side of a target
locus. In the event that sufficient polymorphic markers are not
identified for one or more of the target loci, additional primers
will be acquired through mutual primer exchanges. Also, the use of
random primer pairs has recently been demonstrated to yield
significant numbers of unique amplification products from tomato
genomic DNA.
[0285] C) Isolation of a Molecular Marker Linked to the rin Locus
using Isogenic DNA Pools: Linkage analysis permitted the placement
of the rin locus between the markers TG503 and TG96. Progeny from
an F2 population (L. esculentum X L. pennellii which were scored as
homozygous for all markers between CT227 and TG318 were used to
target the rin locus. DNA from 7-13 individuals was used to
construct 2 nearly isogenic DNA pools. The region shown in black is
the resulting chromosome target for the isolation of molecular
markers linked to the rin locus. Numbers to the left of the
schematic chromosome designates recombination distances in cM
between markers designated on the right. A total of 100 random 10
base primers were utilized for amplification of nearly isogenic DNA
pools resulting in one polymorphic PCR product, P76. The 0.5 kb P76
amplification product was gel-purified, labeled and mapped via an
EcoRI RFLP to the target region.
Example 2
Physical Mapping and Chromosome Walking to rin.sup.2
[0286] Following identification of DNA markers tightly linked to
the RIN locus, physical mapping with high molecular weight DNA
gel-blots was performed to assess the feasibility of initiating a
chromosome walk. The result was identification of an 800 kb SmaI
restriction fragment which hybridized to 3 DNA markers which
flanked the RIN locus and span a genetic distance of 4.2 cM. Based
on this physical mapping data it was estimated that one cM in the
RIN region of chromosome 5 corresponds to approximately 191 kb (800
kb/4.2 cM. =191 kb/cM). This estimate is similar to that for the
Pto locus which is linked to RIN (Martin et al., 1994).
[0287] Given the average 140 kb insert size of the tomato YAC
library (Martin et al., 1992), a chromosome walk was initiated from
the flanking single copy RFLP markers TG503, and CT93 which are
1.24 cM and 2.9 cM from rin, respectively. CT63, although only 0.24
cM from RIN on the TG503 side, was not employed as a probe because
it is a member of a small gene family and unlinked YAC clones would
likely be recovered. However, a 360 kb clone (Yrin2) demonstrated
hybridization to both TG503 and CT63 suggesting that 1) it
contained sequences closer to the target locus than any of the
other clones, and 2) the estimated 191 kb/cM ratio in the region of
RIN was within two fold of accurate (i.e. TG503 and CT63 are
separated by 1 cM and reside on a 360 kb YAC). A single copy end of
Yrin2 (Yrin2R) was isolated by inverse PCR and mapped, in a
population of 670 F2 progeny, on the RIN side of CT63 0.2 cM from
rin. This end was subsequently utilized to take a "step" toward RIN
in the tomato YAC library resulting in the isolation of 4
additional YAC clones, Yrin8, Yrin9, Yrin11, and Yrin12. 7 of the 8
possible YAC ends were isolated through inverse PCR and/or plasmid
rescue for generation of a YAC contig via cross-hybridization of
ends with RIN region YACs, and RFLP mapping. Two YAC clones were
determined to be chimeric based on RFLP mapping (Yrin9R) and
sequencing of YAC ends (Yrin8R--greater than 95% homology with
tobacco chloroplast DNA). One YAC end, Yrin8L, cosegregated with
RIN and hybridized to none of the other YAC clones, suggesting that
it extended the furthest toward RIN and may harbor the target
gene.
[0288] A) Completion of the chromosome walk to RIN: It was
demonstrated that an end clone of a RIN region YAC designated
Yrin8L cosegregates with RIN in a population of 670 F2 individuals.
Based on high stringency (0.2.times.SSC, 65.degree. C.) DNA
gel-blot analysis and RFLP mapping, Yrin8L, is a single copy
sequence in the tomato genome. Specific PCR primers were generated
which amplify the expected 270 bp fragment of Yrin8L from both the
plasmid clone and the tomato genome. The primers were used to PCR
screen the tomato YAC library of Martin et al. (1992). Screening of
this YAC library via colony hybridization or PCR yielded 1-7
verified (via mapping of end clones and/or cross hybridization to 2
or more probes from the target region) recombinant clones for each
of the 6 RIN or NOR linked markers tested. As an alternate
strategy, random PstI, EcoRI, or HaeIII subclones of Yrin8 could be
employed as probes in the next step toward rin, and the random
subclones tested for copy number, map position, and homology to
Yrin YACs to ensure sequences from the end of the clone near RIN
are used.
[0289] B) Isolation and characterization of cDNAs corresponding to
genes within YACs containing the target locus: Once candidate RIN
containing YAC clones were identified, the clones were used as
probes for isolation of cDNAs which may represent transcripts
derived from genes contained on the YAC. Two cDNAs were isolated
and mapped using Ynor3 as a probe from a "Breaker" fruit cDNA,
yielding numerous additional positive clones. Yrin8 also yielded
numerous cDNA clones which were tightly linked to the rin locus. A
similar strategy was employed by Martin et al. (1993) in
identifying the tomato Pto gene on a 400 kb clone from this same
library.
[0290] cDNA libraries in the vector lambda gt10 and made from
Mature Green, Breaker, and Red Ripe stage fruit are all available
in the laboratory (lambda gt10 does not cross hybridize with the
YAC vector pYAC4). All 3 libraries will be screened because it is
not known which stage will express the highest levels of target
gene product. Construction of a fourth cDNA library may also be
used in lambda gt10 made from mRNA derived from several stages of
immature fruit development. Specifically, mRNA will be combined
from ovaries prior to pollination, 2 days post pollination, and
every 10 days post-pollination up to the Mature Green stage
(approximately 30 days in cultivar Ailsa Craig). This library will
help to minimize the problem of not knowing when during fruit
development the RIN or NOR gene is expressed. Tissue for all but
the unpollinated ovaries are stored in a -80.degree. C.
freezer.
Example 3
Physical Mapping and Chromosome Walking to NOR
[0291] High molecular weight DNA gel-blot analysis was also
utilized to estimate the kb/cM ratio in the region of the NOR
locus. A 1000 kb CspI fragment hybridized to both CT16 and TG313
which flank the NOR locus and are separated by approximately 5 cM.
Based on this observation, it was estimated that 1 cM in the region
of the NOR locus corresponds to approximately 200 kb.
[0292] Prior to initiation of the chromosome walk to nor, the
closest known flanking markers were CT16 (0.9 cM) and CT41 (2.3
cM). Based on the published tomato RFLP map (Tanksley et al.,
1992), it was known that TG395 resided in the interval between CT41
and CT16 and thus represented a closer marker to NOR than at least
one of the two. TG395 was mapped as a sequence tagged site (due to
lack of RFLPs between the two parents of the mapping population) to
1.4 cM from NOR on the CT41 side. PCR screening of the tomato YAC
library with TG395 specific primers resulted in the identification
of 7 YAC clones ranging in size from 50 kb-490 kb. CT16 was not
used for library screening because of difficulty in generating
reliable PCR primers.
[0293] Three YAC ends were isolated via plasmid rescue and inverse
PCR, as described for RIN above. YAC end and RFLP marker cross
hybridizations yielded a YAC contig. Of particular interest was the
observation that the 470 kb YAC, Ynor3, hybridized to both TG395
(which was used to retrieve this clone from the library) and CT16.
Localization of NOR within the interval of chromosome 10 bordered
by TG395 and CT16 suggested that the targeted NOR locus resides on
Ynor3, and confirmed estimates of kb/cM in this region of the
genome. A high titer cosmid library (>500,000 clones) was then
prepared of the yeast containing Ynor3 to use in fine mapping and
walking to NOR within the Ynor3 clone. The, cosmid vector employed,
04541, contains T-DNA borders to permit direct Agrobacterium
transfer into plants. Random Ynor3 PstI and HaeIII subclones were
also isolated to use as fine mapping probes. A "breaker" stage
tomato fruit cDNA library was also screened with Ynor3 as probe,
yielding numerous clones for characterization for linkage to
nor.
Example 4
Characterization of YAC Clones Harboring the rin and nor Loci
[0294] Initial screening with tightly linked RFLP markers yielded 6
YACs linked to the nor locus and 5 YACs linked to rin.
Hybridization to YACs with flanking markers revealed that a single
nor YAC termed Ynor3 harbored the target gene assuming no internal
deletions or other perturbations within the YAC. Similar
hybridization experiments, including hybridization with isolated
YAC ends, revealed that there were a number of alterations in
several of the YACs which presumably occurred during library
construction and represented either deletions relative to genomic
sequences or chimerism with fragments of genomic DNA derived from
unlinked regions of the tomato genome. Extensive characterization
of these clones was performed including RFLP mapping of random
subclones and YAC ends resulting in determination that the
resulting YAC contig did not extend to the point including the rin
locus. The terminal YAC end from the YAC extending closest to rin
was subsequently used to re-screen the tomato YAC library resulting
in identification of 4 additional clones. One of these clones was
extensively characterized due to the presence (via hybridization)
of DNA markers flanking rin on this single YAC. The results
indicated that this particular clone harbored an internal deletion
resulting in the absence of the targeted rin locus. Following a
third screen of the YAC library using a terminal YAC end from a
previous screen as probe, a YAC termed Yrin11 was identified which
harbored the rin locus as determined by random subcloning of Yrin11
restriction fragments and RFLP mapping to determine that sequences
flanking the target locus were contained on this YAC. This YAC also
contained sequences that were absent from the YAC descried above
with an internal deletion (thus confirming the integrity of
Yrin11), and said fragments were tightly linked to the rin
locus.
Example 5
cDNA Library Screening
[0295] A IX amplified breaker fruit cDNA library (cv. Ailsa Craig)
in vector lambda TRIPLEX was screened with whole PFGE gel-purified
YACs corresponding to Ynor3 and Yrin11, respectively. A contig of
tomato BAC clones (library in pBELOBAC11, cultivar LA483-L.
cheesmannii) was also simultaneously constructed across the nor
region. Similar BAC contig efforts were initiated for the rin
region of chromosome 5 but were not completed prior to isolation of
the RIN gene. cDNAs hybridizing to the two candidate gene YACs, and
thus potentially representing target gene transcripts, were
verified for homology via hybridization as probes back to the
respective Yrin11 and Ynor3 YACs. Positives were mapped as RFLPs
and sequenced.
Example 6
NOR Gene Identification
[0296] Ynor3 hybridizing cDNA, CD11, was one of two clones found to
cosegregate with the nor locus and to hybridize to the BAC clone
most likely (via hybridization to nor-linked markers) to harbor the
target locus. Both CD11 and the other co-segregating cDNA (CD5)
were hybridized to RNA gel-blots of normal and mutant fruit RNAs.
CD5 was constitutively expressed throughout fruit development while
CD11 was induced during ripening and by ethylene. CD11 was also
greatly reduced in expression in fruit of the nor mutant. CD11 was
sequenced and found to have homology to the CUC
(cup-shaped-cotyledon) gene of Arabidopsis. This gene also has
homology to two functionally defined Arabidopsis transcription
factors of otherwise unknown function. Based on CD11 sequence,
primers were generated for RT-PCR of CD11 mRNA. CD11 alleles from
normal (Nor/Nor) and mutant (nor/nor) tomato lines were generated
by RT-PCR and sequenced. The mutant allele harbors a 2 bp deletion
relative to the normal CD11 allele resulting in a stop codon
approximately mid-way through the CD11 open reading frame. Based on
this mutation, and gene expression patterns described above, in
addition to the putative role of CD11 as a transcription factor, it
was indicated that the CD11 sequence represents the tomato NOR
gene. An original sequence obtained of the clone is indicated in
SEQ ID NO:1 and a corrected version of the sequence in SEQ ID
NO:6.
Example 7
Confirmation of NOR Target Gene Isolation
[0297] The cDNA identified as representing NOR was be tested for
function in ripening through the use of antisense and sense
expression constructs in normal and mutant tomatoes, respectively.
Antisense gene suppression has proven an effective tool for
determining gene function during tomato fruit ripening (reviewed in
Gray et al., 1994).
[0298] A) Preparation of NOR sense and antisense transformation
constructs: T-DNA constructs were prepared for delivery of sense or
antisense NOR gene cDNA (CD-11) sequences into plant genomes (FIG.
1). First, NOR-pBI121 sense and antisense constructs were made by
replacing the GUS gene of T-DNA binary vector pBI121 with the
full-length NOR cDNA referred to as CD-11 (1180 bp) in sense and
antisense orientations, respectively, relative to the CaMV35S
promoter (35S-P) of pBI121 (FIG. 1). In both constructs, the NOR
cDNA (CD-11 insert) was subcloned between SmaI and SacI restriction
sites resulting from removal of the GUS gene from pBI121.
Specifically, EcoRV and SacI sites of the pBluescript vector
containing the NOR cDNA were employed due to the fact that SmaI and
EcoRV (blunt) ends are compatible for ligation. The resulting
ligated sequence no longer can be digested with SmaI or EcoRV.
Completed sense and antisense constructs were initially transformed
into E. coli DH 10B cells and then resulting plasmid DNA was
isolated and transformed into Agrobacterium tumefaciens strain LBA
4404 for use in transfer into the tomato gene as described
herein.
[0299] The sense and antisense orientations of the NOR cDNA
sequence relative to the EcoRV and SacI restriction sites were
obtained by subcloning the original NOR cDNA bound by EcoRI sites
from the original cDNA library vector (lambda gt10) into the EcoRI
site of plasmid vector pBluescript. Due to the fact that the cDNA
sequence was flanked by identical restriction sites (EcoRI), the
insert could insert in either direction essentially at random.
Several resulting NOR-pBluescript clones were isolated and
sequenced to determine the orientation of the cDNA insert relative
to the EcoRV and SacI sites of pBluescript, and one clone
representing each orientation (sense and antisense) was selected
for transfer into the SamI and SacI sites of pBI121 (following
removal of the GUS gene from pBI121). In addition to the
full-length NOR cDNA 3 bp of pBluescript polylinker (BS) was
included on the SmaI side of the cDNA and 51 bp of pBluescript
polylinker was included on the SacI side of the insert (including
the following restriction sites: SacII, NotI, Xbal, SpeI, BamHI,
SmaI, PstI, EcoRI).
[0300] A sample of some of the classes of vectors that were
prepared by the inventors for studies of the function of the NOR
gene is given below, in Table 2.
2TABLE 2 Constructs for preparation of RIN and NOR transgenic
plants: CONSTRUCT HOST PURPOSE 35s-antisense AC wild-type Phenocopy
nor mutation CD5 35s-sense CD5 AC wild-type Ectopic expression of
nor 35s-sense CD5 MH1 nor/nor Complementation of nor mutant/nor
confirmation Genomic CD5 MH1 nor/nor Complementation of nor
mutant/nor confirmation
[0301] B) Transformation of wild type and mutant tomato plants: The
sense and antisense NOR constructs prepared as described above were
transformed into wild type and nor mutant plants for confirmation
of NOR identity. A modified version of the transformation procedure
described by Fillattii et al. (1987) was used for generation of
transgenic tomato plants (Deikman and Fischer, 1988).
[0302] Transgenic tomato plant were prepared as follows. First, the
explant was prepared by sterilizing seeds with soaking in 20%
bleach +0.1% Tween-20 for 15 minutes. The seeds were rinses 4 times
in sterile distilled H.sub.2O and the seeds sown on MSO medium ((1
Liter): 4.0 g MS Salts (Gibco), 5.0 ml B5 Vitamins, 5.0 ml MS
Iron/EDTA, 20.0 g sucrose, 7.0 g agar (phytagar), pH medium to 6.0
with KOH, autoclave) in sterile glass jars, grow in growth chamber.
Agrobacterium was then prepared by streaking selective fresh plates
with Agrobacterium containing the desired construct for 2-3 days
before it was needed. A single colony was picked from the plate and
grown in tubes containing 2 ml YEP medium (YEP Rich Medium (500
ml): 5.0 g Bacto-peptone, 2.5 g NaCl, 5.0 g Bacto Yeast Extract,
7.5 g Bacto agar, autoclave) with appropriate antibiotics, followed
by incubation on a shaker at 28.degree. C. overnight. Explants were
precultured two days before infection takes place, and 8-10 day old
cotyledons were excised. With sterile forceps, cotyledons were
removed and placed onto an MSO plate. Using forceps and a blade,
the cotyledons were cut in 1-2 pieces. All pieces were then placed
on pre-incubation medium ((500 ml): 500 ml MSO Medium, 0.5 ml BAP,
0.1 ml IAA) in a deep petri dish, wrapped in parafilm, and placed
in growth chamber for 2 days.
[0303] Overnight cultures of Agrobacterium were then precultured
and spun down in a 4.degree. C. centrifuge at 2800 rpm for 10
minutes, the supernatant discarded and pellets suspended in 10 ml
of induction media ((100 ml): 5 ml AB Salts, 2 ml MES buffer, 2 ml
Sodium Phosphate buffer, 91 ml distilled water, 1 g glucose,
autoclave, place in 10 tubes, 10 ml each). Then, for
co-cultivation, the suspension was added to precultured cotyledon
pieces, wrapped in parafilm and shaken gently on roto-shaker for 15
minutes. Using a spatula, cotyledon pieces were placed on
co-cultivation media ((500 ml): 500 ml MSO Medium, 1 ml KH2PO4 (100
mg/ml), 250 (l Kinetin (0.2 mg/ml), 100 (l 2,4 D (1 mg/ml), 735 (l
acetosyringone)), wrapped in parafilm and placed in growth chamber
for 2-3 days.
[0304] For regeneration, after 2-3 days of being on co-cultivation
medium, pieces were transferred to regeneration (2Z) medium ((500
ml): 500 ml MSO Medium, 5.0 ml Carbenicillin stock, 1.0 ml
Kanamycin stock, 1.0 ml Zeatin (1 mg/ml), 0.1 ml IAA), wrapped in
parafilm and placed in a growth chamber for 2-3 weeks. The tissue
was transferred to fresh medium every 2-3 weeks. Generally,
calli/shoots were apparent at 6 weeks. The calli was excised from
cotyledon tissue and placed on fresh medium. Multiple shoots on one
callus were separated and placed on medium, keeping all shoots
together on one plate. The taller shoots were placed on deep petri
dish. After shoots were well structured, another regeneration (1Z)
medium (Regeneration Medium (1Z) (500 ml): 500 ml MSO Medium, 5.0
ml Carbenicillin stock, 1.0 ml Kanamycin stock, 0.5 ml Zeatin (1
mg/ml), 0.1 ml IAA) could be utilized for conserving resources.
[0305] When shoots developed a well established meristem,
individual shoots were excised of any remaining callus and placed
in/on rooting medium ((500 ml): 500 ml MSO medium, 1.0 ml Kanamycin
stock, 0.2 ml IAA) in glass jars and placed in a growth chamber.
The shoots were watched for signs that: 1. callus continued,
therefore suppressing roots to form; in this instance the callus
was cut off and again placed on fresh rooting media, or 2. if roots
appeared, the plant was ready for soil. Transformed plantlets were
then transferred from the glass jar and washed off of any remaining
agar on the roots under tap water gently and transplanted in pot
filled with moistened soil. The plantlets were watered, making sure
soil was thoroughly wet. Plantlets were covered with magenta box to
conserve higher humidity. After 5-7 days, the magenta box was
gradually removed. Plants were transferred to the greenhouse grown
to 10-15 cm.
[0306] Media used included the following: YEB Rich Medium (500 ml):
2.75 g Beef Extract, 0.55 g Yeast Extract, 2.75 g peptone, 2.75 g
sucrose, 1 ml MgSO4 (1M) pH 7.2, 7.5 g Bactoagar, autoclave. B5
vitamins (100 ml): 2.0 g myo-inositol, 0.2 g thiamine-HCl, 20.0 mg
nicotinic acid, 20.0 mg pyridoxine-HCl, mix, then put in autoclaved
bottle. MS Iron/EDTA (100 ml): 556.0 mg FeSO4-7H.sub.2O, 746.0 mg
Na2EDTA-2H.sub.2O, mix, then put in autoclaved bottle.
Benzylamino-purine (BAP): 1 mg BAP/1 ml H.sub.2O, dissolve BAP with
5N KOH dropwise, bring up to volume with ddH2O, filter sterilize in
TC Hood, refrigerate. Zeatin: 1 mg Zeatin/1 ml H.sub.2O, dissolve
with 4N NaOH dropwise, bring up to volume with ddH.sub.2O, filter
sterilize in TC Hood, refrigerate. Indoleacetic acid (IAA): 1 mg
IAA/1 ml ethanol, dissolve with 100% ethanol, filter sterilize in
TC Hood, refrigerate in foil (light sensitive, Good 1 week).
Acetosyringone stock (ACE) (10 mg/ml): weigh out 100 mg of
acetosyringone, add 10 ml 70% ethanol, filter sterilize, put in 1.5
ml tubes, place in -20C freezer. Carbenicillin Stock (50 mg/ml):
weigh out 5 grams Carbenicillin, add 100 ml ddH.sub.2O, filter
sterilize, put in 12 ml tubes, place in -20.degree. C. freezer.
Kanamycin Stock (50 mg/ml): weigh out 5 grams Kanamycin, add 100 ml
ddH.sub.2O, filter sterilize, put in 12 ml tubes, place in -20C
freezer. Tetracycline Stock (3 mg/ml): dissolve 3 mg tetracycline
in 1 ml ddH.sub.2O, filter sterlize, refrigerate in foil (light
sensitive), good 1 day.
[0307] The results showed manipulation of fruit ripening and
carotenoid accumulation with the tomato NOR gene (FIG. 2). Shown in
FIG. 2 are representative control and transformed fruit from tomato
a line of the genotype nor/nor in the cultivar MH1 and transformed
with NOR-pBI121 Sense (FIG. 1). Primary transformants (T0) were
confirmed for transgene integration via DNA gel-blot analysis and
subsequently self-pollinated. Resulting seed were harvested and
grown (T1 generation) and analyzed for transgene segregation.
Representative fully mature fruit from T1 nor/nor individuals that
either harbor the sense NOR transgene (+) or have segregated it out
(-) are shown. In summary, transgene expression in the mutant
background was shown to partially recover the non-ripening
phenotype and confer ripening. In this particular line, relatively
low expression of the transgene was observed as compared to
expression of NOR in normally ripening (Nor/Nor) fruit.
Representative normal (Nor/Nor) and nearly isogenic mutant
(nor/nor) cultivar MH1 tomato fruit are shown as controls. The
partial recovery of ripening in the nor/nor fruit harboring the
NOR-pBI121 (+) transgene verified the isolation of the NOR gene.
Furthermore, the partial ripening phenotype observed in this line
demonstrated that regulated expression of the NOR gene can be used
to create a range of degrees of ripening and ripening-associated
characteristics (e.g., carotenoid accumulation, ripe flavor,
nutrient composition, softness, pathogen susceptibility).
[0308] C) Considerations in complementation testing with genomic
sequences: Several problems can arise when working with CaMV
.sup.35S-cDNA constructs including 1) inappropriate level,
developmental timing, or tissue specificity of chimeric gene
expression resulting in the absence of a measurable phenotype in
antisense or sense plants, and 2) induction of gene expression in
inappropriate cell types resulting in malformation or lethality in
sense transformants. Because RIN represents a developmental
regulator whose activity could potentially prove deleterious to
non-fruit tissues, the ideal transgene would be under the control
of the normal RIN allele promoter, although other promoters with
similar expression profiles could provide similar advantages.
Consequently, the major emphasis in verification of putative cDNAs
was placed on complementation of the mutant with corresponding
genomic counterparts.
[0309] Genomic DNA sequences corresponding to the NOR cDNA were
isolated from the tomato genomic library whose construction is
described below (FIG. 5). Full length cDNAs were sequenced at their
termini, and oligonucleotide primers were be synthesized
corresponding to the 5' and 3' ends. Candidate genomic clones were
then utilized as a template in sequencing reactions with these end
primers. Those genomic clones harboring DNA sequences from both
ends of the corresponding full length cDNA, as determined by
sequencing, were restriction mapped to identify location of the
transcribed region within the genomic clone insert. Restriction
mapping, in combination with cDNA hybridization to genomic clone
fragments, was utilized to identify genomic clones likely to
contain at least 2-3 kb of upstream and downstream sequence, prior
to transformation. The sequence of the genomic DNA of the NOR gene
resulting from the analysis is given in FIG. 5.
[0310] D) Construction of target gene containing libraries in the
cosmid/plant transformation vector: In order to facilitate
generation of a contig spanning a target locus, libraries of
genomic DNA from yeast containing YAC clones harboring the desired
sequence were constructed using the cosmid/plant transformation
vector 04541. The much smaller size of the yeast genome relative to
tomato simplified the screening and contig construction. Libraries
in 04541 were generated from yeast harboring Yrin8 and Ynor3. Test
screening of the Yrin8 library with CT63, Yrin2R, and Yrin8L
demonstrated the presence of clones containing all three probed
sequences. In addition, clones hybridizing to TG395, CT16, CDnor1
and CDnor2 (the only 4 probes tested) were retrieved from the Ynor3
library as well.
[0311] E) Walking in 04541 cosmid libraries from Yrin8L and CDnor2:
DNA markers very tightly linked to both RIN (Yrin8L) and NOR
(CDnor2) were identified as described. No recombinations were
identified between RIN and Yrin8L in 670 F2 progeny, and only one
recombinant between NOR and CDnor2 in 347 F2s. Based on the 200-300
kb/cM estimates for both the RIN and NOR regions of the tomato
genome, it was deemed reasonable to attempt a walk to both target
loci from these linked markers as they are within the criteria set
out for initiating development of a cosmid contig. The walk from
CDnor2 was initiated in the Ynor3 yeast cosmid library, while that
from Yrin8L was performed in the tomato genomic cosmid library
described above because RIN may have been off the end of Yrin8. A
DNA sequence surrounding the 04541 cloning site was generated as
were nested primers for IPCR of insert ends.
Example 8
Introgression of Transgenes Into Elite Crop Varieties
[0312] Backcrossing can be used to improve a starting plant.
Backcrossing transfers a specific desirable trait from one source
to an inbred or other plant that lacks that trait. This can be
accomplished, for example, by first crossing a superior inbred (A)
(recurrent parent) to a donor inbred (non-recurrent parent), which
carries the appropriate gene(s) for the trait in question, for
example, a construct prepared in accordance with the current
invention. The progeny of this cross first are selected in the
resultant progeny for the desired trait to be transferred from the
non-recurrent parent, then the selected progeny are mated back to
the superior recurrent parent (A). After five or more backcross
generations with selection for the desired trait, the progeny are
hemizygous for loci controlling the characteristic being
transferred, but are like the superior parent for most or almost
all other genes. The last backcross generation would be selfed to
give progeny which are pure breeding for the gene(s) being
transferred, i.e. one or more transformation events.
[0313] Therefore, through a series a breeding manipulations, a
selected transgene may be moved from one line into an entirely
different line without the need for further recombinant
manipulation. Transgenes are valuable in that they typically behave
genetically as any other gene and can be manipulated by breeding
techniques in a manner identical to any other gene. Therefore, one
may produce inbred plants which are true breeding for one or more
transgenes. By crossing different inbred plants, one may produce a
large number of different hybrids with different combinations of
transgenes. In this way, plants may be produced which have the
desirable agronomic properties frequently associated with hybrids
("hybrid vigor"), as well as the desirable characteristics imparted
by one or more transgene(s).
Example 9
Marker Assisted Selection
[0314] Genetic markers may be used to assist in the introgression
of one or more transgenes of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers may provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers may be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized.
[0315] In the process of marker assisted breeding, DNA sequences
are used to follow desirable agronomic traits (Tanksley et al.,
1989) in the process of plant breeding. Marker assisted breeding
may be undertaken as follows. Seed of plants with the desired trait
are planted in soil in the greenhouse or in the field. Leaf tissue
is harvested from the plant for preparation of DNA at any point in
growth at which approximately one gram of leaf tissue can be
removed from the plant without compromising the viability of the
plant. Genomic DNA is isolated using a procedure modified from
Shure et al. (1983). Approximately one gram of leaf tissue from a
seedling is lypholyzed overnight in 15 ml polypropylene tubes.
Freeze-dried tissue is ground to a powder in the tube using a glass
rod. Powdered tissue is mixed thoroughly with 3 ml extraction
buffer (7.0 urea, 0.35 M NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA,
1% sarcosine). Tissue/buffer homogenate is extracted with 3 ml
phenol/chloroform. The aqueous phase is separated by
centrifugation, and precipitated twice using 1/10 volume of 4.4 M
ammonium acetate pH 5.2, and an equal volume of isopropanol. The
precipitate is washed with 75% ethanol and resuspended in 100-500
.mu.l TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0).
[0316] Genomic DNA is then digested with a 3-fold excess of
restriction enzymes, electrophoresed through 0.8% agarose (FMC),
and transferred (Southern, 1975) to Nytran (Schleicher and Schuell)
using 10.times.SCP (20 SCP: 2M NaCl, 0.6 M disodium phosphate, 0.02
M disodium EDTA). The filters are prehybridized in 6.times.SCP, 10%
dextran sulfate, 2% sarcosine, and 500 .mu.g/ml denatured salmon
sperm DNA and .sup.32P-labeled probe generated by random priming
(Feinberg & Vogelstein, 1983). Hybridized filters are washed in
2.times.SCP, 1% SDS at 650 for 30 minutes and visualized by
autoradiography using Kodak XAR5 film. Genetic polymorphisms which
are genetically linked to traits of interest are thereby used to
predict the presence or absence of the traits of interest.
[0317] Those of skill in the art will recognize that there are many
different ways to isolate DNA from plant tissues and that there are
many different protocols for Southern hybridization that will
produce identical results. Those of skill in the art will recognize
that a Southern blot can be stripped of radioactive probe following
autoradiography and re-probed with a different probe. In this
manner one may identify each of the various transgenes that are
present in the plant. Further, one of skill in the art will
recognize that any type of genetic marker which is polymorphic at
the region(s) of interest may be used for the purpose of
identifying the relative presence or absence of a trait, and that
such information may be used for marker assisted breeding.
[0318] Each lane of a Southern blot represents DNA isolated from
one plant. Through the use of multiplicity of gene integration
events as probes on the same genomic DNA blot, the integration
event composition of each plant may be determined. Correlations may
be established between the contributions of particular integration
events to the phenotype of the plant. Only those plants that
contain a desired combination of integration events may be advanced
to maturity and used for pollination. DNA probes corresponding to
particular transgene integration events are useful markers during
the course of plant breeding to identify and combine particular
integration events without having to grow the plants and assay the
plants for agronomic performance.
[0319] It is expected that one or more restriction enzymes will be
used to digest genomic DNA, either singly or in combinations. One
of skill in the art will recognize that many different restriction
enzymes will be useful and the choice of restriction enzyme will
depend on the DNA sequence of the transgene integration event that
is used as a probe and the DNA sequences in the genome surrounding
the transgene. For a probe, one will want to use DNA or RNA
sequences which will hybridize to the DNA used for transformation.
One will select a restriction enzyme that produces a DNA fragment
following hybridization that is identifiable as the transgene
integration event. Thus, particularly useful restriction enzymes
will be those which reveal polymorphisms that are genetically
linked to specific transgenes or traits of interest.
Example 10
General Methods for Assays
[0320] DNA analysis of transformed plants is performed as follows.
Genomic DNA is isolated using a procedure modified from Shure, et
al., 1983. Approximately 1 gm callus or leaf tissue is ground to a
fine powder in liquid nitrogen using a mortar and pestle. Powdered
tissue is mixed thoroughly with 4 ml extraction buffer (7.0 M urea,
0.35 M NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1% sarcosine).
Tissue/buffer homogenate is extracted with 4 ml phenol/chloroform.
The aqueous phase is separated by centrifugation, passed through
Miracloth, and precipitated twice using 1/10 volume of 4.4 M
ammonium acetate, pH 5.2 and an equal volume of isopropanol. The
precipitate is washed with 70% ethanol and resuspended in 200-500
.mu.l TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0).
[0321] The presence of a DNA sequence in a transformed cell may be
detected through the use of polymerase chain reaction (PCR). Using
this technique specific fragments of DNA can be amplified and
detected following agarose gel electrophoresis. For example, two
hundred to 1000 ng genomic DNA is added to a reaction mix
containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl.sub.2, 50 mM KCl, 0.1
mg/ml gelatin, 200 .mu.M each dATP, dCTP, dGTP, dTTP, 0.5 .mu.M
each forward and reverse DNA primers, 20% glycerol, and 2.5 units
Taq DNA polymerase. The reaction is run in a thermal cycling
machine as follows: 3 minutes at 94.degree. C., 39 repeats of the
cycle 1 minute at 94.degree. C., 1 minute at 50.degree. C., 30
seconds at 72.degree. C., followed by 5 minutes at 72.degree. C.
Twenty .mu.l of each reaction mix is run on a 3.5% NuSieve gel in
TBE buffer (90 mM Tris-borate, 2 mM EDTA) at 50V for two to four
hours.
[0322] For Southern blot analysis genomic DNA is digested with a
3-fold excess of restriction enzymes, electrophoresed through 0.8%
agarose (FMC), and transferred (Southern, 1975) to Nytran
(Schleicher and Schuell) using 10.times.SCP (20.times.SCP: 2 M
NaCl, 0.6 M disodium phosphate, 0.02 M disodium EDTA). Probes are
labeled with .sup.32P using the random priming method (Boehringer
Mannheim) and purified using Quik-Sep.RTM. spin columns (Isolab
Inc., Akron, Ohio). Filters are prehybridized at 65.degree. C. in
6.times.SCP, 10% dextran sulfate, 2% sarcosine, and 500 .mu.g/ml
heparin (Chomet et al., 1987) for 15 min. Filters then are
hybridized overnight at 65 C in 6.times.SCP containing 100 .mu.g/ml
denatured salmon sperm DNA and .sup.32P-labeled probe. Filters are
washed in 2.times.SCP, 1% SDS at 65 C for 30 min. and visualized by
autoradiography using Kodak XAR5 film. For rehybridization, the
filters are boiled for 10 min. in distilled H.sub.2O to remove the
first probe and then prehybridized as described above.
[0323] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
REFERENCES
[0324] The references listed below are incorporated herein by
reference to the extent that they supplement, explain, provide a
background for, or teach methodology, techniques, and/or
compositions employed herein.
[0325] Abdullah et al., Biotechnology, 4:1087, 1986.
[0326] Abel et al., Science, 232:738-743, 1986.
[0327] Abeles F, Morgan P and Saltveit M (1992) Ethylene in Plant
Biology (San Diego: Academic Press)
[0328] Abeles F. Morgan P and Saltveit M (1992) Ethylene in Plant
Biology (San Diego: Academic Press)
[0329] Aida M, Ishida T, Fukaki H, Fujisawa H, and Tasaka M. (1997)
Genes involved in organ separation in arabidopsis: An analysis of
the cup-shaped cotyledon mutant. Plant Cell. 9: 841-857.
[0330] Anteguera, F., Bird, A. (1989) Unmethylated CpG islands
associated with genes in higher plant DNA. The EMBO J.
7:2295-2299.
[0331] Araki et al., J. Mol. Biol. 225(1):25-37, 1992.
[0332] Armaleo et al., Curr. Genet. 17(2):97-103, 1990.
[0333] Armstrong et al., Maize Genetics Coop Newsletter, 65:92-93,
1991.
[0334] Baile, J. and Young, R. (1981) Respiration and ripening in
fruits-retrospect and prospect. In Recent Advances in the
Biochemistry of Fruits and Vegetables. Friend, J. and Rhodes, M.
(ads.). Academic Press. pp1-39.
[0335] Bansal et al., Proc. Nat'l Acad. Sci. USA, 89:3654-3658,
1992.
[0336] Barkai-Golan et al., Arch. Microbiol., 116:119-124,
1978.
[0337] Bates, Mol. Biotechnol., 2(2):135-145, 1994.
[0338] Battraw and Hall, Theor. App. Genet., 82(2):161-168,
1991.
[0339] Belanger and Kriz, Genet., 129:863-872, 1991.
[0340] Bellus, J. Macromol. Sci. Pure Appl. Chem.,
RS3241(1):1355-1376, 1994.
[0341] Benfey, Ren, Chua, EMBO J., 8:2195-2202, 1989.
[0342] Bernal-Lugo and Leopold, Plant Physiol., 98:1207-1210,
1992.
[0343] Berzal-Herranz et al., Genes and Devel., 6:129-134,
1992.
[0344] Bevan et al., Nucleic Acids Research, 11(2):369-385,
1983.
[0345] Bhattacharjee; An; Gupta, J. Plant Bioch. and Biotech. 6,
(2):69-73. 1997.
[0346] Biggs, M. and Handa, A. (1989) Temporal regulation of
polygalacturonase gene expression in fruits of normal, mutant, and
heterozygous tomato genotypes. Plant Physiol. 89:117-125.
[0347] Blackman et al., Plant Physiol., 100:225-230, 1992.
[0348] Bol et al., Annu. Rev. Phytopath., 28:113-138, 1990.
[0349] Bouchez et al., EMBO Journal, 8(13):41974204, 1989.
[0350] Bower et al., The Plant Journal, 2:409-416. 1992.
[0351] Bowler et al., Ann Rev. Plant Physiol., 43:83-116, 1992.
[0352] Branson and Guss, Proceedings North Central Branch
Entomological Society of America, 27:91-95, 1972.
[0353] Broakaert et al., Science, 245:1100-1102, 1989.
[0354] Buchanan-Wollaston et al., Plant Cell Reports 11:627-631.
1992
[0355] Buising and Benbow, Mol Gen Genet, 243(1):71-81. 1994.
[0356] Burke, D., Carle, G. and Olson, M. (1987) Cloning of large
segments of exogenous DNA into yeast by means of artificial
chromosome vectors. Science. 236:806-812.
[0357] Callis, Fromm, Walbot, Genes Dev., 1:1183-1200, 1987.
[0358] Campbell (ed.), In: Avermectin and Abamectin, 1989.
[0359] Capaldi et al., Biochem. Biophys. Res. Comm., 76:425,
1977.
[0360] Casa et al., Proc. Nat'l Acad. Sci. USA, 90(23):11212-11216,
1993.
[0361] Cashmore et al., Gen. Eng. of Plants, Plenum Press, New
York, 29-38, 1983.
[0362] Cech et al., Cell, 27:487-496, 1981.
[0363] Chandler et al., The Plant Cell, 1:1175-1183, 1989.
[0364] Chau et al., Science, 244:174-181, 1989.
[0365] Chomet et al., EMBO J., 6:295-302, 1987.
[0366] Chowrira et al., J. Biol. Chem., 269:16096-25864, 1994.
[0367] Christou; Murphy; Swain, Proc. Nat'l Acad. Sci. USA,
84(12):3962-3966, 1987.
[0368] Chu et al., Scientia Sinica, 18:659-668, 1975.
[0369] Coe et al., In: Corn and Corn Improvement, 81-258, 1988.
[0370] Conkling et al., Plant Physiol., 93:1203-1211, 1990.
[0371] Cordero, Raventos, San Segundo, Plant J., 6(2)141-150,
1994.
[0372] Cordes, S., Deikman, J., Margossian, L. and Fischer, R.
(1989) Interaction of a developmentally regulated DNA-binding
factor with sites flanking two different fruit-ripening genes from
tomato. The Plant Cell. 1: 1025-1034.
[0373] Coxson et al., Biotropica, 24:121-133, 1992.
[0374] Cretin and Puigdomenech, Plant Mol. Biol. 15(5):783-785,
1990
[0375] Cuozzo et al., Bio/Technology, 6:549-553, 1988.
[0376] Cutler et al., J. Plant Physiol., 135:351-354, 1989.
[0377] Czapla and Lang, J. Econ. Entomol., 83:2480-2485, 1990.
[0378] Davies et al., Plant Physiol., 93:588-595, 1990.
[0379] De Block et al., The EMBO Journal, 6(9):2513-2518, 1987.
[0380] De Block, De Brouwer, Tenning, Plant Physiol., 91:694-701,
1989.
[0381] DellaPenna, D. and Giovannoni, J. (1991) Regulation of gene
expression in ripening tomatoes. In Developmental Regulation of
Plant Gene Expression Vol.2. Grierson, D. (ed.). Blackie and Son
Ltd. pp182-216.
[0382] DellaPenna, D., Alexander, D. and Bennett, A. (1986)
Molecular cloning of tomato fruit polygalacturonase: Analysis of
polygalacturonase mRNA levels during ripening. PNAS USA.
83:6420-6424.
[0383] DellaPenna, D., Lincoln, J., Fischer, R. and Bennett, A.
(1989) Transcriptional analysis of polygalacturonase and other
ripening associated genes in Rutgers, rin, nor, and Nr tomato
fruit. Plant Physiol. 90:1372-1377.
[0384] Dellaporta et al., In: Chromosome Structure and Function:
Impact of New Concepts, 18th Stadler Genetics Symposium,
11:263-282, 1988.
[0385] Dennis et al., Nucl. Acids Res., 12(9):3983-4000, 1984.
[0386] Depicker et al., Plant Cell Reports, 7:63-66, 1988.
[0387] D'Halluin et al., Plant Cell, 4(12): 1495-1505, 1992.
[0388] Didierjean et al., Plant Mol Biol 18(4):847-849, 1992.
[0389] Dure et al., Plant Molecular Biology, 12:475-486, 1989.
[0390] Ebert et al., 84:5745-5749, Proc. Nat'l Acad. Sci. USA,
1987
[0391] Ellis et al., EMBO Journal, 6(11):3203-3208, 1987.
[0392] Enomoto, et al., J. Bacteriol., 6(2):663-668, 1983.
[0393] Erdmann et al., Mol. Jour. Gen. Micro., 138:363-368,
1992.
[0394] Feinberg and Vogelstein, Anal. Biochem., 132:6-13, 1983.
[0395] Finkle etal., Plant Sci., 42:133-140, 1985.
[0396] Fischer, R. and Bennett, A. (1991) Role of cell wall
hydrolases in fruit ripening. Ann. Rev. Plant. Physiol. Plant
Molec. Biol. 42:
[0397] Fitzpatrick, Gen. Engineering News, 22:7, 1993.
[0398] Forster and Symons, Cell, 49:211-220, 1987.
[0399] Fraley et al., Bio/Technology, 3:629-635, 1985.
[0400] Franken et al., EMBO J., 10(9):2605-2612, 1991.
[0401] Fransz, de Ruijter, Schel, Plant Cell Reports, 8:67-70,
1989.
[0402] Fromm et al, Nature, 312:791-793, 1986.
[0403] Gallie et al., The Plant Cell, 1:301-311, 1989.
[0404] Ganal, M. and Tanksley, S. (1989) Analysis of tomato DNA by
pulsed field gel electrophoresis. Plant Mol. Biol. Rep.
7:17-27.
[0405] Ganal, M., Martin, G., Messeguer, R. and Tanksley, S. (1990)
Application of RFLPs, physical mapping, and large DNA technologies
to the cloning of important genes from crop plants. AgBiotech News
and Info. 2:835-840.
[0406] Ganal, M., Young, N. and Tanksley, S. (1989) Pulsed field
gel electrophoresis and physical mapping of large DNA fragments in
the Tm-2a region of chromosome 9 in tomato. Mol. Gen. Genet.
215:395-400.
[0407] Gatehouse et al., J. Sci. Food Agric., 35:373-380, 1984.
[0408] Gelvin et al., In: Plant Molecular Biology Manual, 1990.
[0409] Gerlach et al., Nature 328:802-805, 1987.
[0410] Ghosh-Biswas, Iglesias, Datta, Potrykus, J. Biotechnol.,
32(1): 1-10, 1994.
[0411] Giovannoni, J. (1993) Molecular biology of fruit development
and ripening. In Methods In Plant Molecular Biology. (Bryant, J.
ed.) Academic Press. Vol. 10: 253-287.
[0412] Giovannoni, J., DellaPenna, D., Bennett, A. and Fischer, R.
(1989) Expression of a chimeric polygalacturonase gene in
transgenic rin (ripening inhibitor) tomato fruit results in
polyuronide degradation but not fruit softening. The Plant Cell.
1:53-63.
[0413] Giovannoni, J., DellaPenna, D., Bennett, A. and Fischer, R.
(1991) Polygalacturonase and tomato fruit ripening. Horticultural
Reviews. 13:67-103.
[0414] Giovannoni, J., Noensie, E., Ruezinsky, D., Lu, X, Tracy,
S., Ganal, M., Martin, G., Pillen, K. and Tanksley, S. (1995)
Molecular genetic analysis of the ripening-inhibitor and
non-ripening loci of tomato: a first step in genetic map-based
cloning of fruit ripening genes. Molecular and General Genetics
248(2): 195-206.
[0415] Giovannoni, J., Wing, R., Ganal, M. and Tanksley, S. (1991)
Isolation of molecular markers from specific chromosomal intervals
using DNA pools from existing mapping populations. Nuc. Acids Res.
19:6553-6558.
[0416] Golic and Lindquist, Cell, 59:3, 499-509. 1989.
[0417] Gomez et al., Nature 334:262-264. 1988
[0418] Goring et al., Proc. Nat'l Acad. Sci. USA, 88:1770-1774,
1991.
[0419] Grierson, D. (1986) Molecular biology of fruit ripening. In
Oxford Surveys of Plant Molecular and Cell Biology Vol.3. Milan, B
(ad.). Oxford University Press. pp363-383.
[0420] Grierson, D., Tucker, G., Keen, J., Ray, J., Bird, C. and
Schuch, W. (1986). Sequencing and identification of a cDNA clone
for tomato polygalacturonase. Nuc. Acids Res. 1 4:8595-8603.
[0421] Guerrero et al., Plant Molecular Biology, 15:11-26,
1990.
[0422] Gupta et al., Proc. Natl. Acad. Sci. USA, 90:1629-1633,
1993.
[0423] Hagio, Blowers, Earle, Plant Cell Rep., 10(5):260-264,
1991.
[0424] Hamilton et al., Proc. Nat'l Acad Sci. USA,
93(18):9975-9979, 1996.
[0425] Hamilton, A., Lycett, G. and Grierson, D. (1990) Antisense
gene that inhibits synthesis of the hormone ethylene in transgenic
plants. Nature. 346:284-287.
[0426] Hammock et al., Nature, 344:458461, 1990.
[0427] Harriman, R. and Handa, A. (1991) Molecular cloning of
tomato pectin methylesterase gene and its expression in Rutgers,
ripening inhibitor, nonripening and Never ripe tomato fruits. Plant
Physiol. 97:
[0428] Haseloff and Gerlach, Nature, 334:585-591, 1988.
[0429] Haseloff et al., Proc. Nat'l Acad Sci. USA 94(6):2122-2127,
1997.
[0430] He et al., Plant Cell Reports, 14 (2-3): 192-196, 1994.
[0431] Hemenway et al., The EMBO J., 7:1273-1280, 1988.
[0432] Henikoff, S. (1984) Unidirectional digestion with
exonuclease lit creates targeted breakpoints for DNA sequencing.
Gene. 28:351-359.
[0433] Hensgens et al., Plant Mol. Biol., 22(6): 1101-1127,
1993.
[0434] Hiei et al., Plant. Mol. Biol., 35(1-2):205-218, 1997.
[0435] Hilder et al., Nature, 330:160-163, 1987.
[0436] Hinchee et al., Bio/technol., 6:915-922, 1988.
[0437] Hobson, G. (1968) Cellulase activity during the maturation
and ripening of tomato fruit. J. Food Sci. 33:588-592.
[0438] Hou and Lin, Plant Physiology, 111:166, 1996.
[0439] Hudspeth and Grula, Plant Mol. Biol., 12:579-589, 1989.
[0440] Ikeda et al., J. Bacteriol., 169:5615-5621, 1987.
[0441] Ikuta et al, Bio/technol., 8:241-242, 1990.
[0442] Ishida et al., Nat. Biotechnol., 14(6):745-750, 1996.
[0443] Jefferson R. A., Plant Mol. Biol. Rep., 5:387-405, 1987.
[0444] John I, Hackett R, Cooper W, Drake R, Farrell A, and
Grierson D. Cloning and characterization of tomato leaf
senescence-related cDNAs. Plant Molecular Biology. 33: 641-651
[0445] Johnson et al., Proc. Nat'l Acad. Sci. USA, 86:9871-9875,
1989.
[0446] Joshi, Nucleic Acids Res., 15:6643-6653, 1987.
[0447] Joyce, Nature, 338:217-244, 1989.
[0448] Kaasen et al., J. Bacteriology, 174:889-898, 1992.
[0449] Kaeppler et al., Plant Cell Reports 9: 415418, 1990.
[0450] Kaeppler, Somers, Rines, Cockburn, Theor. Appl. Genet.,
84(5-6):560-566, 1992.
[0451] Karsten et al., Botanica Marina, 35:11-19, 1992.
[0452] Katz et al., J. Gen. Microbiol., 129:2703-2714, 1983.
[0453] Keller et al., EMBO J., 8(5):1309-1314, 1989.
[0454] Kim and Cech, Proc. Nat'l Acad. Sci. USA, 84:8788-8792,
1987.
[0455] Kinzer, S., Schwager, S. and Mutschler, M. (1990) Mapping of
ripening-related or -specific cDNA clones of tomato. Theor. Appl.
Genet. 79:489-496.
[0456] Klee, Yanofsky, Nester, Bio-Technology, 3(7):637-642,
1985.
[0457] Knittel, Gruber; Hahne; Lenee, Plant Cell Reports,
14(2-3):81-86, 1994.
[0458] Kohler et al., Plant Mol. Biol., 29(6):1293-1298, 1995.
[0459] Koster and Leopold, Plant Physiol., 88:829-832, 1988.
[0460] Kramer, M., Sanders, R., Sheehy, R., Melis, M., Kuehn, M.
and Hiatt, W. (1990) Field evaluation of tomatoes with reduced
polygalacturonase by antisense RNA. In Horticultural Biotechnology.
Bennett, A. and O'Neill, S. (eds.) Alan R. Liss. pp347-355.
[0461] Kriz, Boston, Larkins, Mol. Gen. Genet., 207(1):90-98,
1987.
[0462] Kunkel et al., Methods Enzymol, 154:367-382, 1987.
[0463] Langridge and Feix, Cell, 34:1015-1022, 1983.
[0464] Langridge et al., Proc. Nat'l Acad Sci. USA, 86:3219-3223,
1989.
[0465] Laufs et al., Proc. Nat'l Acad. Sci., 7752-7756, 1990.
[0466] Lawton et al., Plant Mol. Biol. 9:315-324, 1987.
[0467] Lazzeri, Methods Mol. Biol., 49:95-106, 1995.
[0468] Lee and Saier, J. of Bacteriol., 153-685, 1983.
[0469] Lee; Suh; Lee, Korean J. Genet., 11(2):65-72, 1989.
[0470] Levings, Science, 250:942-947, 1990.
[0471] Lieber and Strauss, Mol. Cell. Biol., 15: 540-551, 1995.
[0472] Lincoln, J. and Fischer, R. (1988) Regulation of gene
expression by ethylene in wild-type and rin tomato (Lycopersicon
esculentum) fruit. Plant Physiol. 88:370-374.
[0473] Lincoln, J., Cordes, S., Read, E. and Fischer, R. (1987)
Regulation of gene expression by ethylene during Lycopersicon
esculentum (tomato) fruit development. PNAS USA. 84: 2793-2797.
[0474] Lindstrom et al., Developmental Genetics, 11:160, 1990.
[0475] Loomis et al., J. Expt. Zoology, 252:9-15, 1989.
[0476] Lorz et al., Mol Gen Genet, 199:178-182, 1985.
[0477] Ma et al., Nature, 334:631-633, 1988.
[0478] Maeser et al, Mol. Gen. Genet., 230(1-2):170-176, 1991.
[0479] Marcotte et al., Nature, 335:454, 1988.
[0480] Margossian, L., Federman, A., Giovannoni, J. and Fischer, R.
(1988) Ethylene-regulated expression of a tomato fruit ripening
gene encoding a proteinase inhibitor I with a glutamic residue at
the reactve site. PNAS USA. 85:8012-8016.
[0481] Mariani et al., Nature, 347:737-741, 1990.
[0482] Martin, G., Ganal, M. and Tanksley, S. (1992) Construction
of a yeast artificial chromosome library of tomato and
identification of clones linked to two disease resistance loci.
Mol. Gen. Genet In press.
[0483] Martinez, Martin, Cerff, J. Mol. Biol., 208(4):551-565,
1989.
[0484] Maunders, M., Holdsworth, M., Slater, A., Knapp, J., Bird,
C., Schuch, W. and Grierson, D. (1987) Ethylene stimulates the
accumulation of ripening-related mRNAs in tomatoes. Plant Cell
Environ. 10: 177-184.
[0485] McCabe, Martinell, Bio-Technology, 11(5):596-598, 1993.
[0486] McCormac et al., Euphytica, v. 99 (1) p. 17-25, 1998.
[0487] McCormick, S., Neidermeyer, J., Fry, J., Barnason, A.,
Horsch, R. and Frayley, R. (1986) Leaf disk transformation of
cultivated tomato (Lycopersicon esculentum) using Agrobacterium
tumefaciens. Plant Cell Rep. 5:81-84.
[0488] McElroy et al., Mol Gen. Genet., 231:150-160, 1991.
[0489] McElroy, Zhang, Cao, Wu, Plant Cell, 2:163-171, 1990.
[0490] Meagher, Int. Rev. Cytol., 125:139-163, 1991.
[0491] Messeguer, R., Ganal, M., deVicente, M., Young, N., Bolkan,
H. and Tanksley, S. (1991) Characterization of the level, target
sites and inheritance of cytosine methylation in tomato nuclear
DNA. Plant Mol. Biol.
[0492] Michel and Westhof, J. Mol. Biol., 216:585-610, 1990.
[0493] Miller, J. and Tanksley, S. (1990) RFLP analysis of
phylogenetic relationships and genetic variation in the genus
Lycopersicon. Theor. Appl. Genet. 80:437-448.
[0494] Mundy and Chua, The EMBO J., 7:2279-2286, 1988.
[0495] Murakami et al., Mol. Gen. Genet., 205:42-50, 1986.
[0496] Murashige and Skoog, Physiol. Plant., 15:473-497, 1962.
[0497] Murdock et al., Phytochemistry, 29:85-89, 1990.
[0498] Nagatani, Honda, Shimada, Kobayashi, Biotech. Tech.,
11(7):471-473, 1997.
[0499] Napoli, Lemieux, Jorgensen, Plant Cell, 2:279-289, 1990.
[0500] Odell et al., Nature, 313:810-812, 1985.
[0501] Oeller, P., Min-Wong, L., Taylor, L., Pike, D. and
Theologis, A. (1991) Reversible inhibition of tomato fruit
senescence by antisense RNA. Science. 254:437439.
[0502] Ogawa et al., Sci. Rep., 13:42-48, 1973.
[0503] Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993.
[0504] Ow et al., Science, 234:856-859, 1986.
[0505] Palukaitis et al., Virology, 99:145-151, 1979.
[0506] Pear, J., Ridge, N., Rasmussen, R., Rose, R. and Houck, C.
(1989). Isolation and characterization of a fruit-specific cDNA and
the corresponding genomic clone from tomato. Plant Mol. Biol.
13:639-651.
[0507] Perlak et al., Proc. Nat'l Acad Sci. USA, 88:3324-3328,
1991.
[0508] Perriman et al., Gene, 113:157-163, 1992.
[0509] Phi-Van et al., Mol. Cell. Biol., 10:2302-2307, 1990.
[0510] Piatkowski et al., Plant Physiol., 94:1682-1688, 1990.
[0511] Poszkowski et al., EMBO J., 3:2719, 1989.
[0512] Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985.
[0513] Poulsen et al., Mol. Gen. Genet., 205(2):193-200, 1986.
[0514] Prasher et al., Biochem. Biophys. Res. Commun., 126(3):
1259-1268, 1985.
[0515] Prody et al., Science, 231:1577-1580, 1986.
[0516] Quigley, Brinkman, Martin, Cerff, J. Mol. Evol.,
29(5):412421, 1989.
[0517] Ralston, English, Dooner, Genet., 119(1):185-197, 1988.
[0518] Reece, "The actin gene family of rice (Oryza sativa L),"
Ph.D. thesis, Cornell University, Ithaca, N.Y., 1988.
[0519] Reece, McElroy, Wu, Plant Mol. Biol., 14:621-624, 1990.
[0520] Reed et al., J. Gen. Microbiology, 130:1-4, 1984.
[0521] Reichel et al., Proc. Nat'l Acad. Sci. USA, 93 (12) p.
5888-5893. 1996
[0522] Reina et al., Nucl. Acids Res., 18(21):6426, 1990.
[0523] Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.
[0524] Rensburg et al., J. Plant Physiol., 141:188-194, 1993.
[0525] Rhodes et al., Methods Mol. Biol., 55:121-131, 1995.
[0526] Rick C M (1980) Tomato linkage survey. Rep Tomato Genet Coop
30:2-17
[0527] Ritala et al., Plant Mol. Biol., 24(2):317-325, 1994.
[0528] Robinson, R. and Tomes, M. (1968) Ripening inhibitor: A gene
with multiple effects on ripening. Rep. Tomato Genet. Coop.
18:36-37.
[0529] Rochester, Winer, Shah, EMBO J., 5:451-458, 1986.
[0530] Rogers et at, Methods Enzymol., 153:253-277, 1987.
[0531] Rommens, J., Iannuzi, M., Kerem, B., Drumm, M., Melmer, G.,
Dean, M., Rozmahel, R., Cole, J., Kennedy, D., Hidaka, N., Zsiga,
M., Buchwald, M., Riordan, J., Tsui, L. and Collins, F. (1989).
Identification of the cystic fibrosis gene: Chromosome walking and
jumping. Science. 45:1059-1065.
[0532] Sambrook, Fritsch, and Maniatis, In Molecular Cloning: A
Laboratory Manual, Second edition, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y., 1989.
[0533] Sauer, Mol. and Cell. Biol., 7: 2087-2096. 1987.
[0534] Schwob et al., Plant J 4(3):423-432, 1993.
[0535] Seymour B. Taylor E, Tucker A (eds) (1993) Biochemistry of
Fruit Ripening. Chapman and Hall, London.
[0536] Shagan and Bar-Zvi, Plant Physiol., 101:1397-1398, 1993.
[0537] Shapiro, In: Mobile Genetic Elements, 1983.
[0538] Sheehy, R., Kramer, M. and Hiatt, W. (1988) Reduction of
polygalacturonase activity in tomato fruit by antisense RNA. PNAS
USA. 85:8805-8809.
[0539] Sheehy, R., Pearson, J., Brady, C. and Hiatt, W. (1987)
Molecular characterization of tomato fruit polygalacturonase. Mol.
Gen. Genet. 208:30-36.
[0540] Sheen et al., Plant Journal, 8(5):777-784, 1995.
[0541] Shure et al., Cell, 35:225-233, 1983.
[0542] Simpson, Filipowicz, Plant Mol. Bio., 32:1-41, 1996.
[0543] Simpson, Science, 233:34, 1986.
[0544] Singsit et al., Transgenic Res., 6(2):169-176, 1997.
[0545] Slater, A., Maunders, M., Edwards, K., Schuch, W. and
Grierson, D. (1985) Isolation and characterization of cDNA clones
for tomato polygalacturonase and other ripeningrelated proteins.
Plant Mol. Biol. 5:137-147.
[0546] Smith, C., Watson, C., Ray, J., Bird, C., Morris, P.,
Schuch, W. and Grierson, D. (1988) Antisense RNA inhibition of
polygalacturonase gene expression in transgenic tomatoes. Nature.
334:724-726.
[0547] Smith, Watson, Bird, Ray, Schuch, Grierson, Mol. Gen.
Genet., 224:447-481, 1990.
[0548] Souer E, Van Houwelingen A, Kloos D, Mol J, and Koes R.
(1996) The no apical meristem gene of petunia is required for
pattern formation in embryos and flowers and is expressed at
meristem and primordia boundaries. Cell. 85: 159-170
[0549] Southern, J. Mol. Biol., 98:503-517, 1975.
[0550] Spencer et al., Plant Molecular Biology, 18:201-210,
1992.
[0551] Sprague and Dudley, eds., Corn and Improvement, 3rd ed.,
1988.
[0552] Stalker et al., Science, 242:419-422, 1988.
[0553] Stief et al, Nature 341:343 1989.
[0554] Sullivan, Christensen, Quail, Mol. Gen. Genet.,
215(3):431-440, 1989.
[0555] Sutcliffe, Proc. Nat'l Acad Sci. USA, 75:3737-3741,
1978.
[0556] Symons, Annu. Rev. Biochem., 61:641-671, 1992.
[0557] Tanksley et al., Bio/Technology, 7:257-264, 1989.
[0558] Tarczynski et al., Proc. Nat'l Acad. Sci. USA, 89:1-5,
1992.
[0559] Tarczynski et al., Science, 259:508-510, 1993.
[0560] Thillet et al., J. Biol. Chem., 263:12500-12508, 1988.
[0561] Thompson et al., The EMBO Journal, 6(9):2519-2523, 1987.
[0562] Thompson, Drayton, Frame, Wang, Dunwell, Euphytica,
85(1-3):75-80, 1995.
[0563] Tian, Sequin, Charest, Plant Cell Rep., 16:267-271,
1997.
[0564] Tigchelaar, E., McGlasson, W. and Buescher, R. (1978).
Genetic regulation of tomato fruit ripening. HortSci.
13:508-513.
[0565] Tigchelaar, E., Tomes, M., Kerr, E. and Barman, R. (1973) A
new fruit ripening mutant, non-ripening (no0. Rep. Tomato Genet.
Coop. 23:33.
[0566] Tingay et al., The Plant Journal v. 11 (6) p. 1369-1376.
1997.
[0567] Tomes et al., Plant. Mol. Biol. 14(2):261-268, 1990.
[0568] Tomic et al., Nucl. Acids Res., 12:1656, 1990.
[0569] Torbet, Rines, Somers, Crop Science, 38(1):226-231,
1998.
[0570] Torbet, Rines, Somers, Plant Cell Reports, 14(10):635-640,
1995.
[0571] Toriyama et at, Theor Appl. Genet., 73:16, 1986.
[0572] Tsukada; Kusano; Kitagawa, Plant Cell Physiol.,
30(4)599-604, 1989.
[0573] Tucker, M. and Laties, G. (1984) Interrelationship of gene
expression, polysome prevalence, and respiration during ripening of
ethylene and/or cyanide-treated avocado fruit. Plant Physiol.
74:307-315.
[0574] Twell et al., Plant Physiol 91:1270-1274, 1989.
[0575] Uchimiya et al., Mol. Gen. Genet, 204:204, 1986.
[0576] Ugaki et al., Nucl. Acid Res., 19:371-377, 1991.
[0577] Upender, Raj, Weir, Biotechniques 18(1):29-30, 1995.
[0578] Van der Krol, Mur, Beld, Mol, Stuitje, Plant Cell, 2:291-99,
1990.
[0579] Van Eck; Blowers; Earle, Plant Cell Reports, 14(5):299-304,
1995.
[0580] Van Tunen et al, EMBO J., 7:1257, 1988.
[0581] Vasil et al., Plant Physiol., 91:1575-1579, 1989.
[0582] Vernon and Bohnert, The EMBO J., 11:2077-2085, 1992.
[0583] Vodkin et al., Cell, 34:1023, 1983.
[0584] Vogel, Dawe, Freeling, J. Cell. Biochem., (Suppl. 0) 13:Part
D, 1989.
[0585] Walker et al., Proc. Nat'l Acad. Sci. USA, 84:6624-6628,
1987.
[0586] Wandelt and Feix, Nucl. Acids Res., 17(6):2354, 1989.
[0587] Wang et al., Molecular and Cellular Biology,
12(8):3399-3406, 1992.
[0588] Watrud et al., In: Engineered Organisms and the Environment,
1985.
[0589] Watson and Ramstad, eds., Corn: Chemistry and Technology,
1987.
[0590] Wenzler et al., Plant Mol. Biol., 12:41-50, 1989.
[0591] Williams, J., Kubelik, A., Livak, K., Rafalski, J. and
Tingey, S. (1990) DNA polymorphisms amplified by arbitrary primers
are useful as genetic markers. Nucleic Acids Res. 1
8:6531-6535.
[0592] Withers and King, Plant Physiol., 64:675-678, 1979.
[0593] Wolter et al., The EMBO J., 4685-4692, 1992.
[0594] Xiang and Guerra, Plant Physiol., 102:287-293, 1993.
[0595] Xu et al., Plant Physiol., 110:249-257, 1996.
[0596] Yamada et al., Plant Cell Rep., 4:85, 1986.
[0597] Yamaguchi-Shinozaki et al., Plant Cell Physiol., 33:217-224,
1992.
[0598] Yang and Russell, Proc. Nat'l Acad Sci. USA, 87:4144-4148,
1990.
[0599] Yen, H., Shelton, A., Howard, L. Vrebalov, J. and
Giovannoni, J. (1997) The tomato high pigment (hp) locus maps to
chromosome 2 and influences plastome copy number and fruit quality.
Theoretical and Applied Genetics 95:1069-1079
[0600] Yuan and Altman, Science, 263:1269-1273, 1994.
[0601] Yuan et al., Proc. Nat'l Acad. Sci. USA, 89:8006-8010,
1992.
[0602] Zhang, McElroy, Wu, The Plant Cell, 3:1155-1165, 1991.
[0603] Zheng and Edwards, J. Gen. Virol., 71:1865-1868, 1990.
[0604] Zhou; Stiff; Konzak, Plant Cell Reports, 12(11) 612-616,
1993.
[0605] Zukowsky et al., Proc. Nat'l Acad. Sci. USA, 80:1101-1105,
1983.
Sequence CWU 1
1
7 1 1211 DNA Tomato 1 aggtcaactc aaacatcgta aattgtgatt tctttatgga
aagtacggat tcatcaaccg 60 ggacacgtca tcagcctcaa ctcccaccgg
ggtttcgatt ccacccgacg gacgaagaac 120 tcatcgtcca ctacctcaaa
aaaccagtcg ccggcgctcc gattccggtg gatattattg 180 gtgaaattga
tctttataag tttgatccat gggaactccc tgctaaggca atattcggag 240
agcaagaatg gttctttttt agtccaagag atagaaaata tcctaacggg gcgaggccaa
300 atcgggctgc aacatcgggt tattggaagg ctaccggaac cgacaagccg
gtttttactt 360 ccggtggaac acaaaaggtt ggggtaaaaa aggcgctcgt
tttttacggc ggtaaaccac 420 caaaaggggt aaaaactaat tggatcatgc
atgaatacag agttgtagaa aataaaacaa 480 ataacaagcc acttggttgt
gataatattg ttgccaacaa aaaaggatct ttgaggctag 540 atgattgggt
tttatgtcga atttacaaga agaataacac acaaaggtcc atagatgatt 600
tgcatgatat gttgggatcg ataccacaaa atgtaccaaa ttcaatatta caaggaataa
660 agccttcaaa ctatggtaca atattgctcg aaaatgaatc gaatatgtac
gatggaatta 720 tgaataacac gaacgatatt atcaacaata ataatagatc
cattccacaa atatcgtcaa 780 agagaacgat gcatggaggt ttgtattgga
ataacgacga agcaacaaca acaacaacaa 840 ctattgatag gaaccattct
ccaaatacaa aaaggtttcc ttgttgagaa caacgaggac 900 gatggactta
acatgaataa tatttcgcga attacaaatc atgaacaaag tagctccatt 960
gccaatttcc tgagccagtt tcctcaaaat ccttggattc aacaacaaca acaacaacaa
1020 gaagaagtat tgggatctct taatgatggg gtcgtctttc gacaacctta
taatcaagtt 1080 actggcatga attggtactc ttaaagatat aaaaaggcaa
aaaatagtta gccctgtaaa 1140 atcaatcgat caatcaatca tagatatatt
atatatggat ttcgttaaaa aaaaaaaaaa 1200 aaaaaaaaaa a 1211 2 1097 DNA
Tomato 2 aaaaggagct aagtttaata attttttttt ataaaaaaaa aaaaaacttt
tttgaagatg 60 ggaagaggaa aagttgaatt aagaaaaata gagaataaaa
taaatagaca agtaacattt 120 tcaaagagaa gaggtggatt agtgaaaaaa
gctcatgaaa tttcagtttt atgtgatgct 180 gaagttgctt taattgtttt
ctctcaaaag ggaaaaatct ttgagtattc ttctgattca 240 tgtatggaac
aaattcttga acgatatgaa agatactcat atgcagagag acgtttgctt 300
gcaaataatt ctgaatcacc ggtgcaggaa aactggagct tggaatatac taaactcaag
360 gctaggattg atctccttca aaggaaccac aagcattata tgggggaaga
tcttgattca 420 atgagcttga aggacttgca aaacttggaa caacagcttg
attctgctct taagctaaat 480 tcgatcgaga aagaaccact catgcatgaa
tcaatctctg aactgcagaa aaaggaaaga 540 gctatcctag aggagaataa
catgctaacc aagaagatta aggagaagga taagatagta 600 gaacagcaag
gtgaatggca ccagcaaact aatcaagttt ctacttcaac atctttcctc 660
ttacaaccac atcaatgcct aaatatggga ggtaattacc aagatgaagt agcagaagca
720 aggaggaata atgagcttga cctaaatctt gattcattat atccacttta
caacatgaat 780 aaacatctat gaataatttc actctttgct aatcgcttga
aacgttgaaa ggagctcact 840 atcaggacag acaaatgagt ataagcgatt
agcgataaaa actctatgcg agaggaaatt 900 atatatgatg ttaattaatc
tatgcttgag aaattcttaa ttatatatat tgagtgtctt 960 tatattgata
tgcatgtata gaaccttatt attatgaatt tctatgtatt aatgtttaag 1020
tatgttaaaa cttaattgtt aatggaatca agtccattct ctttgtatcc aaaaaaaaaa
1080 aaaaaaaaaa aaaaaaa 1097 3 1138 DNA Tomato 3 ttttcttctt
gactagggaa ccattagatt ttaaagacat taaatctatt acccttaccc 60
taagaaataa gaagatgtaa agtagaagag aaaacaacca aaaccatata tatacatata
120 tataattaca ttatattgtc ttataacatg tagtctttta aggaaaaaca
aatttagaaa 180 aaaaataata ttattttaca tttttttttc ttcatacaat
atgggtagag ggaaagtaga 240 attgaagaga attgagaaca aaataaatag
acaagttacc tttgcaaaga gaagaaatgg 300 actcctaaag aaagcttatg
aactttctat actttgtgat gctgaaattg ctcttattat 360 ttcctctagt
cgtggcaagc tttatgaatt ttgcagcaat tcaagtatgt ccaagacatt 420
ggagagatac cacagataca attatggtac acttgaagga acccaaactt catcagattc
480 acagaacaac taccaggagt atttgaagct taaaacaaga gtggaaatgt
tacaacagtc 540 tcaaaggcat ttgctaggtg aggatttggg acaattgggc
acaaaagact tggaacagct 600 tgaacgtcaa ttggattcat cattgaggca
aattaggtca acaaagacac aacacattct 660 tgatcaactt gctgaacttc
aacaaaagga acaatctctt actgaaatga acaaatcttt 720 gagaataaag
ttggaagaac ttggtgttac ctttcaaaca tcatggcatt gtggtgagca 780
aagtgtacaa tatagacatg aacagccttc tcatcatgag ggattttttc aacatgtaaa
840 ttgcaataat acattgccta taagcaccat caacacatgg atgctactgg
agttgtacct 900 ggatggatgc tttgaatttg gagtatatgg agagaaaaaa
tcctcttagt atacaagtta 960 tttattttta ttaaaaaaat aagttagatg
gagaattata tatatcatac tttaaagaac 1020 ttatattgtt tgaatgtttt
agctagcaaa cactttggat tatatataat attgtgatat 1080 atttattgtc
aagaagatat ggcaatattg ataacactat atttttgaaa aaaaaaaa 1138 4 1119
DNA Tomato 4 gggaaccatt agattttaaa gacattaaat ctattaccct taccctaaga
ataagaagat 60 gtaaagtata agagaaaaca accaaaacca tatatataca
tatatataat tacattatat 120 tgtcttataa catatagtct tttaaggaaa
aacaaattta gaaaaaaaat aatattattt 180 tacatttttt tttcttcata
caatatgggt agagggaaag tagaattgaa gagaattgag 240 aacaaaataa
atagacaagt tacctttgca aagagaagaa atggactcct aaagaaagct 300
tatgaacttt ctatactttg tgatgctgaa attgctctta ttattttctc tagtcgtggc
360 aagctttatg aattttgcag caattcaagt atgtccaaga cattggagag
ataccacaga 420 tacaattatg gtacacttga aggaacccaa acttcatcag
attcacagaa caactaccaa 480 gagtatttga agcttaaaac aagagtggaa
atgttacaac agtctcaaag gcatttgcta 540 ggtgaggatt tgggacaatt
gggcacaaaa gacttggaac agcttgaacg tcaattggat 600 tcatcattga
ggcaattagg tacacaagac acacccattc ttgatcaact tgctgaactt 660
caccaaaagg aacaatctct tactgaaatg aacaaatctt tgagaataaa gttggaagaa
720 cttggtgtta cctttccaac atcatggcat tgtggtgagc aaagtgtaca
atatagacat 780 gaacagcctt tccatcatga gggatttttc aacatgtaca
ttgcaataat acattgccta 840 tacgcaccat caacacatga tgctactgga
gttgtacctg gatggatgct ttgaatttgg 900 agtatatgga gagaaaaaat
cctcttagtt atacaagtta tttattttta ttaaaaaaat 960 aagttagatg
gagaattata tatatcatac tttaaagaac ttatattgtt tgaatgtttt 1020
agctagcaaa cactttggat tatatataat attgtgatat atttattgtc aagaagagta
1080 tggcaatatt gataacacta tatttttgaa aaaaaaaaa 1119 5 1191 DNA
Tomato 5 gtcgacggga accattagat tttaaagaca ttaaatctat tacccttacc
ctaagaataa 60 gaagatgtaa agtagaagag aaaacaacca aaaccatata
tatacatata tataattaca 120 ttatattgtc ttataacata tagtctttta
aggaaaaaca aatttagaaa aaaaataata 180 ttattttaca tttttttttc
ttcatacaat atgggtagag ggaaagtaga attgaagaga 240 attgagaaca
aaataaatag acaagttacc tttgcaaaga gaagaaatgg actcctaaag 300
aaagcttatg aactttctat actttgtgat gctgaaattg ctcttattat tttctctagt
360 cgtggcaagc tttatgaatt ttgcagcaat tcaagtatgt ccaagacatt
ggagagatac 420 cacagataca attatggtac acttgaagga acccaaactt
catcagattc acagaacaac 480 taccaagagt atttgaagct taaaacaaga
gtggaaatgt tacaacagtc tcaaaggcat 540 ttgctaggtg aggatttggg
acaattgggc acaaaagact tggaacagct tgaacgtcaa 600 ttggattcat
cattgaggca aattaggtca acaaagacac aacacattct tgatcaactt 660
gctgaacttc aacaaaagga acaatctctt actgaaatga acaaatcttt gagaataaag
720 ttggaagaac ttggtgttac ctttcaaaca tcatggcatt gtggtgagca
aagtgtacaa 780 tatagacatg aacagccttc tcatcatgag ggattttttc
aacatgtaaa ttgcaataat 840 acattgccta taagttacgg atacgataat
gtacaacccg aaaatgcagc accatcaaca 900 catgatgcta ctggagttgt
acctggatgg atgctttgaa tttggagtat atggagagaa 960 gaaatcctct
tagttataca agttatttat ttttattaaa aaaataagtt agatggagaa 1020
ttatatatat catactttaa agaacttata ttgtttgaat gttttagcta gcaaacactt
1080 tggattatat ataatattgt gatatattta ttgtcaagaa gagtatggca
atattgataa 1140 cactaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag
gaaaaaaaaa a 1191 6 1209 DNA Tomato 6 aggtcaactc aaacatcgta
aattgtgatt tctttatgga aagtacggat tcatcaacca 60 ggacacgtca
tcagcctcaa ctcccaccgg ggtttcgatt ccacccgacg gacgaagaac 120
tcatcgtcca ctacctcaaa aaacgagtcg ccggcgctcc gattccggtg gatattattg
180 gtgaaattga tctttataag tttgatccat gggaactccc tggtaaggca
atattcggag 240 agcaagaatg gttctttttt agtccaagag atagaaaata
tcctaacggg gcgaggccaa 300 atcgggctgc aacatcgggt tattggaagg
ctaccggaac cgacaagccg gtttttactt 360 ccggtggaac acaaaaggtt
ggggtaaaaa aggcgctcgt tttttacggc ggtaaaccac 420 caaaaggggt
aaaaactaat tggatcatgc atgaatacag agttgtagaa aataaaacaa 480
ataacaagcc acttggttgt gataatattg ttgccaacaa aaaaggatct ttgaggctag
540 atgattgggt tttatgtcga atttacaaga agaataacac acaaaggtcc
atagatgatt 600 tgcatgatat gttgggatcg ataccacaaa atgtaccaaa
ttcaatatta caaggaataa 660 agccttcaaa ctatggtaca atattgctcg
aaaatgaatc gaatatgtac gatggaatta 720 tgaataacac gaacgatatt
atcaacaata ataatagatc cattccacaa atatcgtcaa 780 agagaacgat
gcatggaggt ttgtattgga ataacgacga agcaacaaca acaacaacaa 840
ctattgatag gaaccattct ccaaatacaa aaaggttcct tgttgagaac aacgaggacg
900 atggacttaa catgaataat atttcgcgaa ttacaaatca tgaacaaagt
agctccattg 960 ccaatttcct gagccagttt cctcaaaatc cttcgattca
acaacaacaa caacaacaag 1020 aagaagtatt gggatctctt aatgatgggg
tcgtctttcg acaaccttat aatcaagtta 1080 ctggcatgaa ttggaatcac
aaagatataa aaaggcaaaa aatagttagc cctgtaaaat 1140 caatcgatca
atcaatcata gatatattat atatggattt cgttaaaaaa aaaaaaaaaa 1200
aaaaaaaaa 1209 7 2680 DNA Tomato 7 aggtcaactc aaacatcgta aattgtgatt
tctttatgga aagtacggat tcatcaacca 60 ggacacgtca tcagcctcaa
ctcccaccgg ggtttcgatt ccacccgacg gacgaagaac 120 tcatcgtcca
ctacctcaaa aaacgagtcg ccggcgctcc gattccggtg gatattattg 180
gtgaaattga tctttataag tttgatcctg ggaactccct ggtactattt tcaccactat
240 actatatttt cttgccctat aacttatata taggggaaaa agatcggagt
cagcgatgaa 300 caattattgt gtctaaatta aattttaaat atgcaataga
ttggtgacga atttcgttgc 360 taattaattt tttagtgata aattaatatt
tttccccttt ttaatcttca tgttttttat 420 cacaaagttt tctatgacca
acttataaag atttgaactc gatcaatttt ttttttagaa 480 tgaatgaact
tatgttatat atagtgatat tttaaatgct tttttatatt ttcaaaagat 540
atccacgata acgtgtaaaa agtgaatttg caaaaaaaaa atgtagtacc ttttatttaa
600 ttttattgta gataatttag attttaattt tgaatttgtt taatttaaat
tctgaatcgt 660 ataatattta tttaatttct attttttgag tttttttttg
gagggtgctt aaaaagtagt 720 attcacaaat ataaagtagt ggacaaacat
aaagtagtgg acccataatt tattttttta 780 aaaattatat taaaactatt
tgttaagttt aaattctgaa ttatcttctt atcatgtgtt 840 taacgcagct
aaggcaatat tcggagagca agaatggttc ttttttagtc caagagatag 900
aaaatatcct aacggggcga ggccaaatcg ggctgcaaca tcgggttatt ggaaggctac
960 cggaaccgac aagccggttt ttacttccgg tggaacacaa aaggttgggg
taaaaaaggc 1020 gctcgttttt tacggcggta aaccaccaaa aggggtaaaa
actaattgga tcgtgcatga 1080 atacagagtt gtagaaaata aaacaaataa
caagccactt ggttgtgata atattgttgc 1140 caacaaaaaa ggatctttga
gggtaagtcc taaattttgc atcgaaacta atttctctat 1200 cgtatcagat
agggataaga tatacgtata ctctaatctc cttgaaccac acaagtacta 1260
tactagatat gttgttgttg tagatgactt gattcaactt tcaaattttt gatgaaaatg
1320 tttaagttat atataccata tatatatagg cgtagctaaa aatttcgata
agggggttta 1380 aatctgaaaa aatggatata cgaaatagcc gaaagaggtt
cgacatagat tattttaacc 1440 atataaaaat aatacaattt tcatatatat
atacgccgtg gttaatatga ggaatatttt 1500 atactattaa tgtactttaa
ccaggggcgg ctctagagtt gatgaaccct ctcagcgaaa 1560 atttacgttg
tatatttaag gtacctttta ataatttttg tatttatata ttaattttga 1620
acctcttgaa tataagatta gacgttgact tagtggtttc aggggttcaa atcactattc
1680 tttttttcct aaccccctta atgaaaatcc tgaatcggcc actaacttta
actggttata 1740 gaaggttaat cttactagaa aaaagcatga aattctaacc
gacaaagatg tagtcgccca 1800 gttagataag acgtttaaat tgggcggata
gagttacttt atttttcact gtcatatgtt 1860 actatatatt gacacttcac
ttaaagagtt atcatatcga tatttttact attagtgtac 1920 ataacacaaa
ctcgaataaa ttcaatgttt cattagctag ttaattagtc taactttttt 1980
aaaaaaaaat atttttctta ctccacacta ttttatttta tttttttgca gctagatgat
2040 tgggttttat gtcgaattta caagaagaat aacacacaaa ggtccataga
tgatttgcat 2100 gatatgttgg gatcgatacc acaaaatgta ccaaattcaa
tattacaagg aataaagcct 2160 tcaaactatg gtacaatatt gctcgaaaat
gaatcgaata tgtacgatgg aattatgaat 2220 aacacgaacg atattatcaa
caataataat agatccattc cacaaatatc gtcaaagaga 2280 acgatgcatg
gaggtttgta ttggaataac gacgaagcaa caacaacaac aacaactatt 2340
gataggaacc attctccaaa tacaaaaagg ttccttgttg agaacaacga ggacgatgga
2400 cttaacatga ataatatttc gcgaattaca aatcatgaac aaagtagctc
cattgccaat 2460 ttcctgagcc agtttcctca aaatccttcg attcaacaac
aacaacaaca acaagaagaa 2520 gtattgggat ctcttaatga tggggtcgtc
tttcgacaac cttataatca agttactggc 2580 atgaattgga atcactaaag
atataaaaag gcaaaaaata gttagccctg taaaatcaat 2640 cgatcaatca
atcatagata tattatatat ggatttcgtt 2680
* * * * *