Note: Descriptions are shown in the official language in which they were submitted.
1~3g~29
5-15883/+/CGC 1198/CIP
Herbicidal Tolerant Plants Containing Gluthathione S-Transferase
Gene
This invention relates to the use of recombinant DNA technology for
the transformation of plants to confer herbicide tolerance to plants
by detoxification of the herbicide. More specifically, the invention
concerns the construction and use of a recombinant DNA molecule that
includes a glutathione S-transferase (GST) gene that upon expression
in a plant increases the levels of GST enzymatic activity in the
plant.
Glutathione S-transferases (EC 2.5.1.18) are a class of enzymes
involved in the detoxification of xenobiotics. These enzymes are
ubiquitous to most living organisms, including microorganisms,
plants, insects, and animals. Each gluthatione S-transferase ~GST)
enzyme within this class is distinct; however, the enzymes do
exhibit some overlapping substrate specificity. Jakoby et al., "Rat
-Glutathione S-transferases: Binding and Physical Properties," in
Glutathione: Metabolism and Function, edited by I. Arias and
W. Jakoby (Raven Press, New York, 1976); Reddy et al., "Purification
and Characterization of Individual Glutathione S-Tran~ferace from
Sheep Liver," Archives of Biochem. and Biophys., 224: 87-101 (1983).
Of the multiple GST functions, GST's catalysis of the conjugation of
glutathione to electrophilic compounds is of particular interest. H.
Rennenberg, "Glutathione Metabolism and Possible Biological Roles in
Higher Plants," Phytochemistry, 21: 2771-2781 (1982); Meister and
Tate, "Glutathione and Related Gamma-Glutamyl Compounds: Bio-
synthesis and Utilization," in Ann. Rev. Biochem., 45: 560-604
(1976). Many xenobiotics, including herbicides, pesticides, and
insecticides are electrophilic compounds. In the conjugation of the
1 3 ~
-- 2
glutathlone and the electrophlllc center of the compound, the
sulfhydryl group of glutathlone reacts wlth the electrophllic
center of the compound. Glutathlone partlclpates as a
nucleophlle by con~ugatlon wlth the electrophlle compound.
Thls con~ugatlon ls catalyzed by a speclflc GST enzyme.
(Rennenberg, supra).
In plants, thls reactlon ls lmportant as lt provldes
a mechanlsm for detoxlflcatlon of the xenoblotlc compound. The
con~ugated electrophlllc, xenoblotlc compound ls rendered
water-soluble and non-toxlc to the plant.
It would therefore be deslrable to develop plants
that are tolerant to herblcldes by lncreaslng the levels of
glutathlone S-transferase enzymatlc actlvlty ln sald plants
uslng genetlc englneerlng technlques. In such a manner, lt
would be posslble to confer herblclde tolerance to a plant.
Thls lnventlon ls dlrected to recomblnant DNA
molecules that confer herblclde tolerance to a plant by
produclng protelns that detoxlfy herblcldes. Known
detoxiflcatlon mechanlsms lnclude the con~ugatlon of
glutathlone to an electrophlllc compound catalyzed by
glutathlone S-transferase; D-amlno acld con~ugatlon to
2,4-dlchlorophenoxyacetlc acld (2,4-D) and hydroxylatlon and
carbohydrate con~ugatlon of sulfonylurea. More partlcularly,
the lnventlon of a dlvlslonal appllcatlon ls dlrected to
herblclde tolerant plants transformed wlth a recomblnant DNA
molecule encodlng an enzyme that detoxlfles herblcldes.
Speclfically, thls lnventlon further relates to the
1339~29
-- 3
recombinant DNA molecules comprlslng genetlc sequences codlng
for glutathlone S-transferase polypeptldes and to herblclde
tolerant, transgenic plant cells wlth lncrea~ed levels of
glutathlone S-transferase enzymatlc actlvlty. In thls
lnventlon, the plant cell ls transformed by a glutathione
S-transferase (GST) gene, whlch, upon expre~slon or
overexpresslon, confers herblclde tolerance.
The lnventlon of the dlvlslonal applicatlon also
relates to plants regenerated from the transformed plant cells
and the seed thereof as well as to progeny of plants
regenerated from the transgenlc plant cells, lncludlng mutant
and varlant progeny.
The lnventlons also relate to chlmerlc genetlc
constructs contalnlng the glutathlone S-transferase gene,
clonlng vectors and hosts, and methods for conferring
herblclde tolerance to plants.
In the followlng the drawlngs should be descrlbed
briefly:
Flgure 1 shows the sequencing strategy for the
pGTR200 cDNA insert of Yb200, a rat llver glutathlone
S-transferase gene.
Flgure 2 deplcts plasmld pCIB710, an E. coll
repllcon, whlch lncludes the promoter for the CaMV 35S DNA
transcrlpt and lts termlnator and polyA addltlon slgnal.
Flgure 3 shows the constructlon of plasmld pCIB12,
an E. coll repllcon, containing the chlmerlc gene wlth the
CaMV 35S promoter llnked to the rat llver glutathlone
133~623
- 3a -
S-transferase cDNA gene, Yb200, and the CaMV termlnator.
Flgure 4A shows the constructlon of plasmld pCIB14,
a broad host range repllcon, contalnlng two chlmerlc genes
lnslde a T-DNA border, wlth the chlmerlc gene with the CaMV
35S promoter llnked wlth the rat llver glutathlone
S-transferase gene, Yb200, the CaMV termlnator and the
kanamycln reslstant gene, nos-neo-nos.
Flgure 4B shows the completlon of a partlal clone by
the ln vlvo recomblnatlon method.
Flgure 4C shows the completlon of a partlal clone by
the ln vltro llgatlon method.
~3
_ 4 _ 1 3 ~ 9 3~ 2 9
Figure 5 shows fluorescence induction patterns typical of non-
transgenic tobacco leaves. The upper curve is seen after the leaves
have imbibed 10 M atrazine for 48 hours. The lower curve is seen
after inhibltion with buffer solution alone.
Figure 6 shows fluorescence induction patterns seen in leaves of
pCIB14 transgenic tobacco plants after imbibing 10 M atrazine for
48 hours. Three classes of responses are seen: (i) no detoxification
of atrazine, top curve; (ii) significant detoxification of atrazine,
bottom curve; and (iii) some intermediate detoxification, middle
curve.
Figure 7a shows the construction of pCIB5,
Figure 7b shows the construction of pCIB4,
Figure 7c shows the construction of pCIB2,
Figure 7d/e describes the construction of pCIB10,
Figure 7f/g describes the construction of pCIBlOa.
In the detailed description that follows, a number of terms used in
recombinant DNA and plant genetics technology are utilized. In order
to provide a clear and consistent understanding of the specification
and claims, including the scope to be given such terms, the follow-
ing definitions are provided:
Heterologous Gene or DNA: A sequence of DNA encoding a specific
product, products, or biological function that is obtained from a
different species than that species into which the gene is intro-
duced, also called a foreign gene or DNA.
Homologous Gene or DNA: A se~uence of DNA encoding a specific
product, products, or biological function that is obtained from the
same species into which the gene is introduced.
133~29
-- 5 --
Plant Promoter: A DNA expression control sequence that is capable of
causing the transcription in a plant of any homologous or hetero-
logous DNA genetic sequence operably linked to such promoter.
Overproducing Plant Promoter (OPP): A plant promoter capable of
causing the expression in a transgenic plant cell of any operably
linked functional genetic sequence or sequences to levels (measured
by mRNA or polypeptide quantities) that are substantially higher
than the levels naturally observable in host cells not transformed
with said OPP.
Glutathione S-Transferase: The definition of this enzyme is func-
tional, and includes any glutathione S-transferase (GST) capable of
functioning in a given desired plant to catalyze the conjugation of
glutathione and an electrophilic compound. The term, therefore,
includes not only the enzyme from the specific plant species
involved in the genetic transformation, but may include GST from
other plant species or microbial or mammalian cells, if such GST is
capable of functioning in the transgenic plant cells. The term GST
includes amino acid sequences longer or shorter than the length of
natural GSTs, such as functional hybrid or partial fragments of
GSTs, or their analogues.
Plant: Any photosynthetic member of the kindom Planta that is
characterized by a membrane-bound nucleus, genetic material orga-
nized into chromosomes, membrane-bound cytoplasmic organelles, and
the ability to undergo meiosis.
Plant Cell: The structural and physiological unit of plants,
consisting of a protoplast and cell wall.
Plant Tissue: A group of plant cells organized into a structural and
functional unit.
1~39~2'3
Plant Organ: A distlnct and vlslbly dlfferentlated part of a
plant such as root, stem, leaf or embryo.
Detalled Descrlptlon of the Inventlon
Herblclde Tolerant Plants wlth Increased GST Enzymatlc
Actlvlty Level
Thls lnventlon ls dlrected to recomblnant DNA
molecules that confer herblclde tolerance to a plant by
detoxlfylng herblcldes. Known detoxlflcatlon mechanlsms
lnclude hydroxylatlon and carbohydrate con~ugatlon of
sulfonylurea lHutchlson, et al., Pestlclde Blochem. and
Physlol., 22: 243-249(1984)], D-amlno acld con~ugatlon to
2,4-D-lDavldonls, et al., Plant Phys., 70: 357-360 (1982)]
and the con~ugatlon of glutathlone to an electrophlllc
compound catalyzed by glutathlone S-transferase.
The lnventlon of the dlvlslonal appllcatlon ls
dlrected to herblclde tolerant plants transformed wlth a
recomblnant DNA molecule encodlng an enzyme that detoxlfles
herblcldes. Speclflcally, thls lnventlon further relates to
recomblnant DNA molecules comprlslng a genetlc sequence codlng
for a glutathlone S-transferase polypeptlde and to herblclde
tolerant, transgenlc plant cells and plants wlth lncreased
levels of glutathlone S-transferase enzymatlc actlvlty. The
glutathlone-contalnlng plant cell and plants are transformed
by a glutathlone S-transferase (GST) gene that, upon
expresslon ln sald plant cell and plant, lncreases the level
of GST enzymatlc actlvlty and thus confers herblclde tolerance
to the plant. The lnventlons use genetlc
13:~9623
- 6a -
englneerlng technlques ln the modlfication of these plants.
The invention is also dlrected to a process of
producing an herblcide tolerant plant cell comprlsing
transformlng a plant cell wlth a recomblnant DNA molecule
capable of detoxlfylng herblcldes whereln the recomblnant DNA
molecule comprlses a genetlc sequence codlng for a
glutathlone S-transferase polypeptlde, sald genetlc sequence
belng operably llnked to a promoter and addltlonal genetlc
sequences capable of induclng expresslon of the GST codlng
region ln said plant cell.
The lnventlon further comprlses a recombinant DNA
molecule that confers herbiclde tolerance to a plant
comprlslng a genetlc sequence codlng for a rat glutathlone S-
transferase polypeptlde, sald genetlc sequence belng operably
linked to a promoter and addltlonal genetic sequences capable
of inducing expression of a GST coding region in a plant
cell.
The invention also comprlses a recomblnant DNA
molecule that confers herbicide tolerance to plant by
detoxlfylng herblcldes, comprlslng a genetlc sequence of rat
origin, codlng for a glutathione S-transferase polypeptide,
sald genetlc sequence belng operably linked to a plant
promoter and addltlonal genetlc sequences capable of lnduclng
expression of a GST codlng reglon in a plant cell.
The term "herbicide tolerant plant" as used herein
ls deflned as a plant that survlves and preferably grows
13~9~;29
- 6b -
normally at a usually effective dose of a herblclde.
Herblclde tolerance ln plants accordlng to the lnventlon of
the dlvlslonal applicatlon refers to detoxlflcatlon mechan-
i,~. .
133~629
-- 7 --
isms in a plant, although the herbicide binding or target site isstill sensitive. Resistance is the m~xi ~ tolerance that can be
achieved.
Detoxification should be distinguished from another mechanism for
conferring herbicide tolerance in which the herbicide binding or
target site is changed so that it is no longer sensitive. In the
present invention, the herbicide binding site remains sensitive but
the herbicide never binds to it because the herbicide is detoxified
by, for example the GST enzyme. Thus, the term "herbicide tolerance"
as used herein is meant to include tolerance and resistance to
herbicides due to detoxification of the herbicide by, for example,
increased GST enzymatic activity levels. The herbicide tolerant
plants of the present invention survive without damage in the
presence of certain herbicides that are lethal or that damage the
growth or vigor of herbicide sensitive plants.
The herbicides that are contemplated in this invention include all
those that are capable of being detoxified by, for example, forming
conjugates with plant glutathione or analogues or homologues of
glutathione, typically any electrophilic compound. Of particular
interest in this invention are those herbicides that have chlorine
residues. Herbicides that are contemplated in this invention
include, but are not limited to, triazines, including chlorotria-
zines, acetamides including chloroacetanilides, sulfonylureas,
imidazolinones, thiocarbamates, chlorinated nitrobenzenes, diphenyl
ethers and the like. Some specific examples of herbicides include
atrazine, alachlor, S-ethyl dipropylthiocarbamate, and diphenyl-
ethyls. See also, Herbicide Resistance in Plants (H. LeBaron and
J. Gressel, editors, 1982).
Some of the herbicides are typically potent inhibitors of photo-
synthesis. Frear et al., Phytochemistry, 9: 2123-2132 (1970)
(chlorotriazines); Frear et al., Pesticide Biochem. and Physiol.,
20: 299-310 (1983) (diphenylether); Lay et al., Pesticide Biochem.
13.~2~
-- 8 --
and Physiol., _: 442-456 (1976) (thiocarbamates); and Frear et al.,
Pesticide Biochem. and Physiol., 23: 56-65 (1985) (metribuzin).
Others deactivate enzymes necessary for amino acid biosynthesis.
Although the term "herbicides" is used to describe these compounds,
the use of this term herein is not meant to be limiting. For
example, many insecticides and pesticides (for controlling diseases,
parasites, and predators) that are applied to plants have dele-
terious effects on plant vigor. The insecticidal or pesticidal agent
may be absorbed into the plant tissues either through leaves and
stems, or from the soil, through the plant's root system. Moreover,
there are many xenobiotics that are electrophilic compounds capable
of being conjugated by glutathione, a reaction, which is catalyzed
by the GST enzymatic activity. These xenobiotic compounds ar.e within
the scope of this invention.
In one embodiment of this invention, the herbicides contemplated are
sensitizers, that is, inhibitors of the GST enzymatic activity.
These sensitizers inhibit the endogenous detoxification mechanism by
forming a GST-sensitizer conjugate. The application of an herbicide
and a sensitizer, for example, tridiphane, to a plant will inhibit
the enzymatic conjugation of glutathione and a herbicide. Ezra
et al., "Tridiphane as a Synergist for Herbicides in Corn (Zea mays)
and Proso Millet (Panicum miliaceum), Weed Science, 33: 287-290
(1985).
Thus, in this invention, the genetic engineering techniques used to
confer GST enzymatic activity, or increased levels of GST enzymatic
activity, to a plant results in the transgenic plant being more
tolerant or resistant to sensitizers such as tridiphane. In this
manner, for instance, a sensitizer and an herbicide in combination
can be applied to herbicide sensitive plants and simultaneously to
herbicide tolerant plants according to this invention. The combina-
tion of sensitizer and herbicide will be typically toxic to the
herbicide sensitive plants, but not to the transgenic tolerant
plants.
1~3!~2~
g
Any plant that contalns glutathlone or analogles,
such as homoglutathlone, and that ls capable of undergolng
genetic manlpulatlon by genetlc engineering technlques may be
used. The transgenlc plant should also be capable of
expresslng the GST gene. As used hereln, the term "plant"
includes plant cells, plant protoplasts, plant-tlssue culture
that can be cultured and lnduced to form plants, plant calll,
plant clumps and plant cells that are lntact ln plants or
parts of plants. "Plant" also refers to pollen that may be
transformed by genetlc englneerlng technlques.
Glutathlone ln plants ls typlcally found ln hlghest
concentrations ln the subcellular compartments. The highest
concentratlon of glutathlone ls ln the plant plastlds,
typlcally ln the chloroplasts. [Rennenberg, H.,
Phytochemlstry, 21: 2771-2781 (1982)]. Glutathlone
has a structure of gamma-L-glutamyl-L-cystelnyl-glyclne. A
homologous form of glutathlone, homoglutathlone, has been
identified ln some plants, wlth the structure of
gamma-L-glutamyl-L-cystelnyl-beta-alanlne. [Carnegie, P.,
Biochem. J., 89: 459-471 (1963) and Carnegie, P., Blochem. J.,
89: 471-478 (1963)~. Plants contaln varylng amounts of
glutathlone or homoglutathlone. For example, several legumes
contaln malnly homoglutathlone, whlle other legumes contaln
mainly glutathione. Typically, where either homoglutathione
or glutathione is predominant in a plant, only reduced amounts
of the other compound are found. (Rennenberg, supra.)
The codlng reglon for the glutathlone S-transferase
'~'
133~29
- 9a -
(GST) gene that may be used ln thls lnventlon may be
homologous or heterologous to the plant cell or plant belng
transforme~. It is necessary, however, that the genetlc
sequence codlng for GST be expressed, and produce a functional
enzyme or polypeptlde ln the resultlng plant cell. Thus, the
lnventlon of the dlvlslonal appllcatlon comprlses plants
contalnlng elther homologous GST genes or heterologous GST
genes that express the GST enzyme. Further, the heterologous
GST may be from other plant specles, or from organlsms of
dlfferent klngdoms, such as mlcrobes or mammals.
':~
~3~9~29
~o
As previously described, the CST enzymefi are a class of enzymes that
are multlfunctionsl. Thu~, lt 18 al~o nece~sary ~o choo~e a GST gene
that will catalyze the con~ugatlon of glutathione and an electro-
philic compound. Since GST recognizes glutathione afi a substrate, it
was uncertsin prlor to this invention whether the GST enzyme
specific for glutathione conjugation would sccept homoglutathione in
transformed plants. Frear et al., Phytochemistry, 9: 2123-2132
(1970) (indicating that glutathione S-transferase wa~ specific for
reduced glutathione). The requisite GST enzyme 6pecific for gluta-
thione can be identified and chosen using an assay that will
determine substrate specificity to differentiate the various
glutathlone S-transfera~es. In a typical a8~ay~ the glutathlone
specific GST can be characterized by affinlty chromatography. I TU
et al., Biochem. snd Biophys. Research Comm., 108: 461-467 (1982);
lu et al., J. Biol. Chem., 258: 4659-4662 (1983); and Jakoby et al.,
in Glutathione; Metabolism and Function, (Raven Press, New York,
1976)].
In one embodiment of this invention, the GST comprises a plant GST
that is homologous to the plant to be transformed. In another
embodiment of this invention, the CST comprlses n plant GST that is
heterologous to the plant to be transformed. Plants that contain an
abundance of GST include corn and sorghum. In still another embodi-
ment of this invention, the GST comprises a mammalian GST. Mammalian
GSTs are known and are described in Reddy, et al., Archives of
Biochem. and Biophysics, 224: 87-101 (1983) (sheep liver); Tu
et al., J. Biol. Chem., 258: 4659-4662 (1983) (rat ti~sue including,
heart, kidney, liver, lung, spleen, and testis). The preferred GST
gene comprises the coding region of A rat liver GST gene, and
especiAlly Yb200 described in Example IA and the completed Yb187 as
described in Example IB. A further embodiment of the present
inventlon comprlses the codlng reglon of a rat braln CST, and
especially the cDNA clone GlYb as described in Example IE. However,
other GST genes are known and may be used in this invention. See,
Mannervik, B., Adv. Enzymol. Relat. Areas Mol. Biol., 57: 357-417
(1985),
11 13~9~23
The DNA sequence coding for glutathione S-transferase may be
constructed entirely of genomic DNA, or entirely of cDNA. Alter-
natively, the DNA sequence may be a hybrid construction of both cDNA
and genomic DNA, in which case the cDNA may be derived from the same
gene as the genomic DNA, or the cDNA and the genomic DNA may be
derived from different genes. In either case, both the genomic DNA
and/or the cDNA separately may be constructed from the same gene, or
from different genes. If the DNA sequence comprises portions from
more than one gene, the portions of genes may all come from the same
organism; from organisms of more than one strain, variety or species
of the same genus; or from organisms of more than one genus of the
same or of different kingdoms.
Portions of DNA sequences may be joined together to form the total
glutathione S-transferase coding sequence by methods that are known
in the art. Some suitable methods include, for example, in vivo
recombination of DNA sequences having regions of homology in vitro
ligation of appropriate restriction fragments.
There are a variety of embodiments encompassed in the broad concept
of the invention. In one of its embodiments, this invention com-
prises chimeric genetic sequences containing:
~a) a first genetic sequence coding for the glutathione S-trans-
ferase polypeptide that, upon expression of the gene in a given
plant cell, is functional for glutathione S-transfersse activity;
and
(b) one or more additional genetic sequences operably linked on
either side of the GST coding region. These additional genetic
sequences contain promoter and/or terminator regions. The plant
regulatory sequences may be heterologous or homologous to the host
cell.
- 12 - 1 3 3 9 6 2'3
Any promoter and any terminator capable of inducing expression of a
GST coding region may be used in the chimeric genetic sequence. Some
suitable examples of promoters and terminators include those from
nopaline synthase (nos), octopine synthase (ocs) and cauliflower
mosaic virus (CaMV) genes.
One type of efficient plant promoter that may be used is an over-
producing plant promoter. Such promoters, in operable linkage with
the genetic sequence for GST, should be capable of promoting expres-
sion of said GST such that the transformed plant is tolerant to an
herbicide due to the presence of, or increased levels of, GST
enzymatic activity. Overproducing plant promoters that may be used
in this inventlon include the promoter of the small subunit (ss) of
the ribulose-1,5-bisphosphate carboxylase from soybean [Berry-Lowe
et al., J. Molecular and Appl. Gen., 1: 483-498 (1982)~, and the
promoter of the chlorophyll a/b binding protein. These two promoters
are known to be light induced in eucaryotic plant cells [see, for
example Genetic Engineering of Plants, an Agricultural Perspective,
A. Cashmore, Plenum, New York 1983, pages 29 - 38, Coruzzi G.
et al., The Journal of Biological Chemistry, 258: 1399 (1983), and
Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:
285 (1983)].
The chimeric genetic sequence comprising the glutathione S-trans-
ferase gene operably linked to a plant promoter can be ligated into
a suitable cloning vector. In general, plasmid or viral (bacterio-
phage) vectors containing replication and control sequences derived
from species compatible with the host cell are used. The cloning
vector will typically carry a replication origin, as well as
specific genes that are capable of providing phenotypic selection
markers in transformed host cells, typically resistance to anti-
biotics or resistance to selected herbicides. The transforming
vectors can be selected by these phenotypic markers after trans-
formation in a host cell.
1339~29
- 13 -
Host cells that may be used in this invention include procaryotes,
including bacterial hosts such as A. tumefaciens, E. coli, S. typhi-
murium, Serratia marcescens and cyanobacteria. Eucaryotic host
cells such as yeast, filamentous fungi, and plant cells may also be
used in this invention.
The cloning vector and host cell transformed with the vector are
used in this invention typically to increase the copy number of the
vector. With an increased copy number, the vectors containing the
GST gene can be isolated and, for example, used to introduce the
chimeric genetic sequences into the plant cells.
The introduction of DNA into host cells may be accomplished by
methods known in the art. Bacterial host cells can be transformed,
for example, following treatment of the cells with calcium chloride.
DNA may be inserted-into plant cells by contacting protoplasts of
the cells directly with the DNA. Alternatively, DNA may be inserted
into plant cells by contacting the cells with viruses or with
Agrobacterium. Contact with viruses and Agrobacterium may occur
through infection of sensitive plant cells or through co-cultivation
of protoplasts of plant cells with Agrobacterium. These methods are
discussed in greater detail below.
There are a number of methods for- the direct insertion of DNA into
plant cells. For example, the genetic material contained in the
vector can be microinjected directly into plant cells by use of
micropipettes to mechanically transfer the recombinant DNA. The
genetic material may also be transferred into plant protoplasts
following treatment of the protoplasts with polyethylene glycol.
~Paszkowski et al., EMB0 J., _: 2717-22 (1984)].
In an alternate embodiment of this invention, the GST gene may be
introduced into the plant cells by electroporation. [Shillito
et al., Biotechnology, 3: 1099-1103 (1985); Fromm et al., Proc.
_
Nat'l Acad. Sci. USA, 82: 5824 (1985)]. In this technique, plant
- 14 - ~ 3 39 6 2 9
protoplasts are electroporated in the presence of plasmids con-
taining the GST genetic construct. Electrical impulses of high field
strength reversibly render biomembranes permeable, allowing the
introduction of the plasmids. Electroporated plant protoplasts
reform cell walls, divide, and form plant calli. Selection of the
transgenic plant cells with the expressed GST enzyme can be ac-
complished using the phenotypic markers as described above.
Cauliflower mosaic virus (CaMV) may also be used as a vector for
inrorduclng the GST gene into plant cells in this invention. (Hohn
et al., in "Molecular Biology of Plant Tumors", Academic Press, New
York, 1982 pages 549-560; Howell, United States Patent
No. 4,407,956). The entire CaMV viral D~A genome is inserted into a
parent bacterial plasmid creating a recombinant DNA molecule that
can be propagated in bacteria. The recombinant plasmid is cleaved
with restriction enzymes either at random or at unique non-vital
sites in the viral portion of the recombinant plasmid, for example,
at the gene for aphid transmissability, for insertion of the GST
genetic sequence. A small oligonucleotide, described as a linker,
having a unique restriction site may also be inserted. The modified
recombinant plasmid again is cloned and further modified by intro-
duction of the GST genetic sequence thereof into a unique restric-
tion site. The modified viral portion of the recombinant plasmid is
then excised from the parent bacterial plasmid, and used to inocu-
late the plant cells or plants.
Another method of introducing the GST gene into the cells is to
infect a plant cell with Agrobacterium tumefaciens transformed with
the GST gene. Under the appropriate conditions known in the art, the
transgenic plant cells are grown to form shoots and roots, and to
develop further into plants. The GST genetic sequences can be
introduced into appropriate plant cells, for example, by means of
the Ti plasmid of Agrobacterium tumefaciens. [DeCleene et al., Bot.
Rev., 47: 147-194 (1981); Bot. Rev., 42,: 389-466 (1976)~. The Ti
plasmid is transmitted to plant cells on infecton by Agrobacterium
- 15 - 13 3 9 ~ 29
tumefaciens and is stably integrated into the plant genome. [Horsch
et al., Science, 233: 496-498 (1984); Fraley et al., Proc. Nat'l
Acad. Sci. USA, 80: 4803 (1983)].
For plants whose cells are not sensitive to infection by Agrobacte-
rlum, one can resort to co-cultivation of the Agrobacterium with the
corresponding protoplast.
Ti plasmids contain two regions essential for the production of
transformed cells. One of these, the transfer DNA (T-DNA) region, is
transferred to plants and induces tumor formation. The other, the
virulent (vir) region, is essential for the formation but not
maintenance of tumors. The transfer DNA region can be increased in
size by the insertion of the GST genetic sequence without its
transferring ability being affected. By removing the tumor-causing
genes so that transgenic plant cells are non-tumorous, and adding a
selectable marker, the modified Ti plasmid can be used as vector for
the transfer of the gene constructs of the invention into an
appropriate plant cell.
The vir region causes the T-DNA region to be transferred from
Agrobacterium to the genome of a plant cell irrespective of whether
the T-DNA region and the vir region occur on the same vector or on
different vectors in the Agrobacterium cell. A vir region on a
chromosome also induces transfer of T-DNA from a vector into a plant
cell.
The preferred system for transferring a T-DNA region from Agrobacte-
rium into plant cells comprises a vir region on a vector other than
the vector containing the T-DNA region. Such a system is known as a
binary vector system and the T-DNA-containing vector fs known as a
binary vector.
1~39fi23
Any T-DNA-contalnlng vector that can be transferred
lnto plant cells and that allows the transformed cells to be
selected ls sultable for use ln this lnventlon. A vector
constructed from a promoter, a codlng se~uence and pCIB10 ls
preferred.
Any vlr reglon-contalning vector that causes the
transfer of a T-DNA reglon from Agrobacterlum to plant cells
may be used ln thls lnventlon. The preferred vlr
region-containlng vector ls pCIB542.
Plant cell or plants transformed with DNA ln
accordance wlth this lnventlon can be selected by an
approprlate phenotyplc marker that ls present ln the DNA ln
addltlon to the GST gene. These phenotypic markers lnclude,
but are not llmlted to, antlblotlc reslstance markers, such as
kanamycln and hygromycin genes, or herblclde reslstance
markers. Other phenotypic markers are known ln the art and may
be used in thls lnventlon.
All plants whose cells can be transformed by dlrect
lnsertlon of DNA or by contact wlth Aqrobacterlum and
regenerated into whole plants can be sub~ected to the methods
of the inventlon of the dlvlsional appllcatlon so as to
produce transgenlc whole plants that contaln the transferred
GST gene. There ls an lncreaslng body of evldence that
practlcally all plants can be regenerated from cultured cells
or tlssues, lncludlng but not llmlted to all ma~or cereal crop
specles, sugarcane, sugar beet, cotton, frult and other trees,
legumes and vegetables.
.~
~.s
13~S~29
- 16a -
Addltlonally lncluded wlthln the scope of the
present lnventlon, target crops are, for example, those of the
group consisting of Fragaria, Lotus, Medlca~o, Onobrychls,
Trifollum, Trlgonella, Vlgna, Cltrus, Llnum, Geranium,
Manlhot, Daucus, Arabldopsl~, Brasslca, Raphanus, Slnapls,
Atropa, Capslcum, Datura, HyoscYamus~ LYcoperslcon~ Nlcotlana,
Solanum, Petunla, Dlgltalls, Ma~orana, Clchorlum, Hellanthus,
Lactuca, Bromus, Asparagus, Antlrrhlnum, Hemerocallls,
Nemesia, Pelargonium, Panlcum, Pennlsetum, Ranunculus,
Seneclo, Salplglossls, Cucumls, Browallla, Glyclne, Lollum,
Zea, Trltlcum and Sorghum, as well as those of the group
conslstlng
!
." ~'
1339629
- 17 -
of Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus,
Populus, Santalum, Allium, Lilium, Narcissus, Ananas, Arachis,
Phaseolus and Pisum.
Limited knowledge presently exists on whether all of these plants
can be transformed by Agrobacterium. Even species that are not
natural plant hosts for Agrobacterium may be transformable in vitro.
For example, monocotyledous plants and, in particular, cereals and
grasses are not natural hosts to Agrobacterium. There is growing
evidence now that certain monocots can be transformed by Agro-
bacterium. Using novel experimental approaches that have now become
available, cereal and grass species may be transformable [Grims-
ley, N. et al., Nature, 325: 177-179 (1987)].
Plant regeneration from cultural protoplasts is described in Evans,
et al., "Protoplast Isolation and Culture," in Handbook of Plant
Cell Culture, 1: 124-176 (MacMillan Publishing Co. New York 1983);
M.R. Davey; "Recent Developments in the Culture and Regeneration of
Plant Protoplasts," Protoplasts, 1983 - Lecture Proceedings,
pp. 19-29, (Birkhauser, Basel 1983); P.J. Dale, "Protoplast Culture
and Plant Regeneration of Cereals and Other Recalcitrant Crops," in
Protoplasts 1983 - Lecture Proceedings, pp. 31 - 41, (Birkhauser,
Basel 1983); and H. Binding, "Regeneration of Plants," in Plant
Protoplasts, pp. 21-37, (CRC Press, Boca Raton 1985).
Regeneration varies from species to species of plants, but generally
a suspension of transformed protoplasts, cells or tissue containing
multiple copes of the GST gene is first provided. Embryo formation
can then be induced from the suspensions, and allowed to develop to
the stage of ripening and germination as natural embryos. The
culture media will generally contain various amino acids and
hormones, such as auxin and cytokinins. It is also advantageous to
add glutamic acid and proline to the medium, especially for such
species as corn and alfalfa. Shoots and roots normally develop
simultaneously. Efficient regeneration will depend on the medium, on
- 18 - 13 39 ~29
the genotype, and on the history of the culture. If these three
variables are controlled, then regeneration is fully reproducible
and repeatable.
Some suitable plants for use in this invention include, for example,
species from the genera Lotus, Medicago, Onobrychis, Trifolium,
Trigonella, Citrus, Linum, Manihot, Daucus, Arabidopsis, Brassica,
Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lyco-
persicon, Nicotiana, Solanum, Petunia, Majorana, Cichorium, Helian-
thus, Lactuca, Asparagus, Antirrhinum, Panicum, Pennisetum, Ranun-
culus, Salpiglossis, Glycine, Gossypium, Malus, Prunus, Rosa,
Populus, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus, and
Pisum.
Since it has been found that Oryza (rice~ can be regenerated to
whole plants from protoplasts, it should be possible to regenerate
other plants belonging to the Gramineae family. Therefore, the
following plants may be used in the present invention: Lolium, Zea,
Triticum, Sorghum and Bromus.
The preferred plants according to this invention are from the genera
Nicotiana spp, ~e.g. tobacco), Glycine spp. (especially Glycine max,
soybean) and Gossypium spp. (cotton).
The mature plants grown from the transformed plant cells are selfed
to produce seeds, some of which contain the gene for the increased
GST enzymatic activity level in proportions that follow well
established laws of inheritance. These seeds can be grown to produce
plants that are herbicide tolerant. The tolerance of these seeds can
be determined, for example, by growing the seeds in soil containing
an herbicide. Alternatively, the herbicide tolerance of the trans-
formed plants can be determined by applying an herbicide to the
plant.
- 19 - 1 3 3 9 6 2 9
Homozygous lines can be produced by repeated selfing to give
herbicide tolerant inbreds. These inbreds can be used to develop
herbicide tolerant hybrids. In this method an herbicide tolerant
inbred line is crossed with another inbred line to produce an
herbicide resistant hybrid.
Parts obtained from the regenerated plant, such as flowers, seeds,
leaves, branches, fruit and the like are covered by the invention
provided that these parts comprise the herbicide tolerant cells.
Progeny (including hybrid progeny), variants, and mutants of the
regenerated plants are also included within the scope of this
invention.
Uses For The GST Genetic Constructs and Herbicide Tolerant Plants
The GST genetic constructs, as previously described, may be used in
vectors as intermediates in the preparation of herbicide tolersnt
plant cells, plant organs, plant tissues, and plants.
The importance of herbicide tolerant plants according to the
invention is apparent. Such plants would enable farmers to plant an
herbicide tolerant crop and then treat the field for weeds without
adversely affecting the crop. Further, an herbicide tolerant plant
would enable farmers to grow crops in fields that have been treated
with herbicides, for example, during a crop rotation cycle in which
a naturally tolerant plant is rotated with a naturally sensitive
plant. These herbicidally treated fields may contain a certain
amount of "herbicide carryover" in the soil. Rotational crops that
are naturally sensitive to the herbicide can be injured by such
herbicide carryover unless they are rendered tolerant [Sheets, T.
Residue Reviews, 32: 287-310 (1970); Burnside, et al., Weed Science,
19: 290-293 (1971)].
For example, farmers typically plant corn and soybean crops in
alternating succession. While the corn crop may naturally be
tolerant to certain herbicides, for example certain triazine herbi-
; 2 '3
- 20 -
cides such as atrazine, the more sensitive soybean crop,
planted after the corn fields have been treated with
herbicides, may be damaged. By the use of a herbicide tolerant
plant damage due to herbiclde carryover ls avoided. [Fink, et
al., Weed Sclence, 17: 35-36 (1969) (soybeans); Khan, et al.,
Weed Research, 21: 9-12 (1981) (oats and timothy plants);
Brinkman, et al., croP Science, 20: 185-189 (1980) (oats);
Eckert, et al, J. Range Mgmt., 25: 219-224 (1972)
(wheatgrass)].
The invention of the dlvlslonal applicatlon also
encompasses a method of plant control whlch comprises
contacting a mixed population consisting of a herblcide
sensltlve plant, such as a weed, and a herblcide tolerant
plant of the lnvention with plant controlllng amounts of a
herblcide, which are sufflcient to control herblclde sensltlve
plants. Thus, foliar herbicldal treatment of plants ln a fleld
with both herblclde tolerant plants and herblclde sensltive
weeds, and wherein both plant types are simultaneously
contacted with the herblcide during the treatment operation,
ls a method lncluded ln the lnventlon of the dlvlsional
appllcation.
Also, as previously described, the invention of the
dlvlslonal appllcatlon lncludes the method of plant control
comprlsing applying a herbicide and a sensltizer
simultaneously to both herblcldal tolerant plants and to
herblcide sensitive plants.
The term "plant controlling amounts of herbiclde"
.~
~33~2~
- 21 -
lncludes, functlonally, an amount of herblclde that is capable
of affectlng the growth or development of a glven plant. Thus,
the amount may be small enough to slmply retard or suppress
the growth or development, or the amount may be large enough
to lrreversibly destroy the sensltlve plant.
The actual amount of the herblclde depends on the
herblclde and on the plant belng controlled. For example,
dlcotyledonous plants and weeds are often controlled at
concentratlons of herblcldes between 0.5 and 1.5 kg/ha. For
monocotyledonous plants, concentratlons of herblcldes between
0.5 kg/ha and about 2.0 kg/ha are typlcal. Some herblcldes,
such as sulfonylurea herblcldes, are known to control plants
at signlflcantly lower rates.
The herblclde can, of course, be brought lnto
contact wlth the approprlate plant uslng well known spraylng
or spreadlng methods. For example, follate admlnlstratlon used
ln the prlor art for control of weeds by atrazlne can be used
wlth atrazlne-tolerant plants falllng wlthln the lnventlon.
Havlng now generally descrlbed the lnventlons the
same wlll be better understood by reference to speclflc
examples, whlch are lncluded hereln for purposes of
lllustratlon only, and are not lntended to be llmltlng unless
otherwlse speclfled.
Examples
The procedures of the followlng examples may be
generally found ln Manlatls et al., Molecular Clonlng, Cold
Sprlng Harbor Laboratory, 1982. Enzymes, unless otherwlse
.~.~
1~962~
- 21a -
noted, can be obtalned from New England Blolabs, and are used
ln accord wlth the manufacturers recommendatlons unless
otherwlse lndlcated.
Example IA: Isolatlon of GST cDNA Clone Yb 200
Antlbodles:
Antlsera agalnst homogeneous rat hepatlc glutathlone
S-transferases (GSTs) (afflnlty chromatography fractlon) are
ralsed as descrlbed [Tu et al., Nuclelc Aclds Res., 10:
5407-5419 (1982)]. The IgG fractlon ls purlfled from proteln
A-Sepharose* (Pharmacla) column and concentrated by
ultraflltratlon (Amlcon*, XM--50 membrane) as descrlbed by
Kraus and Rosenberg [Kraus & Rosenberg, Proc. Nat'l Acad. Scl.
USA, 79: 4015-4019 (1982)]. It ls stored ln 50 % glycerol
and 0.2 mg/ml heparln at -18~C.
1339~i2~3
- 22 -
Isolation of Polysomes:
Livers (ca. 26 g) from two male Sprague-Dawley rats (body weight
ca. 300 g) are homogenized with a Potter-Elvehjem homogeni~er in
150 ml (final volume) of 50 mM Tris-HCl, pH 7.5, 25 mM MgCl2, 0.25 M
sucrose containing bentonite (1 mg/ml), heparin (0.2 mg/ml) and
cycloheximide (l microgram/ml) in several aliquots to a 15 ~O
(wt/vol) homogenate. Polysomes are isolated exactly according to
published procedures, Kraus & Rosenberg, ~ . The yield is 1389
A260 units before, and 1208 A260 units after, dialysis.
Immobilization of Polysome-Antibody Complexes and Elution of
Specific mRNA:
1130 A260 units of polysomes were recovered for~immunoabsorption
with anti-GST IgG (7.1 mg). Protein A-Sepharose affinity chromato-
graphy and elution of bound RNAs are carried out as described by
Kraus & Rosenberg ~ . The eluted RNAs are immediately adjusted to
0.5 M NaCl and 0.5 % sodium dodecylsulfate and purified further by
oligo(dT)-cellulose column [Aviv & Leder, Proc. Nat'l Acad. Sci.
~SA, 69: 1408-1412 (1972); Bantle et al., Anal. Biochem., 72:
413-417 (1976)]. The purified poly (A+) RNAs are assayed by
in vitro translation and immunoprecipitation [Tu et al., Nucleic
Acids Res., 10: 5407-5419 (1982); Pelham and Jackson, Eur. J.
Biochem., 67: 247-256 (1976)] before cDNA synthesis. The immuno-
precipitated materials are separated on sodium dodecylsulfate-poly-
acrylamide gels and visualized after fluorography [Laemmli, Nature,
227: 680-684 (1970); Swanstrom and Shenk, Anal. Biochem., 86:
184-192 (1978)].
Isolation of GST cDNA Clones:
cDNA synthesis is performed according to the method of Okayama and
Berg [Okayama and Berg, Mol. Cell Biol., 2: 161-170 (1982)] as
modified by Gubler and Hoffman, [Gubler and Hoffman, Gene,
25: 263-269 (1983~] with minor modifications. Approximately
100-500 ng of poly(A+) RNA from immunoprecipitated polysomes are
used for cDNA synthesis. Reverse transcription of the mRNA into cDNA
is in 40 microliters containing 50 mM Tris-Hcl pH 8.3, 100 mM NaCl,
~ Ir~le~ .fk
- ~33g~29
- 23 -
10 mM MgCl2, 10 mM DTT, 4 mM sodium pyrophosphate, 1.25 mM of the
four dNTPs, 1800 U/ml RNAsin in (Promega Biotec), 100 micrograms/ml
oligo-(dT)12 18 (Pharmacia/PL) and 3,000 U/ml of Reverse Transcrip-
tase (Molecular Genetics).
The reaction is incubated 25 minutes at 43~C, then terminated by
addition of 2 microliters of 0.5 M EDTA. The reaction is extracted
with 1 volume of phenol:chloroform and the aqueous phase back-
extracted with one-half volume of chloroform. The organic phase
again back-extracted with TE buffer.
To the combined aqueous phases, 1 volume of 4 M ammonium acetate is
added and the nucleic acid is precipitated by addition of 2 volumes
of ethanol and chilling on dry ice for 20-30 minutes. The solution
is then warmed to room temperature for 5 minutes and centrifuged for
15 minutes in an Eppendorf microfuge at 4~C. The resulting pellet is
dissolved in 25 microliters of TE buffer. Ammonium acetate
(25 microliters of 4 M) and 100 microliters of ethanol are added and
the nucleic acid precipitated and recovered as before. The pellet is
washed with 70 ~/O ethanol, dried and dissolved in 20 microliters of
water.
Replacement of the mRNA strand of the mRNA:cDNA hybrid is accom-
plished in 50 microliters containing 20 mM Tris-HCl pH 7.5, 5 mM
magnesium chloride, 10 mM ammonium sulfate, 100 mM KCl, 0.15 mM
beta-NAD, 0.04 mM of the four dNTPs, 20 microliters of the first
strand product, 10-20 microCuries 32P-dATP, 10 U/ml E. coli DNA
ligase (New England Biolabs~, 230 U/ml DNA polymerase (Boehringer
Mannheim) and 8.5 Ulml E. coli RNAse H (Pharmacia/PL). The reaction
is incubated 90 minutes at 12-14~C, then one hour at room tempera-
ture. The double stranded cDNA is purified by phenol:chloroform
extraction and recovered by ethanol precipitation exactly as
described for the first strand product. The final dried pellet is
dissolved in 20 microliters of water.
~-1 rade~ rk
- 24 - 13 3~ b 23
Ten microliters of the double-stranded cDNA are tailed with dCTP in
a total volume of 20 microliters containing 100 mM potassium caco-
dylate pH 7.0, 1 mM CoCl~, 0.2 mM DTT, 0.1 mM dCTP and 500 U/ml
terminal deoxynucleotidyl transferase (Pharmacia/PL). The reaction
is incubated 1-2 minutes at 37~C after which 20 microliters of 4 mM
EDTA is added and the enzyme is heat inactivated by incubation at
6-5~C for lO minutes.
PstI-digested, dG-tailed pBR322 (Bethesda Research Labs, Inc.) is
added in approximately 1:1 molar ratio to the dC-talled, double-
stranded cDNA (i.e. about 5-10 fold excess by molecular weight of
vector over the estimated amount of cDNA). The DNA solution
(cDNA + vector) is diluted to give a final total DNA concentration
of 0.5-2.0 ng/microliter (0.5 is optimal) in the presence of 10 mM
Tris-HCl pH 7.5, 1 mM EDTA and 150 mM NaCl. The mixture is incubated
for 5 minutes at 65~C, then the DNA annealed at 55-58~C for
90 minutes.
The annealed cDNA:vector is transformed into E. coli strain MM2g4
using 5 microliters of DNA per 200 microliter aliquot of trans-
formation competent cells [Hanahan J. Mol. Biol., 155: 557-580
(1983)]; the control transformation frequency is 1-2 x 108 trans-
formants per microgram of covalently closed circular pBR322 DNA. The
transformed cells are plated on LM plates (without magnesium)
containing 17 microgram/ml tetracycline (Hanahan, supra). Resulting
colonies are tested for ampicillin sensitivity. Those colonies which
are tetracycline resistant and ampicillin sensitive (ca. 50%~ are
picked for further analysis.
Hybrid-Selected in Vitro Translation of pGTR200.
Plasmid DNAs are purified using an alkaline lysis procedure [Birn-
boim and Doly, Nucleic Acids Res., 7: 1513-1523 (1979)] from 354
ampicillin-sensitive transformants and these DNAs digested with PstI
to determine cDNA insert sizes. Among them, 134 contain visible cDNA
inserts by agarose gel electrophoresis. Those with inserts greater
than 800 nucleotides are analyzed further by Southern blot hybridi-
1339629
- 25 -
zation [Southern, J. Mol. Biol., 98: 503-517 (1975)] using Y
(pGTR261) and Y (pGTR262) as probes ~Lai et al., J. Biol. Chem.,
259: 5536-5542 (1984); Tu et al., J. Biol. Chem., 259: 9434-9439
(1984)3.
Twelve clones which do not hybridize to these probes are then
characterized further by hybrid-selected in vitro translation
[Cleveland et al., Cell, 20: 95-105 (1980)]. One of these negative
clones is designated pGTR200. Rat liver poly(A+) RNAs selected by
pGTR200 DNA immobilized on activated aminophenylthioether cellulose
(APT-paper) are eluted at 75~C and 100~C and used to program 1n
vitro translation in the rabbit reticulocyte lysate system. The
ln vitro translation products are immunoprecipitated by antisera
against total rat liver G~Ts followed by sodium dodecylsulfate-
polyacrylamide gel electrophoresis. The immunoprecipitated product
is of Yb mobility; no other class of GST subunits is selected by
pGTR200. Earlier hybrid-selected in vitro translation experiments
with Y and Y clones do not reveal any Y subunit products (Lai
a c b
et al. supra; Tu et al. supra).
Nucleotide Sequence of pGTR200 cDNA Insert
The DNA sequence of the cDNA in pGTR200 is determined according to
the strategy given in Figure 1 by the chemical method of Maxam and
Gilbert [Maxam and Gilbert, Methods Enzymol., 65: 499-560 (1980)].
The DNA fragments generated by the various restriction endonuclease
cleavages were labeled at the 3' ends. Each determination was
repeated at least once.
The nucleotide sequence is given below. The single letter code of
amino acids is used for the 218 residue open reading frame. Old and
Primrose, Principles of Gene Manipulation, (1985), Blackwell's
Publications, London, p. 346. The poly(A) addition signal, AATAAA is
underlined.
~339~i29
- 26 -
60~TGAAGCCAAATTGAGAAGACCACAGCGCCAGAACCATGCCTATGATACTGGGATACTGG
M P M I L G Y W
100 110 120
AACGTCCGCGGGCTGACACACCCGATCCGCCTGCTCCTGGAATACACAGACTCAAGCTAT
N V R G L T H P I R L L L E Y T D S S Y
130 140 150 160 170 180
GAGGAGAAGAGATACGCCATGGGCGACGCTCCCGACTATGACAGAAGCCAGTGGCTGAAT
E E K R Y A M G D A P D Y D R S Q W L N
190 200 210 220 230 240
GAGAAGTTCAAACTGGGCCTGGACTTCCCCAATCTGCCCTACTTAATTGATGGATCGCGC
E K F K L G L D F P N L P Y L I D G S R
250 260 270 280 290 300
AAGATTACCCAGAGCAATGCCATAATGCGCTACCTTGCCCGCAAGCACCACCTGTGTGGA
K I T Q S N A I M R Y L A R K H H L C G
310 320 330 340 350 360
GAGACAGAGGAGGAGCGGATTCGTGCAGACATTGTGGAGAACCAGGTCATGGACAACCGC
E T E E E R I R A D I V E N Q V M D N R
370 380 390 400 410 420
ATGCAGCTCATCATGCTTTGTTACAACCCCGACTTTGAGAAGCAGAAGCCAGAGTTCTTG
M Q L I M L C Y N P D F E K Q K P E F L
430 440 450 460 470 480
AAGACCATCCCTGAGAAGATGAAGCTCTACTCTGAGTTCCTGGGCAAGCGACCATGGTTT
K T I P E K M K L Y S E F L G K R P W F
490 500 510 520 530 540
GCAGGGGACAAGGTCACCTATGTGGATTTCCTTGCTTATGACATTCTTGACCAGTACCAC
A G D K V T Y V D F L A Y D I L D Q Y H
550 560 570 580 S90 600
ATTTTTGAGCCCAAGTGCCTGGACGCCTTCCCAAACCTGAAGGACTTCCTGGCCCGCTTC
I F E P K C L D A F P N L K D F L A R F
610 620 630 640 650 660
GAGGGCCTGAAGAAGATCTCTGCCTACATGAAGAGCAGCCGCTACCTCTCAACACCTATA
E G L K K I S A Y M K S S R Y L S T P
670 680 690 700 710 720
TTTTCGAAGTTGGCCCAATGGAGTAACAAGTAGGCCCTTGCTACACTGGCACTCACAGAG
F S K L A Q W S N K *
730 740 750 760 770 780
AGGACCTGTCCACATTGGATCCTGCAGGCACCCTGGCCTTCTGCACTGTGGTTCTCTCTC
790 800 810 820 830 840
CTTCCTGCTCCCTTCTCCAGCTTTGTCAGCCCCATCTCCTCAACCTCACCCCAGTCATGC
850 860 870 880 890 900
CCACATAGTCTTCATTCTCCCCACTTTCTTTCATAGTGGTCCCCTTCTTTATTGACACCT
910 920 930 940 950 960
TAACACAACCTCACAGTCCTTTTCTGTGATTTGAGGTCTGCCCTGAACTCAGTCTCCCTA
970 980 990 1000 1010 1020
GACTTACCCCAAATGTAACACTGTCTCAGTGCCAGCCTGTTCCTGGTGGGGGAGCTGCCC
- 27 - ~ 3 3 3i~ 2 g
1030 1040 1050 1060 1070
CAGGCCTGTCTCATCTTTAATAAAGCCTGAAACACAA~ 4A~~A~- A
Example IB
Isolation of Partial GST cDNA Clone Ybl87
By essentially the same procedure, cDNA clone Ybl87 was obtained.
Ybl87 is a partial cDNA clone lacking 96 nucleotides at the 5' end
of the coding sequence. The nucleotide and the corresponding amino
acid sequences for the missing 96 nucleotides and Ybl87 are given
below:
1 0 30
ATG CCT ATG ACA CTG GGT TAC TGG GAC ATC CGT GGG CTG GCT CAC
Met Pro Met Thr Leu Gly Tyr Trp Asp Ile Arg Gly Leu Ala His
7 0 90
GCC ATT CGC CTG TTC CTG GAG TAT ACA GAC ACA AGC TAT GAG GAC
Ala Ile Arg Leu Phe Leu Glu Tyr Thr Asp Thr Ser Tyr Glu Asp
1 10 13 0
AAG AAG TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG
Lys Lys Tyr Ser Met Gly Asp Ala Pro Asp Tyr Asp Arg Ser Gln
150 1 70
TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG
Trp Leu Ser Glu Lys Phe Lys Leu Gly Leu Asp Phe Pro Asn Leu
19 0 210
CCC TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC
Pro Tyr Leu Ile Asp Gly Ser His Lys Ile Thr Gln Ser Asn Ala
2 30 25 0 . 270
ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA
Ile Leu Arg Tyr Leu Gly Arg Lys His Asn Leu Cys Gly Glu Thr
2 90 31 0
GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC CAG GCT ATG
Glu Glu Glu Arg Ile Arg Val Asp Val Leu Glu Asn Gln Ala Met
330 3 50
GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT
Asp Thr Arg Leu Gln Leu Ala Met Val Cys Tyr Ser Pro Asp Phe
37 0 390
GAG AGA AAG AAG CCA GAG TAC TTA GAG GGT CTC CCT GAG AAG ATG
Glu Arg Lys Lys Pro Glu Tyr Leu Glu Gly Leu Pro Glu Lys Met
1339~2~
- 28 -
4 10 43 0 450
AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG
Lys Leu Tyr Ser Glu Phe Leu Gly Lys Gln Pro Trp Phe Ala Gly
4 70 49 0
AAC AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTC CTT GAT
Asn Lys Ile Thr Tyr Val Asp Phe Leu Val Tyr Asp Val Leu Asp
510 5 30
CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC
Gln His Arg Ile Phe Glu Pro Lys Cys Leu Asp Ala Phe Pro Asn
55 0 570
CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT
Leu Lys Asp Phe Val Ala Arg Phe Glu Gly Leu Lys Lys Ile Ser
5 90 61 0 630
GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA
Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro Ile Phe Ala
6 50
AAG ATG GCC TTT TGG AAC CCA AAG TAG
Lys Met Ala Phe Trp Asn Pro Lys End
Example IC
Entire Clone Corresponding to Ybl87
The missing nucleotides are added to Yb187 either in vivo by
recombination or in vitro by ligating appropriate restriction
fragments. These methods are illustrated in Figures 4B and 4C,
respectively.
Recombination can be effected by introducing into E. coli strain
SR43 (ATCC accession number 67217 deposited September 22, 1986) the
genomic DNA clone carried on a colE1 plasmid (eg pUC8 or pBR322) and
the cDNA clone carried on an inc P1 pla~mid (eg pRK290). Strain SR34
bears polA1 supF183 mutations fTacon, W. & Sherratt, D., Mol. Gen.
Genet., 147: 331-335 (1976)] such that replication of colE1 plasmids
is prohibited at the restrictive temperature (42 degrees). Shifting
of the SR34 bacteria containing the two plasmids from permissive
temperature ~28 degrees) to 42 degrees with maintenance of the
selection for the antibiotic resistance carried by the colE1 plasmid
(eg Ap resistance of pBR322) allows selection of bacteria containing
cointegrated forms of the two plasmids. Such cointegrate plasmids
- 13;~9~23
-- 29 --
.
are formed by recombination at regions of DNA homology. Isolation of
cointegrate plasmids allows cloning of the full length cDNA sequence
and upstream genomic DNA as a single PstI or BamHI fragment. Bal31
resection of upstream DNA followed by BamHI linker addition allows
isolation of a clone containing the full length coding region
(confirmed by DNA sequence analysis).
The full length coding region is cloned as a BamHI fragment into
pCIB710 as described in Example IA for transfer to plant cells. This
same fragment is cloned into pDR540 as described in Example IV below
for expression in E. coli.
Alternatively, the entire clone corresponding to Ybl87 can be
constructed by ligating appropriate restriction fragments of cDNA
and genomic clones together (Figure I). Plasmids pUC19 containing
the 5' end of the genomic clone inserted as a PstI-HindIII fragment
contains approximately the first 400 bases of the coding sequence.
The 3' end of the cDNA coding sequence can be isolated as a 630 base
HinfI fragment from the partial cDNA clone in BR325. The pUC plasmid
cut at its unique HinfI site and the HinfI fragment containing the
3' end of the gene are ligated together, recreating the complete
coding sequence of the GST gene. The complete gene is isolated as a
BglI-PstI fragment. Bal31 resection of upstream DNA followed by
BamHI linker addition allows isolation of the full length coding
region without upstream sequences. This BamHI fragment is inserted
into pCIB710 and into pDR540 as described above.
Example ID
Hvbrid Clones: Hybrid clones derived from coding sequences for
different genes are constructed in essentially the same manner as
described above for combining the partial cDNA clone Ybl87 and the
missing nucleotides (Example IC) using heterologous genes as the
sources of the two DNA fragments.
- 30 - i3.~ ~23
Example IE
Isolation of the GST-cDNA clone GlYb
A cDNA library in the phage expression vector ~ gtll [Young, R.A.
and Davis, R.W., Proc. Natl. Acad. Sci. USA, 80: 1194 (1983)] is
constructed from poly(A) RNA isolated from rat brains, according to
methods previously described (see Example IA). The cDNA is isolated
by antibody screening procedures using antibodies raised against rat
brain GST. The isolation of RNA and the construction and isolation
of the corresponding cDNA are accomplished by methods described in
the prior art, such as those in Young and Davis [Young, R.A. and
Davis, R.W., Science, 222; 778-782 (1983)].
The nucleotide and the corresponding amino acid sequences of the
resulting cDNA clone GlYb are given below:
A GAC CCC AGC ACC ATG CCC ATG ACA CTG GGT TAC TGG GAC ATC
Met Pro Met Thr Leu Gly Tyr Trp Asp Ile
5 0 70
CGT GGG CTA GCG CAT GCC ATC CGC CTG CTC CTG GAA TAC ACA GAC
Arg Gly Leu Ala His Ala Ile Arg Leu Leu Leu Glu Tyr Thr Asp
11 0 130
TCG AGC TAT GAG GAG AAG AGA TAC ACC ATG GGA GAC GCT CCC GAC
Ser Ser Tyr Glu Glu Lys Arg Tyr Thr Met Gly Asp Ala Pro Asp
1 50 17 0
TTT GAC AGA AGC CAG TGG CTG AAT GAG AAG TTC AAA CTG GGC CTG
Phe Asp Arg Ser Gln Trp Leu Asn Glu Lys Phe Lys Leu Gly Leu
190 2 10
GAC TTC CCC AAT CTG CCC TAC TTA ATT GAT GGA TCA CAC A-AG ATC
Asp Phe Pro Asn Leu Pro Tyr Leu Ile Asp Gly Ser His Lys Ile
23 0 250 2
ACC CAG AGC AAT GCC ATC CTG CGC TAT CTT GGC CGC AAG CAC AAC
Thr Gln Ser Asn Ala Ile Leu Arg Tyr Leu Gly Arg Lys His Asn
29 0 310
CTG TGT GGG GAG ACA GAA GAG GAG AGG ATT CGT GTG GAC ATT CTG
Leu Cys Gly Glu Thr Glu Glu Glu Arg Ile Arg Val Asp Ile Leu
3 30 35 0
GAG AAT CAG CTC ATG GAC AAC CGC ATG GT& CTG GCG AGA CTT TGC
Glu Asn Gln Leu Met Asp Asn Arg Met Val Leu Ala Arg Leu Cys
- 31 - 1~ 39 ~2 ~9
370 3 90
TAT AAC CCT GAC TTT GAG AAG CTG AAG CCA GGG TAC CTG GAG CAA
Tyr Asn Pro Asp Phe Glu Lys Leu Lys Pro Gly Tyr Leu Glu Gln
41 0 430 4
CTG CCT GGA ATG ATG CGG CTT TAC TCC GAG TTC CTG GGC AAG CGG
Leu Pro Gly Met Met Arg Leu Tyr Ser Glu Phe Leu Gly Lys Arg
4 7 0 490
CCA TGG TTT GCA GGG GAC AAG ATC ACC TTT GTG GAT TTC ATT GCT
Pro Trp Phe Ala Gly Asp Lys Ile Thr Phe Val Asp Phe Ile Ala
5 10 53 0
TAC GAT GTT CTT GAG AGG AAC CAA GTG TTT GAG GCC ACG TGC CTG
Tyr Asp Val Leu Glu Arg Asn Gln Val Phe Glu Ala Thr Cys Leu
550 5 70
GAC GCG TTC CCA AAC CTG AAG GAT TTC ATA GCG CGC TTT GAG GGC
Asp Ala Phe Pro Asn Leu Lys Asp Phe Ile Ala Arg Phe Glu Gly
59 0 610 6
CTG AAG AAG ATC TCC GAC TAC ATG AAG TCC AGC CGC TTC CTC CCA
Leu Lys Lys Ile Ser Asp Tyr Met Lys Ser Ser Arg Phe Leu. Pro
65 0 670
AGA CCT CTG TTC ACA AAG ATG GCT ATT TGG GGC AGC AAG TAG GAC
Arg Pro Leu Phe Thr Lys Met Ala Ile Trp Gly Ser Lys End Asp
6 90 71 0
CCT GAC AGG TGG GCT TTA GGA GAA AGA TAC CAA ATC TCC TGG GTT
Pro Asp Arg Trp Ala Leu Gly Glu Arg Tyr Gln Ile Ser Trp Val
7 30 7 50
TGC CAA GAG CCC TAA GGA GCG GGC AGG ATT CCT GAG CCC CAG AGC
Cys Gln Glu Pro End Gly Ala Gly Arg Ile Pro Glu Pro Gln Ser
77 0 790 8
CAT GTT TTC TTC CTT CCT TCC ATT CCA GTC CCC AAG CCT TAC CAG
His Val Phe Phe Leu Pro Ser Ile Pro Val Pro Lys Pro Tyr Gln
83 0 850
CTC TCA TTT TTT GGT CAT CM ATT CCT GCC AAA CAC AGG CTC TTA
Leu Ser Phe Phe Gly His Gln Ile Pro Ala Lys His Arg Leu Leu
8 70 89 0
AAA GCC CTA GCA ACT CCT TTC CAT TAG CAA AAT AGC CTT CTA AAG
Lys Ala Leu Ala Thr Pro Phe His End Gln Asn Ser Leu Leu Lys
910 9 30
TTA AAG TGC CCC GCC CCC ACC CCT CGA GCT CAT GTG ATT GGA TAG
Leu Lys Cys Pro Ala Pro Thr Pro Arg Ala His ~al Ile Gly Lnd
95 0 970 9
TTG GCT CCC AAC ATG TGA TTA TTT TGG GCA GGT CAG GCT CCC CGG
Leu Ala Pro Asn Met End Leu Phe Trp Ala Gly Gln Ala Pro Arg
101 0 1 030
CAG ATG GGG TCT ATC TGG AGA CAG TAG ATT GCT AGC AGC TTT GAC
Gln Met Gly Ser Ile Trp Arg Gln End Ile Ala Ser Ser Phe Asp
- 13;39~2~3
- 32 -
10 50 107 0
CAC CGT AGC CAA GCC CCT CTT CTT GCT GTT TCC CGA GAC TAG CTA
His Arg Ser Gln Ala Pro Leu Leu Ala Val Ser Arg Asp End Leu
1 090 11 10
TGA GCA AGG TGT GCT GTG TCC CCA GCA CTT GTC ACT GCC TCT GTA
End Ala Arg Cys Ala Val Ser Pro Ala Leu Val Thr Ala Ser Val
113 0 1 150 11
ACC CGC TCC TAC CGC TCT TTC TTC CTG CTG CTG TGA GCT GTA CCT
Thr Arg Ser Tyr Arg Ser Phe Phe Leu Leu Leu End Ala Val Pro
119 0 1 210
CCT GAC CAC AAA CCA GAA TAA ATC ATT CTC CCC TTA AAA AAA AAA
Pro Asp His Lys Pro Glu End Ile Ile Leu Pro Leu Lys Lys Lys
AAA AAA AAA A
Lys Lys Lys
Example II
Construction of Plasmid pCIB710: The plasmid pLW111, ATCC No. 40235,
consists of the three smaller EcoRI fragments of the BJI strain of
Cauliflower Mosaic virus (CaMV) [Franck et al., Cell, 21: 285-294
(1980)] cloned into pMB9. pLWlll is digested with BglII and a 1149
bp fragment (base pairs #6494-7643) isolated. This fragment is
ligated into the BamHI site of pUC19. This restriction fragment
codes for both the promoter for the CaMV 35S RNA and the polyA
addition signal (i.e. the terminator) for that transcript.
In Vitro Mutagenesis: A unique BamHI site is inserted between this
promoter and terminator via in vitro mutagenesis. A 36-base oligo-
nucleotide is synthesiszed which is identical to the CaMV sequence
in that region except that a BamHI restriction site is inserted at
base pair #7464 in the sequence.
The 1149 bp BglII fragment from above is cloned into M13mpl9 and
single-stranded phage DNA isolated. This single-stranded DNA is
annealed with the synthetic oligonucleotide, a new strand is
synthesized using the oligonucleotide as a primer; and the DNA
transfected into E. coli strain JM101 [Zoller & Smith, DNA 3:
479-488 ~1980)]. M13 phage having the BamHI site inserted are
isolated as described in Zoller & Smith, supra.
~33~i23
Selection of Desired Mutant Phage: The 36-base oligonucleotide is
labeled by kinasing with 32P-ATP. A set of the transfected M13 phage
plaques is localized on a nitrocellulose filter. This filter is
hybridized with the labelled 36-mer. The filter is washed at
increasing temperatures. The labeled 36-mer bound to mutated phage
is stable at higher wash temperature. One of these phages stable at
higher temperature is isolated and sequenced to confirm the presence
of the BamHI site.
Construction of pCIB710: Double-stranded DNA isolated from this
phage is digested with HindIII and EcoRI. pUC1~ is cleaved with
HindIII and EcoRI. These two digested DNA's are ligated. Trans-
formants are selected by ampicillin resistance. One transformant
frDm this ligation is pCIB710. This plasmid is shown in Figure 2.
Example III:
Construction of Plasmids pCIB11, pCIB12, pCIB13 and pCIB14
1. The plasmid pGTR200 is digested with HaeII, PstI and PvuII and
the 678 bp HaeII/PstI fragment containing the GST coding sequence is
isolated from an agarose gel.
2. This HaeII/PstI fragment is rendered blunt-ended by treatment
with T4 DNA polymerase, and BamHI linkers (dCGGATCCG-New England
Biolabs) are ligated on to the blunt ends using T4 DNA ligase.
3. The plasmid pCIB710 is cut with BamHI and trated with calf
intestinal alkaline phosphatase (Boehringer Mannheim).
4. The BamHI-linkered GST fragment from above is ligated into
BamHI-digested pCIB710, the ligation mixture transformed into
E. coli strain HB101 and the desired transformants selected by
resistance to ampicillin. Transformants bearing the GST coding
1339~)2~
~ 34 -
qequences in the appropriate orientation for transcription from the
CeMV promoter (pCIB12) a8 well as in the opposite orientation
(pCIB11) are characterized.
5. The plasmid pCIB10 (see Example V) is digested with XbaI and
EcoRI.
6. The plasmid pCIB12 is digested with XbaI and EcoRI and the
smaller fragment isolated from an agarose gel.
1 0
7. The isolated XbaI/EcoRI fragment, which bears the chimeric gene,
is ligsted into the digested pCIB10, the ligation tr~nsformed into
E. coli HB 101 and transformants selected by kanamycin resistance.
These transformants, which besr the CST coding sequence in the
appropriate orientation for transcription from the CaMV promoter,
are designated pCIB14 (see Flgure 3).
8. The plasmid pCIB11 is similarly manipulated to construct pCIB13,
a plasmid having the CST coding sequence in an orientation opposlte
that appropriate for transcription from the CaMV promoter.
Introduction of pCIB13 and pCIB14 into Agrobacterium:
Purified plasmid DNA of pCIB13 or pCIB14 was introduced by trans-
formation into ARrobacterium tumefaciens A136 [Watson et al.,
J. Bacteriol., 123: 255-264 (1975)] bearing pCIB542 (see Example VII
below). Transformants were ~elected on kanamycin (50 ~g/ml) and
spectinomycin (25 ~g/ml).
ExAmple IV
Insertion of ~b200 GST Gene in trp-lac Expression Vector:
Ihe plasmid pCIB12 was digested with BamHI and the 708 bp Bam-
linkered GST gene fragment isolated. The plasmid pDR540 ~Pharmacia
P-L Biochemicals), a trp-lac expression vector [Russel, D.R. and
Bennett, G.N., Gene, 20: 231-243 (1982)1 was digested with BamHI and
the GST fragment ligated in. The resulting recombinant plasmid was
_ 35 _ 13 3 9 ~ 29
transformed into E. coli strain JM103. Cultures of the resulting
strain were induced at the desired time by addition of 1.0 mM
isopropyl beta-D-thiogalactoside ( IPTG).
After induction with IPTG and expression of the GST Yb200 gene, the
bacterial host cells are pelleted. The pellets formed are resuspen-
ded in 59 ml 0.2 M Tris HCl, pH 8.0, and 1 mM EDTA. To this sus-
pension 0.5 ml 100 mM phenylmethylsulfonylfluoride (PMSF) in ethanol
and 5 ml 10 mg/ml lysozyme in buffer was added. The suspension was
incubated for approximately 15 minutes at 37~C until the bacterial
cells were lysed. The lysed bacterial cell suspension was pelleted
and the supernatant collected. The supernatant was dialy~ed against
25 mM Tris HCl, pH 8.0 with 1/100 volume of PMS~.
The dialyzed supernatant was assayed for GST enzymatic activity.
Following this, the dialyzed supernatant was loaded onto the
S-hexylglutathion-agarose affinity column at a flow rate of
0.5 ml/minute. The column was washed with 25 mM Tris HCl, 0.2 M KCl
until the absorbance at 280 nm was less than 0.005. After all
unbound material had been washed from the column the bound material
was eluted with 25 mM Tris HCl, 0.2 M KCl (pH 8.0) containing 5 mM
S-hexylglutathione, and 2.5 mM glutathione. Eluted fractions were
monitored at an absorbance of 280 nm. Those fractions which were
believed to containing the purified enzyme were individually
dialyzed against 0.1 M NaPO~ (pH 6.5) containing PEG to concentrate
the fractions, and then in the same buffer without PEG.
The volumes in each of the dialyzed fractions were recorded. For
each fraction, the degree of concentration and amount of sample was
calculated and then diluted such that the same proportion relative
to the starting fraction was present in each fraction. Each fraction
was assayed for enzymatic activity.
GST activity was assayed using l-chloro-2,4-dinitrobenzene (ClDNB)
as a substrate. For each reaction, 1 mM ClDNB (20.2 mg/ml in
ethanol) was added to 5 mM reduced glutathione (30.8 mg/ml in
13~29
- 36 -
buffer, O.lM sodium phosphate, pH 6.5) and GST enzyme. ClCNB
and reduced glutathione were prepared fresh dail~. The
reactlon had a flnal total volume of 1 ml. The reaction was
run at room temperature and inltlated by the addition of
ClDNB. The reaction was monltored on Gilford
spectrophotometer at 340 nm. A typical reaction contained 10
to 50 units of GST, where 1 unit is equivalent to 1 nanomole
substrate converted per minute per ml.
The proteln concentratlon of the enzymatically
active fractions was determlned in the starting material
(Blorad, BSA standard). Each of the enzymatically active
fractions was analyzed on a 15 percent Laemmli gel using
dilutions of standard enzyme (1.0 ~g, 0.3 ~g, and 0.1 ~g) as
~uantitative standards. Purifled enzyme was detected by
immunoblottlng using specific antibody.
Example V
Construction of pCIB10 and pCIBlOa:
The constructlon of plasmld pCIB10 and pCIBlOa ls
described below (see Figures 7a to 7g).
Construction of PCIB10
15. A T-DNA fragment containing the left border from pTiT37
is lsolated from pBR325 (EcoRI29) [Yadav et al., Proc. Nat'l
Acad. Sci USA, 79 6322 ~1982)]. pBR325 (EcoRI29) ls cut wlth
EcoRI and the 1.1 kb fragment linkered with HindIII linkers
(New England Biolabs).
16. The plasmid pBR322 (Bolivar et al., Gene, 2:75) is cut
with HlndIII.
'
1339~2~3
17. The left-border containing fragment described in step 15 is
ligated into the HindIII digest of pBR322.
18. The left-~order containing pBR322 plasmid of step 17 is digested
with ClaI and HindIII and the 1.1 kb HindIII/ClaI fragment isolated
[Hepburn et al., J. Mol. Appl. Genet., 2: 211 (1983)].
19. The plasmid pUC18 [Norrander et al., Gene, 26: 101 (1983)] is
cut with HindIII and EcoRI; the 60 bp polylinker is end-labeled
using T4 polynucleotide kinase and gamma 32P-dATP and isolated from
an acrylamide gel.
20. The plasmid pBR322 is cut with EcoRI and ClaI and the large
fragment isolated.
21. The 60 bp HindIII/EcoRI polylinker and the 1.1 kb HIndIII/ClaI
fragment of EcoRI29 is ligated into pBR322 cut with ClaI and EcoRI,
constructing pCIB5 (steps 15 to 21 see Figure 7a).
22. A chimeric gene conferring kanamycin resistance (nos-neo-nos) is
taken from Bin 6 [Bevan, Nucleic Acids Res., 12: 8711 (1984)] as a
SalI/EcoRI fragment.
23. The plasmid pUC18 is cut with EcoRI and SalI.
24. The SalI/EcoRI fragment containing the chimeric gene from
step 22 is ligated iinto the pUC18 cut with EcoRI and SalI.
25. The BamHI recognition site in the termination sequence of this
chimeric gene is destroyed by cutting with BamHI, filling in using
T4 DNA polymerase, and ligating.
26. The resulting plasmid is cut wiht SstII (Bethesda Research
Laboratories) and HindIII.
- 38 - 13 3g ~ 23
27. A fragment containing the 5' part of the nos promoter and the
right border of pTiT37 is isolated by cutting pBR325 (Hind23) with
HindIII and SstII and isolating the 1.0 kb fragment.
28. This 1.0 kb HindIII/SstII fragment is ligated into the restric-
ted pUC18 of step 26, constructing pCIB4 (steps 22 to 28 see
Figure 7b).
29. pCIB5, containing the left T-DNA border, is cut with AatII,
rendered blunt-ended by treatment using T4 DNA polymerase, and then
cut with EcoRI.
30. pCIB4 is cut with HindIII, rendered blunt by treatment using
Klenow fragment of E. coli DNA polymerase and cut with EcoRI.
-
31. The restricted pCIB5 of step 29 is ligated with the restrictedpCIB4 (step 30), constructing pCIB2, a colEl replicon containing
left and right T-DNA borders flanking a chimeric kanamycin-resi-
stance gene and a polylinker (steps 29 to 31 see Figure 7c).
32. The plasmid pRZ102 ~Jorgensen et al., Mol. Gen. Genet., 177: 65
(1979)] is digested with BamHI and filled in using Klenow.
33. An AluI partial digest of plasmid pA03 [Oka, J. Mol. Biol.,
174: 217 (1981)] is made.
34. The AluI digest is ligated into the restricted pRZ102 of step 32
above, selecting the desired transformants by resistance to kana-
mycin.
35. The resulting plasmid has the coding sequence of Tn903 present
on a 1.05 kb BamHI fragment which is isolated after BamHI digestion.
This fragment is treated with Klenow DNA polymerase.
~ 39 ~ 1 3 39 ~ 29
36. The plasmid pRK252, a derivative of the broad host range plasmid
RK2, is available from Dr. Don Helinski of the University of
California, San Diego. This plasmid lacks the BglII site present in
the parent plasmid pRK290 [Ditta et al,, Proc. Nat'l Acad. Sci. USA,
77: 7347 (1980)]. pRK252 is digested with SmaI and SalI, filled in
using Klenow, and the large fragment resulting from this digest
isolated.
37. The Tn903-containing fragment, isolated in step 35, is ligated
into the large fragment from pRK252, constructing pRK252Km (steps 32
to 37 see Figure 7d~.
38. The plasmid pRK252Km is cut with EcoRI, blunt-ended using
Klenow, and linkered with BglII linkers (New England Biolabs).
39. The plasmid pCIB2 is cut with EcoRV and the smaller fragment,
containing the right border and the nos-neo-nos, isolated. This
fragment is filled-in using Klenow polymerase and linkered with
BglII linkers (New England Biolabs).
40. The BglII fragment resulting from step 39 is ligated with the
linkered pRK252Km of step 38, producing pCIB10 (steps 38 to 40 see
Figure 7e).
Construction of PCIBlOa:
41. The plasmid pRZ102 [Jorgensen, et al., (1979)] is digested with
BamHI and filled in using Klenow.
42. An AluI partial digest of plasmid pAO3 [Oka, et al., (1978)]
is made.
43. The AluI digest is ligated into the restricted pRZ102, from
step 32, selecting the desired transformants by resistance to
kanamvcin.
1339~29
- 40 -
44. The resulting plasmid has the coding sequence of Tn903 present
on a 1.05 kb BamHI fragment; this fragment is isolated after BamHI
digestion and filling-in with Klenow.
45. The plasmid pRK290, a derivative of the broad host range
plasmid RK2, is available from Dr. Don Helinski of the University of
California, San Diego. pRK290 is digested with SmaI and SalI, filled
in using Klenow, and the large fragment resulting from this digest
isolated.
46. The Tn903 containing fragment, isolated in step 35, is ligated
into the large fragment from pRK290, constructing pRK290Km.
47. The plasmid of step 46 is digested with BglII, filled in using
Klenow and ligated, destroying its BglII site, to construct pRK290Km
(steps 41 to 47 see Figure 7f).
48. The plasmid pRK290Km is cut with EcoRI, blunt-ended using
Klenow, and linkered with BglII linkers (New England Biolabs).
49. The plasmid pCIB2 is cut with EcoRV and is linkered with BglII
linkers (New England Biolabs).
50. The BglII-linkered pCIB2 of step 39 is ligated into vector of
step 47 constructing pCIBlOa (steps 48 to 50 see Figure 7g).
Steps 41 to 50 can be repeated substituting the plasmid pRK290 for
pRK252.
Example VI
Construction of pCIB23 and pCIB24, Vectors Targeting the GST Enzyme
for the Chloroplast of Transformed Plants:
Plasmid pSRS2.1, which contains the 5' sequence of the soybean small
subunit (SSU) of ribulose bis-phosphate carboxylase (RuBPC) [Berry-
Lowe et al., J. Mol. Appl. Genet., 1: 483-498 (1982)] is obtained
- 41 - 1 ~ 39 6 29
from Dr. Richard Meagher of the Department of Genetics, The Uni-
versity of Georgia, Athens, Georgia 30602. This plasmid is digested
with EcoRI. The 2.1 kb fragment containing the soybean SSU 5' region
is isolated from an agarose gel. The 2.1 kb EcoRI fragment is
digested with DdeI. A 471 bp DdeI fragment is isolated. This
fragment contains the transit peptide and a portion of the second
exon.
The 471 bp DdeI fragment is treated with Klenow (New England Biolabs
DNA polymerase). A kinased BglII linker [d(CAGATCTG) New England
Biolabs] is ligated onto this fragment. This BglII fragment is
digested with TaqI. The resulting TaqI fragments are treated with
Klenow (Bethesda Research Labs DNA polymerase). The resulting blunt
fragments are ligated onto kinased BamHI linkers ~d(CGCGGATCCGCG)
New England Biolabs] and purified. These BamHI fragments are
digested with BglII and BamHI. A BamHI/BglII fragment of approxi-
mately 400 bp is purified; this fragment contains the SSU 5' region.
pCIB710 is cut with BamHI and treated with calf intestinal alkaline
phosphatase. The 400 bp BamHI/BglII fragment is ligated into this
pCIB710. This ligation is transformed into E. coli HB101 and trans-
formants selected on ampicillin.
Transformants bearing the BamHI/BglII fragment in both orientations
are found. pSCR2 has the 5' region of the transit peptide adjacent
to the 35S promoter. pSCR1 has the BamHI/BglII fragment in the
opposite orientation.
pCIBl2 is digested with BamHI and the 708 bp BamHI fragment bearing
the GST gene is isolated. The plasmid pSCR2 is cut with BamHI and
treated with calf intestinal alkaline phosphatase. The 708 bp BamHI
fragment from pCIBl2 is ligated into the BamHI treated pSCR2. The
ligation is transformed into HB101 and transformants are selected on
ampicillin.
1~396~9
- 42 -
Transformants bearing the GST gene in both orientations to the 5'
regulatory regions are found. The clone pCIB22 has the GST gene in
appropriate orientation for transcription from the CaMV promoter.
The clone pCIB21 has the GST gene in the opposite orientation.
pCIB22 is digested with XbaI and EcoRI. The fragment carrying the
chimeric gene is purified from a gel.
The plasmid pCIB10 is digested with XbaI and EcoRI. The XbaI/EcoRI
fragment carrying the chimeric gene is ligated into the digested
pCIB10 and transformants are selected by kanamycin resistance. The
resulting plasmid, pCIB24, is a broad host range plasmid which bears
the chimeric GST gene attached to a chloroplast transit peptide
sequence.
Using similar manipulations and beginning with clone pCIB21 in place
of pCIB22, a plasmid pCIB23 is constructed. This plasmid bears the
GST gene in opposite orientation to the GST gene of-pCIB24.-
These plasmids are introduced into Agrobacterium strains in a mannersimilar to pCIB13 and pCIB14 above.
Example VII
Construction of pCIB542, An Agropine Vir Helper Plasmid Bearing a
Spectinomycin Drug Resistance Gene in the Place of the T-DNA: -
The Ti plasmid, pTiBo542 [Sciaky, D., Montoya, A.L. & Chilton, M-D,
Plasmid, 1: 238-253 (1978)], is of interest because Agrobacteria
bearing this Ti plasmid are able to infect the agronomically
important legumes, alfalfa and soybean. [Hood, E.E., et al. 7
Bio/Technology, 2: 702-708 (1984)]. The construction of a pTiBo542
derivative deleted on the T-DNA has been described [Hood, Eliza-
beth E., (1985) Ph.D. thesis; Washington University,
St. Louis, Mo.~. In this construction named EHA101, the T-DNA was
replaced by the kanamycin drug resistance gene. The parent of
EHA101, A281~ is on deposit at the ATCC, designated ATCC No. 53487.
13~ b29
- 43 -
A derivative of EHA101 having the kanamycin drug resistance gene
replaced by a spectinomycin drug resistance gene was constructed.
The plasmid p pi delta 307 [E. Hood, Washington University, thesis
(1985)3 has a 1.7 kb region of homology to the left side of Bam a of
pTiBo542 ~Hood, et al. (1984)] and an 8 kb region of homology to the
right side of Bam2a of pTiBoS42, separated by a unique EcoRI site.
The plasmid pMON30, ATCC No. 67113, bears the spectinomycin/strepto-
mycin drug resistance gene (spc/str) from Tn7 [Hollingshead, S. and
Vapnek, D., Plasmid, 13: 17-30 (1985)]. pMON30 was digested with
EcoRI, the 5.5 kb fragment containing the spc/str gene isolated from
an agarose gel, and ligated into EcoRI-restricted plasmid p pi
delta 307. The desired recombinant is selected as a spectinomycin
resistant (50 ~g/ml) tetracycline resistant (10 ~g/ml) trans-
formant. This plasmid was transformed into Agrobacterium A136/EHA101
and selected by its streptomycin-resistant, tetracycline-resistant,
kanamycin-resistant phenotype. Homogenotes [Matzke, A.J.M. & Chil-
ton, M-D, J. Mol. Appl. Genet., 1: 39-49 (1981)] of EHA101 and the
spectinomycin plasmid were selected after introduction of the
eviction plasmid R751-pMG2 [Jacoby, G. et al., J. Bacteriol., 127:
1278-1285 (1976)] and selection on gentamycin (50 ~g/ml) and
spectinomycin. The desired homogenote had a gentamycin-resistant,
spectinomycin-resistant, tetracycline-sensitive and kanamycine-
sensitive phenotype. The structure of the resulting plasmid was
confirmed by probing Southern blots.
Example VIII
Testing Plants for Atrazine Tolerance:
Regenerated tobacco plants bearing the GST gene constructions pCIB14
are tolerant to atrazine as determinerd by several measures inclu-
ding:
(1) fluorescence induction; and
(2) ability of seedlings to grow on levels of atrazine toxic to
control or wild-type plants.
~3~9~2~
Fluoresence induction assays are an indication of the status of the
photochemical apparatus in the plant. [Voss et al., Weed Science,
32: 675-680 (1984)]. In such assays, leaf tissue is irradiated with
light of characteristic wavelength and the resulting fluorescence at
a second wavelength is recorded. Figure 5 illustrates the fluor-
escence induction pattern typical of an excised tobacco (untrans-
formed) leaf infiltrated with buffer solution by uptake through the
cut petiole (leaf) (lower curve). One sees a sharp rise in fluor-
escence when the light is turned on, then a peak followed by a
smooth decay of the signal over time. This pattern indicates that
the chloroplasts are being excited by the incident light and
fluoresce at a wavelength characteristic of the system. At this
point energy is channeled out of the photosystem as electrons flow
through the electron transport pathway of photosystem II and I
-- this is indicated by the smooth decay curve of the fluorescence
signal. If, however, the leaf is infiltrated with a solution of
M atrazine, one sees a pattern such as the upper curve in
Figure 5. Here, the light energy is absorbed, the chloroplasts are
excited and fluoresce, but no energy channeling occurs because
electron flow is blocked at the quinone binding step of photo-
system II. It is as if the photosystem were frozen in the excited
state. Thus, one sees the sharp rise in fluorescence followed by no
decay at all.
When such measurements were carried out on genetically engineered
tobacco plants according to this invention in the presence of 10 M
atrazine, all control plants show fluorescence induction patterns
identical to the non-transgenic tobacco. When measurements were done
on the experimental plants, they fell into three classes according
to their fluorescence induction patterns (Figure 6).
Some of the transgenic plants showed no evidence of atrazine
detoxification (the top curve), some showed modest (middle curve)
detoxification, and some showed significant (bottom curve) atrazine
- 45 - ~ ~ 3~ ~ 2~
detoxification as evidenced by normal electron channeling through
the photosystem. Of 27 plants characterized in this manner, 5 showed
significant evidence of ability to detoxify atrazine.
Example IX
Agrobacterium Infection of Plant Material:
The different genotypes of Agrobacterium tumefaciens were grown on
AB minimal medium [Watson, B. et al., J. Bacteriol., 123: 255-264
(1975)] plus mannitol for 48 hours at 28~C. Bacteria were pelleted,
resuspended in MSBN medium at a two-fold dilution, and held for
three hours at 25~C. [MSBN medium was prepared with a prepackaged
mix (KC Biologicals) to provide the major and minor salts at
concentrations according to Murashige and Skoog with the following
additions (in final concentrations): benzyladenine (1 mg/l);
naphthylacetic acid (0.1 mg/l; myo-inositol (1 mg/l); nicotinic acid
(1 mg/l); pyridoxine (1 mg/l); thiamine HCl (10 mg); and sucrose
(30 mg/l)]. The pH was adjusted to 5.7 to 5.8. Leaf discs from
in vitro cultured Nicotiana tabacum cv. petite Havana SR1 plants
were floated on the bacterial suspension for 10 minutes in a
modification of the method of Horsch, R. et al., Science, 227:
1229-1231 (1985). They were then transferred to filter paper on MSBN
without antibiotics. At 48 hours the leaf discs were dipped in
liquid MSBN containing 500 mg/l of carbenicillin and transferred to
solid selection medium containing 100 mg/l kanamycin and 500 mg/l
carbenicillin.
Plant Maturation and Self Pollination:
Shoots that arose from calli on selection medium were removed,
transferred to OMS with 100 mg/l kanamycin and 250 mg/l carbenicil-
lin, and development allowed to continue for three weeks. They were
then planted in soil and moved to the greenhouse. Flowers formed
four to eight weeks after transfer to the greenhouse. As a flower
opened and its anthers dihisced, forceps were used to remove the
anthers and to self-pollinate each flower by rubbing the anthers on
the stigma. Seed capsules matured in 40 days.
- 46 - 1339~23
Testing Seed Progreny From Control, Control Transgenic and Experi-
mental Transformed Plants:
Seeds were first removed axenically from mature capsules and stored
in sterile petri dishes. Seeds were then placed on seml-soft seed
germination medium (SGM) comprising the major and minor salts of
Murashige & Skoog (KC Biologicals) at full strength, 1 mg/l of
thiamine hydrochloride and 0.6 % purified agar (Difco). Analytical
grade atrazine was added to the medium at concentrations of 10 M,
3xlO M, 5xlO M, 8xlO M and 10 M. The growth and survival of the
seedlings were assessed on these concentrations and on zero level
atrazine at 14 days; the results are given in Table 1 below. All
control plants and control transgenic plants germinated but failed
to grow past the cotyledone stage of growth at atrazine concen-
tratons of 5xlO M or higher. Among the seedlings that grew from
seeds of every selfed pCIB14 plant, approximately 75% remained green
and produced primary leaves at 5xlO M, while seedlings on concen-
trations of atrazine of 8xlO M or higher formed only cotyledons
before bleaching and dying. Although tolerant seedlings did not grow
as well on the atrazine medium as on atrazine-free medium, they
could easily be distinguished from the sensitive seedlings on the
same medium.
Atrazine Concentration
Seedling Genotype 10 M 3xlO M 5xlO M 8xlO M 10 M
Control + + o o o
LBA 4404 + + o o o
Bin 6 + + o o o
pCIB 13 + + o o o
pCIB14 + + + ~ ~
1339~2~
- 47 -
Table Legend: The difference ln growth and survival between
the progeny from pCIB14 transgenlc plants and from all
controls ls shown. A posltlve growth response ls lndicated by
+ whlle o lndlcates no growth.
Although the foregolng lnventlons have been
descrlbed ln some detall by way of lllustratlon and example
for purposes of clarlty and understandlng, lt wlll be obvlous
that certaln changes and modlflcatlons may be practlced wlthln
the scope of the lnventions as llmlted only by the scope of
the appended clalms.
Wlthln the scope of the present lnventlon not only
DNA molecules havlng the glven concrete nucleotlde sequence
and codlng for a glutathlone S-transferase polypeptlde are an
embodlment of the present lnventlon but also variants and
mutants thereof coding for polypeptldes whlch show glutathione
S-transferase actlvlty.
