Note: Descriptions are shown in the official language in which they were submitted.
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MODIFIC~TION OF SOLUBLE SOLIDS USING SUCROSE
PHOSPHATE SYNTHASE ENCODING SEQUENCE
I~TROllUCTION
T.-rhnir~l Field
The present invention is directed to compositions and mP~ho-lc related to
modif;r~3tion of the sweetness of selected plant tissues. The invention is exemplified by
plants, plant parts, and plant cells l~dr~ro,llled with one or more copies of a transgene
comprising DNA encoding SPS and a Llallsc~ iOnal initiation region functional in plants.
R~rluFro~nl1
Sucrose is one of the primary end products of photosynthesis in higher plants. It is
also the major carbohydrate transported to sucrose arc~m~ ting, or carbon sink, tissues
for plant growth and development. Plant regions, such as leaf tissue, where sucrose is
2 0 synthesized are cornmonly referred to as sucrose source tissue. Plant storage organs, such
as roots or tubers, and fruits are examples of sink tissues. The sucrose translocates from
the mature leaf (source) to any tissue l~ uilillg photo~csimil~e (sink), especially growing
tissues including young leaves, seeds, and roots. Diffir~lties in the purification of sucrose
phosphate ~yllLhase (SPS) from plants have hlh,~.ed with efforts to characterize this
enzyme. SPS catalyses the formation of sucrose phosphate, the sucrose ple~.ul:iol
molecule, from fructose-6 phosphate and UDP-glucose in photosyn~hetir~lly active plant
cells. Sucrose phosphatase then acts on the sucrose phosphate moiety, in an irreversible
reaction. to remove the phosphate and to release sucrose.
SPS is considered a rate limiting enzyme in the pathway providing sucrose to
3 0 growing tissue, therefore the study of SPS and its activity is of special interest. In a recent
publication, Walker and Huber, Plant Phys. (1989) 89:518-524, the purifir~ion and
prelimin~ry characLc~.~aLion of spinach (Spinachia oleracea) SPS was reported. However,
monoclonal antibodies specific to the spinach SPS were found to be non-reactive with all
other plants tested, "closely related" and "relatively unrelated species", including corn (Zea
3 5 maize), soybean (Glycine max), barley (Hordeum vulgare), and sugar beet (Beta vulgaris).
Thus, additional purified sources of SPS enzyme are needed for effective characterization
of this factor. Especially of interest is the characterization of the corn SPS because of its
~ very high export rates, as compared for example, to SPS levels of activity as found in the
leaves of soybean.
_
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With the advent of biotechnology, the ability to modify various properties of plants,
especially agront~mir~lly i~ olL~ crops, is of interest. In this regard, it would be useful
to determine the coding sequence for an SPS gene to probe other crop sources, to use such
coding sequences to prepare DNA ek~ ,s~ion constructs capable of directing the expression
5 of the SPS gene in a plant cell and to express a DNA seq~nre encoding an SPS enzyme in
a plant to lllea~ e the effects on crop yield due to the increased rate of sucrose
translocation to growing tissues.
E~elev~nt T.it~r~tl-re
1 0 The following references are related to ex.~ression of SPS in transgenic plants:
Sol.lnewdld, et al. (1994) Plant, Cell and Environment 17:649-658; Worrell, et al. (1991)
The Plant Cell 3: 1121-1130; Micallef, et al. (1995) Planta 196:327-334; Foyer, et al.
(1994) Plant Physiol., 105(S), 23; Galtier et al. (1993) Plant Physiol. 101:535-543; and
PCT Application No. WO 94/00563. The following references are related to isolation of
DNA encoding SPS: Valdez-Alarcon et al., (1996) Gene 170(2):217-222; Sakamoto et al.,
(1995) Plant Science (Shannon) 112(2):207-217; Heese et al., (1995) Mol. Gen. Genet.,
247(4):515-520; Klein et al., (1993) Planta 190(4):498-510; Salvucci et al., (1993) Plant
Physiol., 102(2):529-536; Sonnewald et al., (1993) 189(2):174-181; and Herrera-Estrella
et al., (1991) J. Cell Biochem. Suppl. 0 (15 Part A) 148. PCT Application WO 94/00563
2 0 discloses ~ el-~e potato SPS placed behind a tuber promoter and used to alter the sucrose
levels in potato. Acid invertase encoding seq~len~es are described by Klann et al., (Plant
Phys. (1992) 99:351-353).
SUMl~ARY OF l~il~, IlW~l~TION
2 5 Methods for modifying the sweetness of plant sink tissues are provided in which
sucrose phosphate synthase (SPS) activity and/or invertase activity in plant tissues are
manipulated Also provided are nucleic acid constructs, vectors, plant cells, plant parts and
plants cont~ining at least one exogenously supplied copy of an SPS gene. The invention
finds use in modifying carbohydrate partitioning in plant tissues and/or parts, which in turn
3 0 can be used to alter plant growth, soluble solid content and/or ~w~tLlless, and/or to alter
the sensitivity of plant growth to temperature and/or to levels of carbon dioxide and
oxygen.
RRni ~ nF~SCRTPrION OF TT~ FICT'UR~.S
3 5 Figure 1 shows an SDS-PAGE profile of corn SPS at various stages of SPS
purification and the quality of the final preparation. Using an 8.5 % acrylamide gel~
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reducing conditions and staining with silver nitrate. The abbreviations used are as follows:
M: Standard of molecular weight B-Galactosidase (116 kd), bovine Albumin (68 kd), Egg
Albumin (45 kd), carbonic anhydrase (29 kd); H: Heparin fraction, 30 micrograms of
proteins per well; FP: Final Preparation, 7.5 micrograms of L)lotehls per well; FE: Final
~ 5 Extract, 7.5 micrograms of ~lot~ s per well; D: Fast-Flow DEAE fraction, 78.5
micrograms of proteins per well.
Figure 2 shows the results of a Western analysis of SPS using monoclonal
antibodies. In Fig. 2A, .llellll,ldlle is inrllhatPd in the ~lesGllce of the SPB3-2-19 antibody;
in Fig. 2B, lll~"llbldlle is inrl-hatP~l in the presence of an antibody not directed against SPS
(negative control anti-neomycin monoclonal antibody); in Fig. 2C, membrane is incubated
in the ~l~sence of the SPB13-2-2 antibody. The abbreviations used are as follows: M:
standards of molecular weight radio-labeled by I-125, (NEX-188 NEN) B-(Jal~rtosidase
(116 kd), bovine albumin (68 kd), carbonic anhydrous (29 kd), trypsin inhibitor (20.1 kd),
Alpha-Lactalbumin (14.4 kd), 150,000 cpm per lane; PA: proteins obtained after
immnn~ffinity chromatography (see below) with the SPB13-2-2 monoclonal antibody,about 40 micrograms of plOL~illS per lane; H: Heparin fraction, about 40 micrograms of
protein per lane.
Figure 3 shows peptide seq~lenres (SEQ ID NOS: 1-5) derived from SPS protein.
All peptides are typed N~C tPrTnin~l.
Figure 4 shows oligonucleotides used for the PCR reactions CD3 (SEQ ID NOS:
10-11) and CD4 (SEQ ID NOS: 12-13) in relation to the peptides (~ P..ce sequenrPs are
pr~:sent~d upside down). Arrows point to the direction the oligonucleotides will prime the
polymerase.
Figure 5 shows the chara~;L~l~aLion of CD3 and CD4 PCR reactions. Figure 5A
2 5 shows agarose gel electrophoresis of CD3 and CD4 PCR reactions. The sizes are given in
kb. Figure SB shows autoradiograph of Southern blot of CD3 and CD4 PCF reactionsprobed with oligonucleotides 4k5 (SEQ ID NO: 14).
Figure 6 shows schPm~rir diagrams ~ sf..~ g SPS cDNA and selected clones.
The upper bar lc~resell~s the entire 3509 bp combined map. Translation stop and start
paints are in~lir~tP(I
Figure 7 shows the assembled SPS cDNA sequence (SEQ ID NO: 6). The
sequences of clones SPS 90, SPS 61 and SPS 3 were fused at the points in~lir~tP~ in Fig. 2.
The SPS reading frame is tr~nci~ted (SEQ ID NOS: 6-7). All SPS protein derived peptide
seqnPnres are in~ tP~.
3 5 Figure 8 shows Western blots demonstrating the characteristics of rabbit SPS 90 and
SPS 30 antisera. The abbreviations used are: pAS** = plei.",....n~ serum, SPS 30 rabbit
; AS** = il"~..nts serum anti-SPS 90. Molecular weight markers at left, where in~liratPcl.
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S = SPS 120 kd polypeptide; S* = SPS 90 kd polypeptide; S** = SPS 30 kd
polypeptide.
Figure 9 shows analysis of protein from a 30-day old corn plant. Figure 9A showsa Comassie Blue-stained gel of total protein isolated from a 30 day old corn plant. M =
5 size marker; R = roots; 1-8 = leaf numbers counting from the bottom of the plant. Leaf
5 has been cut into 5 segmtontc from the leaf tip (Sa) to the end of the sheath (Sc). PEP =
phosphoenolpyruvate carboxylase. Figure 9B shows the results of Western blot analysis
using a mixture of antiSPS 30 and antiSPS 90 antisera against total plant protein isolated
from a 30 day old corn plant. The signal corresponding to SPS appears at 120-140 kd.
1 0 Figure 10 shows a sch~om~tir ~u~ a~y of a construction of plasmids pCGN627,
pCGN639 and pCGN986. Figure 10A shows construction of pCGN627; Figure 10B
shows construction of pCGN639; and Figure 10C shows construction of pCGN986.
Figure 11 shows partitioning between starch and sucrose as a function of
temperature. The squares are data from control UC82B plants while triangles are data
1 5 from transgenic tomatoes e~ ,s~ g SPS on a Rubisco small subunit promoter
(pCGN3812) .
Figure 12 shows m~imllm rates of photosynthesis for regencldted control (solid
bars) and pCGN3812-24 transgenic (open bars) potatoes at three weeks after (panel A) and
seven weeks after (panel B) planting.
2 0 Figure 13 shows tuber dry mass for regenc.d~cd control (solid bars) and
pCGN3812-24 tr~n~genic (open bars) potatoes add 35 and 70 Pa carbon dioxide in
highlight growth ~ lhtls (Figure 13A) and open top ch~llbels in the field (Figure 13B).
l~T~.~CI2TPTION OF T~F, SPFCIFIC ~l~nOnTl~F~TS
2 5 Methods for modifying the solids content of plant sink tissue which use a construct
encoding SPS for example as a way of increasing the sweetness of fruit. The soluble solids
include simple sugars, but also can include certain soluble polymers, and other soluble cell
components. Total solids include more complex carbon compounds, such as starches and
cellulose. The method provides for increasing the total solids in a plant sink tissue so that
3 0 total solids are morlifi~l from a given ratio of total solids per unit weight of sink tissue, as
measured in control plant cells, to a dirr.,~ ll ratio of total solids per unit weight of sink
tissue. The amount of sucrose available to growing tissues in the plant is hl.;lcased, and
the increased sucrose results in increased total solids per unit weight in the sink tissues of
the plant.
The method generally colll~lises growing a plant having integrated into its genome
a construct comprising as operably linked components in the 5' to 3' direction of
transcription. a transcription initiation region functional in a plant cell and a DNA encoding
SPS. The transcription initiation region may be col~liLuLive or tissue specific. By tissue
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speci~lc is intended that the region is ~.efe.~,l,Lially e~r~,ssed in cells of a particular plant
tissue or part, for inct~n~e fruit or leaf as compared to other plant tissues. In one
embodiment the method produces sink tissue having i.lcleased carbon as soluble solids, as
an increased ratio of soluble solids per unit weight of sink tissue, as compared to that
5 Il,ea~ d in control plant cells. This results from the hl~ ased levels of sucrose
generating an increased rate of L d.~l,o,L~Lion of the available sucrose into the carbon sink
tissue. In another embodiment, a method is provided to modify the soluble solids ratios in
sink tissue. such as the ratio of sucrose to fructose, as colllpaled to that measured in control
plant cells or tissue. ~f the i~l~lcased soluble solids in said sink tissue col.l~lises fructose, a
10 phello~y~e will result having an increased sweetness as opposed to the control tissue. A
method is also disclosed, however, wh.,l~l)y a decreased ratio of fructose to sucrose, and
whereby a reduced sweetness phenotype may be produced.
The use of constructs Colll~Jlisillg encoding seque~es to other sucrose metabolizing
el~ylllcs, such as acid invertase, or the utilization of such enzymes which are endogenous
15 to the plant sink cells, can be advantageously used with this invention. For in.ct~n(~e, acid
invertase can be expressed in the cells or sink tissue from an expression construct. or,
alternatively, the sink tissue can be prevented from converting sucrose to fructose and
glucose by the use of an ~nficçnce acid invertase col~llucl, wL.,lcl)y cells of the sink tissue
will have a decreased acid invertase activity, and thereby a decreased ratio of fructose to
2 0 sucrose as CUIII~a1~ to cells in a control sinlc tissue. Fruit having increased total soluble
solids and/or modified or in.;l._ased fructose levels, as nlea~ul~d per unit weight are
provided and include fruits such as tomato, ~L~dwl,~ y and melon. The fruit has a
modifit-d sweetness phenotype, either from a total increase in ~ .ess by percentage of
fruit weight, or from an increased ratio of fructose to sucrose in the soluble solids in the
2 5 fruit.
Transgenic plants and plant parts are provided which have altered carbon
partitioning and end-product synthesis through expression of a transgene required for
sucrose synthesis. The transgenic plants, cells and plant parts such as leaf, fruit and root
are characterized by modified levels of SPS activity c-~--pa-.,d to controls. By "modified
3 0 SPS activity" is int~n-led an increase or decrease in sucrose synthesis. Mo~ifi.-~ti- n of SPS
activity according to the subject invention alters the carbon partitioning beLweell source
tissue and sink tissue through an increase or decrease in sucrose synthesis. Altered carbon
partitioning is Illal~ir~Led by one or more cll~n~es in development, growth and yield
through mo~1ific~tinn of end-product synthesis and conversion in general. The protein and
3 5 DNA encoding SPS of the subject invention is obtainable from any llunl~cl of sources
which contain an endogenous SPS. Among the ~lel~l-ed SPSs are those obtainable from
corn or derived from corn SPS protein or nucleic acid using antibody and/or nucleic acid
probes for SPS ic~entifil~tion, amplification and isolation. The subject invention also
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provides a variety of SPS transgenes which have different promoter regions to regulate the
transcription and level of SPS activity in plants or plant parts in a tissue-specific and
growth-dependent manner. Among the plefellcd promoter regions are those which provide
for leaf, fruit and/or root specific e~ression of SPS. Preferably, the DNA encoding a SPS
of interest in operably linked in a sense or antisense orientation to a select~ci lldns~ Lion
initiation region to provide for a sufficient level of expression of SPS in the desired tissue
or tissues.
An advantage of inc.easillg or decreasing SPS activity is the modification of sucrose
synthesis, which is a key metabolic product that affects the interface beLweell end-product
synthesis and carbon partitioning for most plant systems. By "end-product" ~y~lLhesis is
intt?n~l~cl the metabolic product interface beLw~ll photosynthesis and plant growth and
development. Information flow across the interface may occur by mass action or by signal
llal~lllis~lon and tr~n~duction. Mass action effects occur when an increase in
photosynthesis leads to faster growth resnlting from an increase in the availability of
photosynthate. Conversely, mass action :Fee~back occurs when accllmlll~tion of end-
products reduce the rates of photosynthetic reactions. Thus, an advantage of the subject
invention is that mo-lifir~tion of sucrose synthesis through SPS activity provides a central
control point for modifying carbohydrate partitioning through end-product synthesis in a
source tissue such as leaf and end-product conversion in a sink tissue such as growing leaf,
2 0 fruit or root. For example, modulation of photosynthetic metabolism through expression of
exogenous SPS is advantageously used to alter the synthesis of end-products such as starch,
sucrose glucose, fructose, sugar alcohols, and glycine and serine from photol~hdt~,ly
metabolism. SPS preferably is used to modulate end product synthesis of non-
phosphorylated products of metabolism.
2 5 Another advantage of the subject invention is that altering SPS activity provides a
means for altering plant growth and yield of specific plant cells, plant tissues, plant parts
and plants. In addition, by mo~ tin~ the ability of a plant to synth~si7~ sucrose, the
growth response of a plant under a variety of dirr.,rc.lL environm~nt~l conditions can be
affected including carbon dioxide ntili7~tion~ oxygen sensitivity, te.l.pe.~.Lure-dependent
3 0 growth responsiveness and expression of endogenous genes responsive to sugar content in
general. Manipulation of growth conditions also permits the modulation of metabolism and
the activity of the SPS transgene, for example, through light-mP~ t.o~l activation or
deactivation of the SPS transgene and its product. Another advantage is the mot~ tion of
overall soluble solids such as starch, sucrose, glucose and fructose in sink tissue such as
3 5 fruit or root. SPS activity and sugar content also permit manipulation of endogenous gene
expression and/or en7yme activity in the plant, such as the endogenous acid invertase found
in ripening fruit to increase glucose and fructose levels as well as acid content. An
additional advantage is that the onset of flowering, fruit number, mass, dimensions, and
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overall morphology can be modified by altering carbon partitioning. Thus, the subject
invention permits the obtention of any transgenic plant or plant part which have any one of
several readily selectable phenotypes related to SPS transgene expression and SPS protein
activity.
In the subject invention, purification of corn SPS protein is exemplified. By
"protein" is intPn~ any amino acid sequence, including a protein, polypeptide, or peptide
fragment, whether obtained from plant or synthetic sources, which demo,~l,dtes the ability
10 to catalyze the formation of sucrose phosphate. An SPS of this invention includes
sequences which are modified, such as seq~lenrçs which have been ml1t~tr~1, Llu..~ d,
increased in size, contain codon snbstitlltions as a result of the degeneracy of the DNA
code, and the like as well as sequences which are partially or wholly artificially
synthesized. so long as the synthetic seqll~onres retain the characteristic SPS activity. SPS
15 from sources in addition to corn are obtainable by a variety of standard protocols
employing protein ~ro~ ies, amino acid and nucleic acid information derived from corn
SPS. For example, antibody or nucleic acid probes derived from seqllenring inforrn~tion
permit isolation of a gene or parts of the gene inr~ ing genomic DNA and cDNA
encoding the target SPS of interest. For this purpose, degen.,.dLe and non-deg~ne.d~t:
2 0 probes from hybridization studies with parts or all of the corn SPS sequenre can be used
for identifir~ri~ n, isolation and amplification of a gene or fr~mrntc encoding the SPS of
interest. The SPS gene or fr~gmrnt~ are assembled and evaluated by conventional
recombinant DNA and bioc~ mir~l techniques, and through nucleic acid and amino acid
sequence d~t~b~e comparisons. As an example, SPSs derivable from corn SPS seqllenres
2 5 in this manner include potato, spinach, rice and sugar beet. In vitro and in vivo t~ r~ssion
systems can be used to produce and test the SPS. The SPS activity can be evaluated by
measuring formation of sucrose phosphate from fructose-6-phosphate and UDP-glucose
substrates.
In order to obtain the nucleic acid sequ~nre~ encoding the SPS, e~peci~lly corn SPS,
3 0 subst~nti~lly purified SPS was required. As de.llo-~Llaled more fully in the examples, corn
SPS purified S00-fold was obtained in small qll~ntiti~s which were then ultim~3tPly used to
obtain the peptide sequence which in turn led to the determination of the cDNA seqllenre.
Among the preferred pro~ s of the invention are the proteins having the above
definition with a molecular weight from about 110 to about 130 kd, having the form of a
35 monomer, a dimer or a LeLlallle. and their derivatives, CollllJlisillg at least one peptide
having the following amino acid sequenre:
Thr-Trp-Ile-Lys (SEQ ID NO: 1)
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Tyr-Val-Val-Glu-Leu-Ala-Arg (SEQ ID NO: 2)
Ser-Met-Pro-Pro-Ile-Trp-Ala-Glu-Val-Met-Arg (SEQ ID NO: 3)
Leu-Arg-Pro-Asp-Gln-Asp-Tyr-Leu-Met-His-Ile-Ser-His-Arg (SEQ ID NO: 4)
Trp-Ser-His-Asp-Gly-Ala-Arg (SEQ ID NO: 5)
The invention also relates to a process to prepare proLei,ls as above defined, having
the following steps: (a) extracting SPS from parts cont~inin~ sps~ which are preserved at
low telllpeldL~lle, by grinding, centrifugation and filtration; (b) increasing the rate of SPS
extraction from the extract so obtained by ~lcci~iLdLion in an applop-iate solvent,
centrifugation and solubilization of the ~l~,ci~iLaL~ in a buffer solution; (c) ~ulifyillg the
protein so obtained by chlulllatugraphy and, if desired, (d) ~ alhlg hybridomas, and
monoclonal antibodies from an antigenic solution obtained at step (a), (b), or (c) above; (e)
S~ ,llillg the hybridomas and raising monoclonal antibodies specifically directed against
SPS; and (f) further purifying the SPS obtained at step (a), (b), or (c) with the monoclonal
antibodies prepared.
The invention more precisely relates to a process of preparation of corn SPS having
the following steps: (a) extracting SPS from parts of corn plants by grinding,
centrifugation, and filtration; (b) increasing the rate of SPS extraction from the extract so
obtained by precipitation in polyethyleneglycol (PEG), centrifugation and solubilization of
the precipitate obtained in a buffer solution; (c) purifying the protein so obtained by low
pressure anion exchange chlulllaLography and by ch.clllatography on heparin sepharose,
then by anion exchange high p~lrolllldllce chl~ulllatography; (d) l~ulifyillg the active pools
by passage on two high ~elrollllallce chlulllatography columns, and if desired; (e)
pl~,pdlillg hybridomas and monoclonal antibodies from an antigenic solution pLcpdl.,d from
steps (a), (b), or (c); (f) screening the hybridomas and raising the monoclonal antibodies
2 5 specifically directed against SPS; and (g) purifying the SPS prepdldLion with the
monoclonal antibodies so obtained.
Preferably the corn is a corn Pioneer corn hybrid strain 3184, the parts of plants are
leaves which are kept at low telllpeldLule, for example beL~weell -50~C and -90~C, and
purification in the polyethyleneglycol is realized first by pleci~iL~Lil1g at a final
cuncelllldLion in PEG about 6%, and then by precil,iL~Lillg at a final concellLlation of about
12%. The various chromato~;l~hies are pelrolllled in the following way: 1st
chromatography, DEAE sepharose; 2nd chromatography, heparin sepharose (at this stage,
the preparation obtained may be kept several days without loss of activity); 3rdchlulllatography, Mono Q chromatography; 4th clllclllatugraphy, HPLC hydroxyapatite;
3 5 and 5th chromatography, HPLC hydroxyapatite.
A variety of additional protein fractionation methûds can be combined to generate a
suitable purification scheme for SPS plOL~ills and peptides from corn and those in addition
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WO 97/15678 PCTAUS96/17351
to corn. If only very small amounts of denatured protein are needed, a high resolution
tecilni-luc may be used such as two--1im~ncional gel cle~;L,ophoresis to obtain the protein in
one step. When retention of activity is desired, a series of purification steps are designed
to take advantage of different plo~,.Lies of the SPS of interest such as precipitation
~ 5 ~lo~e.lies, charge, size, adsorptive pl~elLies and affinity plo~ Lies as demon,ctrated for
corn SPS.
~ In general, purification follows the initial extraction and IJle~ala~ion of total protein.
bulk precipitation followed by chromatographic procedures such as ion exchange,
adsorption, gel filtration, affiniy resins and non-denaLu,illg electrophoresis methods so as
to be subst~nti~lly free from other L-~oLei,ls, particularly proteins of the source tissue. By
'~SU~ 1IY free from other ~loLeills"is meant that the protein has been partially purified
away from ~lOteillS found in the source tissue or o,gan,~"l. Such a protein of this
invention will delllol~L,dL~ a specific enzymatic activity of at least greater than 0.05, more
preferably at least greater than at least 0.30, wherein specific el.~ylllatic activiy (sA) is
measured in units which coll~s~ond to 1 ~lmole (micromole) of sucrose formed per minute
per mg of protein at 37~C. In a more prefelled embo~im~rlt, the protein will demonstrate
even more improved sA and increased purification factors (see, Table 5).The proteins can
be further purified if desired, when retention of activity is less important, byelectrophoretic procedures including native or del~Lulillg polyacrylamide gel
2 0 clecLlol)horesis, icoelectric focusing and two dimensional gel ele.;LloL,horesis. During the
dirr~ steps of purification and thel~,a~Lel, the SPS activity can be measured by two
m.othn-lc: (a) a method based on a colo,i",ellic test or reso,~;i,lol test; and (b) a method
based on the amount of one of the products formed during the tran~r~ Lion reactions
where SPS is involved. Both mPthrrlc are ~let~ l in the e~Le,h,lell~l part ~llot~ilPd
2 5 hereunder. The exemplified invention relates to the enzyme comprising a corn SPS having
a molecular weight from about 110 to 130 kilodalton (kd) and a specific activity of greater
than 0.05 U. The invention relates more particularly to the enzyme c(jnl~lisillg a corn SPS
having a specific activiy of about 25 U. Antibodies to SPS are l,r~all,d as follows, or
by other methods known to those skilled in the art. Mice are i.. ~ (i with several
3 0 injections of enzymatic preparations. Dirr~rc llL kinds of mice may be used, for example
BALB/c. The antigen can be provided in complete Freunds adjuvant then in incomplete
Freunds adjuvant. Several injections in mice are realized: good results have been obtained
with three injections of Mono Q, pools, (~ above purification scheme) followed by three
injections of final pools (days 0, 14, 27, 60, 90 and 105 for example). The first injections
3 5 are ~rlminict~red sub-cutaneously, for example in the cushions, and the feet, the last
injection is ~ L~ ed intravenously, in the tail for example. The preparation of spleen
cellular suspensions from animals i.. ~ l as described above is made in a conventional
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way. The steps of fusion with myeloma cells, of conservation of the hybridoma, of
cloning, of antibodies production are made by conventional ways. To detect the hybridoma
secreting the monoclonal antibodies raised against the antigen, two methods are used to
select antibodies: a method of detection of antibodies as inhibitor of SPS activity; and a
5 method of detection of antibodies p,~cil,ilating SPS activities. In a p.~f~ ,d embodiment,
these methods are the mt-tho~ described in the experimental section ~let~ od hereunder.
Among the objects of the invention, are also provided lines of hybridoma cells, and
in particular hybridoma cells described as: SPA 2-2-3: I-971; SPA 2-2-22: I-970; SPA
2-2-25: I-972; SPB 3-2-19: I-973; SPB 5-2-10: I-974; SPB 54-2: I-975; SPB 13-1-7:
1 0 I-976; and SPB 13-2-2: I-977. Deposits of these hybridoma cells were made at the
C.N.C.M. (Institut Pasteur Paris) on June 11, 1990. The invention relates also to
monoclonal antibodies specifically directed against SPS.
The invention relates also to a process of preparation of proteins as defined above
characterized in that a preparation cont~inin~ the so-called proteins is purified on a
1 5 chromatography column having monoclonal antibodies as defined above specifically raised
against the p-oLei,ls.
The invention relates also to cDNA coding for plol~ills as defined above, especially
cDNA coding for corn SPS. Among the ~lcfc.-ed cDNA, most p,eI~ d is cDNA
COIll~liSillg a nucleotide seqllenre rep,eselll~d in Figure 7 (SEQ ID NO: 6). Thus, this
2 0 invention relates to an extrachromosomal DNA sequence encoding a SPS as defined above.
Any DNA seql~enre which is not incorporated into the genome of a plant is considered
extrachromosomal, i.e., outside of the chromosome, for purposes of this invention. This
includes, but is not limited to cDNA, genomic DNA, truncated sequences, single stranded
and double stranded DNA. In a ple~ed embodiment, the DNA sequence is cDNA. In a
25 different plefc.,~,d embodiment, the DNA sequence is obtainable from corn or is derived
from the corn DNA sequence.
Among the preferred ~-. lehls and nucleic acid sequences of the invention is corn
SPS. The corn SPS is represented in Figure 1, which shows the presence of pioleills at
about 120. 95 and 30 kd. The prolt:hls shown at 95 and 30 kd are considered to be
3 0 breakdown products of the protein shown at 120 kd. The complete protein is believed to
be a di- or lelldlll~lic protein having as the basic sub-unit from about a 110 to about a 130
kd protein. The complete cDNA sequence of the corn SPS is shown in Figure 7 (SEQ ID
NO: 6).
The cDNA coding for sucrose phosphate synthase has been ~lcpal~d in the
35 following way: (1) sequencing of peptide fragm~o~t~ from purified SPS. With the purified
preparations of SPS previously obtained, following separation on an acrylamide gel, a 120
kd minor band (corresponding to the total protein sequence) and two 90 kd and 30 kd
major bands are obtained. Both major polypeptides are separated by electrophoresis and
CA 0223~801 1998-04-24
W o 97~678 PCT~US96/17351
electroeluted. By trypsin digestion and sequencing of the fragmeMs so obtained, the
sequPnre of 5 peptides has been d~L~ ,ed. This amino acid seq~npnre makes it possible
to determine the corresponding degel1~ldte nucleotide seqn~pnre.
(2) Corn leaf isolation. Total RNA is isolated according to Turpen and Griffith
5 (1986, Biotechniques 4~ 15) for poly(A) RNA preparation, the standard oligo dT
cellulose column is used.
- (3) cDNA library construction. cDNA is s~llLhesi~d using the protocol of a kit
supplied by Plulllcga except that M-MLV reverse transcriptase is used instead of AMV
reverse Llanscli~Lase. The length of cDNA obtained is from 500 to several thousand base
pairs. EcoF~ linkers are added to the blunt ended cDNA and this material is cloned into a
second geneldLion lambda GT11 expression vector. Total library size is about l.5x106
plaques.
(4) Utilization of PCR to synthPci7ing a nucleotide sequence specific for SPS. The
oligonucleotides derived from peptides B11 (SPS 30 kd) (SEQ ID NO: 3) and 4K (90 kd)
(SEQ ID NO: 4) described in figure 3 are used as plillleLS in a PCR reaction. It has been
~c$llmfd that peptides derived from SPS 30 and SPS 90 are degradation products of protein
SPS 120 kd, and that the peptides derived from SPS and SPS 90 are enro~Ptl by the same
RNA.
With this hypothesis, by using in proper polarity pairs of oligonucleotides
2 0 corresponding to the peptidic seqnPnrP~s in a PCR re~ctio~, one may obtain the synthesis of
the DNA, Col-l~f-~-L;I~g the two location. Since it is a priori not know in which order the
peptides are located relative to each other, one has to do the two dirr~l~.lL possibilities (Fig.
4). Only the oligonucleotide couple CD3 synthPsi7Ps a cDNA of defined length (1200 bp)
(Fig. 5).
2 5 (5) cDNA library sc~ illg. When 250,000 lambda clones GT11 are sc~ ,ned
using the 1200 bp long PCR cDNA, 16 positives are obtained. Sizes of the inserts ranged
from 0.3 kb to 2.8 kb (see Fig. 6 for the two longest clones). The seq nPn~e is not
complete in 5 ' . In a second round of library scleelling with a 400 bp DNA fragment
corresponding to the most 5' fragment of the clone SPS 3, a SPS 61 clone ~xlPl--l;..g further
3 0 5 ' without having the 5 ' end of the reading frame is obtained (Fig. 6).
(6) Creation and screening of a second cDNA library in order to clone the 5'
sequence of cDNA coding for SPS. A oligonucleotide comph",f."il,.y to the 5' sequence
of clone SPS 61 is used as a primer for cDNA synthesis. After second strand reaction is
completed, the cDNA is cloned into bacteriophage lambda GTll. The library includes
3 5 about one million clones. The SPS 90 and SP 77 were obtained by screening this library
with SPS 61 (Fig. 6).
(7) The assembled SPS reading frame. DNA sequences which encode the SPS may
CA 0223~801 1998-04-24
W O 97/15678 PCTAUS96/17351
be employed as a gene of interest in a DNA construct or as probes in accordance with this
invention. When provided in a host cell, the sequenre can be e~ ssed as a source of
SPS. More pl~ lcd is the SPS seql-rnre in a vegetal cell under the regulatory control of a
lldns~ Lional and translational initiation region functional in plants. Vegetal cell means
5 any plant cell being able to forrn ulldirr.,~ LidLed tissues as callus or dirr~lellLidLed tissues
as embryos, parts of plants, whole plants or seeds. Plants means for example plants
producing grain seeds such as cereals, and inrl~ldes wheat, barley, corn, and oat;
lc~.llllhluus plants such as soybean; oleaginous plants such as turnesol; tuberous plants such
as potato; plants with roots such as beet; and fruit such as tomato. The sucrose phosphate
10 synthase is a key enzyme, in sucrose regulation ...rcl~ mc, but also in carbon partitioning
regulation between starch and sucrose during photosynthesis (~ J. Preiss, Tibs January
1984, page 24, or Stitt and Coll, (1987) Biochemistry of Plants, 10:3-27). Of particular
interest are plants of the night~h~-lr family Sol~n~re~ including the gen~tir,~lly similar but
physiologically disparate plants potato (Solanium tuberosum) and tomato (Hycopersicon
1 5 esculentum).
When provided in a DNA construct for integration into a plant genome, the
sequence can encode a sense strand or an anti-sense strand. By increasing the amount of
SPS available to the photosynthptir~lly active plant cell by the expression of additional
SPS, an increased flow of sucrose can be provided to glv~lvillg tissues res~lting, for
2 0 example, in increased plant yields; by decreasing the amount of SPS available to the
photosynthPtir~lly active plant cell, the rate of sucrose release from the plant cell may be
hindered, reslllting in less new plant growth. Controlling the rate of ~ ol~ and the
amount of sucrose available to growing tissues can be used to increase or decrease the total
solids in a plant sink tissue from a given ratio of total solids per unit weight sink tissue.
2 5 Total solids include soluble solids and insoluble solids such as sugars, starches and
cellulose. Of particular interest are the soluble solids, which include the sugars sucrose,
fructose, and glucose, soluble OlgdlliCS, polymers and other soluble co~ one.~ of cells.
Increased total solids in a plant sink tissue may be in the form of an hl~;l.,ase in glucose
and/or fructose levels. Where the increase comprises fructose, for example, the resulting
3 0 phenotype is increased sweetness. Where fructose levels are lowered a reduced sweetness
phenotype is produced. Of particular interest is fruit having a modified sweetness
phenotype. Increasing or de~ ,asing the flow and/or amount of sucrose available to fruit
tissue increases or decreases the conversion of sucrose to glucose and fructose by acid
invertase, and thus the sweetness of fruit. In tomato fruit, for example, glucose and
3 5 fructose are produced from sucrose by a vacuolar acid invertase that is active during fruit
ripening. As fructose is twice as sweet on a molar basis as glucose, an increase in fructose
levels or a fructose to glucose ratio can result in an increased sweetness of the fruit. Of
CA 0223~801 1998-04-24
WO 97flS678 PCT~US96/17351
particular interest is fruit of the plant family Sol~n~rea~P. Sink tissue solids can be modified
with SPS levels and/or activity in conjullcLion with endogenous sucrose and starch
metabolizing el~yllles, such as acid invertase for sucrose and glycogen synthase for starch.
Mo~lifir~tion can be used to ~nh~nre or inhibit e l~ylll~LiC activity, for example through
5 sense or ~nticence expression. By increasing or decreasing SPS activity in plants, the
interaction bcLween photosynthesis and the synthesis of end products, such as sucrose and
starch. can be modified. Of particular interest is the moAifir~tiQn of the starch to sucrose
ratio in a vegetal cell through the eA~le;,sion of a transgene encoding SPS. Modifying the
starch to sucrose ratio in vegetal cell may tr~n~AIlre the affect through end-product
1 0 ~y-llLllesis, signal tr~nc~ c tion and/or translocation to other vegetal cells, particularly the
vegetal cells of leaf, fruit and root. In some plants, the change in carbohydrate partitioning
can also affect the sensitivity of the altered plant to carbon dioxide and oxygen. Increasing
sucrose synthesis can result in greater capacity for photosynthesis at elevated carbon
dioxide, particularly in the potato. Conversely, de~ asil1g sucrose ~yllLhesis (increasing
1 5 starch synthesis) induces oxygen insensitivity. Such an effect can be obtained by
e~lcsshlg ;.,.lic~ e SPS.
A sucrose metabolizing enzyme can also be modified through sense or ~,,I;ce,,.ceexpression. Seq-nPnres to be transcribed are ligated to the 3' end the plant lldlls~ lion
initiation region. In the sense col~LlucL~, an mRNA strand is produced which encodes the
2 0 desired sucrose metabolizing enzyme, while in anLisellse constructs, an RNA sequence
complemPnt~ry to an enzyme coding sequence is produced. The sense strand is desirable
when one wishes to increase the production of a sucrose metabolizing enzyme in plant
cells, whereas the ~"~i.cP"~e strand may be useful to inhibit production of a related plant
sucrose metabolizing enzyme. The inhibition of acid invertase in tomato fruit, for inct~nrP,
2 5 can lead to fruit having elevated levels of sucrose in the tomato fruit. The seq~lenre to acid
invertase is known (Klann et al., (1992) Plant. P*ys. (1992) 99:351-353). Expression of
other sucrose metabolizing enzymes may result in alterations to other carbon components,
for in.ct~nre the ek~.cssion of starch synthPci7:ing enzymes to act in concert with the
increase availability of sucrose may result in hl~;lcased starch levels in the sink tissue. The
3 0 Lldl~r~ aLion of plants using glycogen ~yllLllci,is enzymes (glgA, glgB and glgC) to
modify starch compositions is described in U. S. Patent No. 5,349,123.
The L,r~el.ce of sucrose metabolizing enzyme sc~lue--ces in the genome of a plant
host cell may be confirmed, for example by a Southern analysis of DNA or a Northern
analysis of RNA seq~lPnrec or by PCR mPthnAc. In addition to sequences providing for
35 llal,s.;li~lional initiation in a plant cell, also of interest are sequences which provide for
Llallsc.ipL~onal and translational initiation of a desired sequence encoding a sucrose
metabolizing enzyme. Translational initiation regions may be provided from the source of
the transcriptional initiation region or from the gene of interest. In this matter, expression
CA 0223~801 1998-04-24
W O 97/15678 PCT~US96/17351
of the sucrose metabolizing enzyme in a plant cell is provided. The presence of the sucrose
metabolizing enzyme in the plant host cell may be confirmed by a variety of methods
inrhl-ling an immnnological analysis of the protein (e.g. Western or ELIZA), as a result of
phenotypic challges observed in the cell, such as altered soluble solids content or by assay
5 for increased enzyme activity, and the like.
Other seqntonrçs may be inrh-cle~l in the nucleic acid construct providing for
expression of the sucrose metabolizing el~y.nes ("e~.c;ssion constructs") of this invention,
including endogenous plant llanscl;~ion termination regions which will be located 3' to the
desired sucrose metabolizing enzyme encoding seqnenre. For in~t~nre, lla~ iOn
10 lt;ll"indtion sequences derived from a patatin gene may be utilized when the sink tissue is
pOtdtO tubers. T,allsclil)lion Lel,-,i-ldlion regions may also be derived from genes other
than those used to regulate the l~dns~,lip~ion in the nucleic acid constructs of this invention.
Tlal s~ui~lion h,l."i"ation regions may be derived from a variety of dirr~ L gene
sequences, including the Agrobacterium, viral and plant genes discussed above for their
15 desirable 5' regulatory seqllrnres. Further constructs are considered which provide for
Lldnscli~Lion and/or expression of more than one sucrose metabolizing enzyme. For
example, one may wish to provide enzymes to plant cells of the sink tissue which provide
for modification of the type of soluble solids to be produced therein, as well as for
enhancing or otherwise modifying the increase or decrease in overall soluble solids
2 0 production. An example of el~y",es which may prove useful in modifying soluble solids
ratios is the acid invertase enzyme.
In developing the nucleic acid constructs of this invention, the various components
of the construct or fr~gmrntc thereof will normally be inserted into a convenient cloning
vector, e.g. a plasmid, which is capable of replication in a bacterial host, e.g. E. coli.
25 Numerous vectors exist that have been described in the li~.d~ule, many of which are
commercially available. After each cloning, the cloning vector with the desired insert may
be isolated and subjected to further manipulation, such as restriction, insertion of new
fr~gmPnt~ or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the
components of the desired sequence. Once the construct has been completed, it may then
30 be Lldl~r~lled to an a~lopliate vector for further manipulation in accordance with the
manner of tran~rc ll-lation of the host cell.
The constructs of this invention providing for L~a-ls~ Lion and/or expression ofsucrose metabolizing enzyme sequences of this invention may be utilized as vectors for
plant cell tran~.lllation. The manner in which nucleic acid sequences are introduced into
3 5 the plant host cell is not critical to this invention. Direct DNA L.dl~re. techniques, such as
electroporation, microinjection or DNA bombardment may be useful. To aid in
identification of transformed plant cells, the constructs of this invention may be further
14
CA 0223~801 1998-04-24
WO 97~n5678 P ~ ~US96~7351
manipulated to include plant selectable lllalh.,ls. The use of plant selectable markers is
pler~,l-ed in this invention as the amount of e,~cli...Pr-t~tion required to detect plant cells is
greatly reduced when a se1ect~ble marker is expressed. Useful selectable ~-.a.h~l~ include
e.~y.ncs which provide for ~~si~ re to an antibiotic such as ge~ ycin, hy~ ~ulllycin,
~ 5 kanamycin, and the like. Similarly, el~yl.. es providing for production of a compound
~ ontifi~hle by color change, such as GUS, or lllmin~srçnre, such as luciferase are useful.
- An alternative method of plant cell Llal~rollllaLion employs plant vectors which
contain additional seql~enres which provide for ~ransfer of the desired sucrose metabolizing
enzyme sequences to a plant host cell and stable integration of these sequences into the
10 genome of the desired plant host. Select~ble malhels may also be useful in these nucleic
acid constructs to provide for dir~l~--Liation of plant cells cont~ining the desired sequences
from those which have only the native genetic material. Seq~lenres useful in providing for
Llan~Ç~r of nucleic acid sequences to host plant cells may be derived from plant pathogenic
bacteria, such as Agrobacterium or Rhizogenes, plant pathogenic viruses, or plant
15 transposable elements.
A sucrose metabolizing enzyme conci~lered in this invention inrl~ es any seqllenre
of amino acids, such as protein, polypeptide, or peptide fragm~nt, which demonstrates the
ability to catalyze a reaction involved in the synthesis or degradation of sucrose or a
~re~ lsol of sucrose. These can be endogenous plant seqluonres~ by which is meant any
2 0 sequence which can be naturally found in a plant cell, including native (indigenous) plant
sequences as well as sequences from plant viruses or plant pathogenic bacteria, such as
Agrobacterium or Rhizobium species that are naturally found and functional in plant cells.
It will be recognized by one of ol-lhlal y skill in the art that sucrose metabolizing enzyme
sequences may also be modified using standard trrhniql~es of site specific mutation or
2 5 PCR, or modification of the seqn~onre may be accomplished in producing a synthetic
nucleic acid sequence and will still be considered a sucrose biosynthesis enzyme nucleic
acid sequence of this invention. For example, wobble positions in codons may be changed
such that the nucleic acid sequence enrodes the same amino acid seq~lenre, or ~Itrrn~tively,
codons can be altered such that conservative amino acid s~lbstih~ticlns result. In either case,
30 the peptide or protein m~int~inc the desired enzymatic activity and is thus considered part
of the instant invention. A nucleic acid seqllenre to a sucrose metabolizing enzyme may be
a DNA or RNA seqllenre, derived from genomic DNA, cDNA, mRNA, or may be
synth,oci7P-l in whole or in part. The structural gene seqnpnres may be cloned, for
example, by isolating genomic DNA from an a~plup.i~ source, and amplifying and
3 5 cloning the sequenre of interest using a polymerase chain reaction (PCR). Alternatively,
the gene sequences may be sy..~ si7~d, either completely or in part, especially where it is
desirable to provide plant-preferred sequences. Thus, all or a portion of the desired
CA 0223~801 1998-04-24
W O 97/1~678 PCT~US96/17351
structural gene may be synthesized using codons p~ d by a selected plant host. Plant-
preferred codons may be determined, for example, from the codons used most frequently in
the proteills expressed in a particular plant host species. Other mor1ifir~tir~ns of the gene
sequences may result in "".~"l~ having slightly altered activity. Once obtained, a sucrose
5 metabolizing enzyme may be utilized wit'n the SPS seqnpnre in a variety of ways.
Other endogenous plant seq-lenres may be useful in nucleic acid constructs of this
invention, for example to provide for Lldnscli~lion of the sucrose metabolizing enzyme
seqllçnre~. Transcriptional regulatory regions are located immr~ tely 5' to the DNA
seqnenres of the gene of interest, and may be obtained from sequences available in the
10 liL~.dlul~, or ic~entifird and characterized by isolating genes having a desirable ll~ns~ lion
pattern in plants, and studying the 5' nucleic acid sequences. Numerous lldnscliplion
initiation regions which provide for a variety of col~LiLuLive or regulatable, e.g. inducible,
ssion in a plant cell are known. Among sequences krlown to be useful in providing
for constitutive gene expression are regulatory regions associated with Agro~acterium
15 genes, such as for nopaline synthase (Nos), mannopine synthase (Mas), or octopine
synthase (Ocs), as well as regions coding for e~ cssion of viral genes, such as the 35S and
l9S regions of cauliflower mosaic virus (CaMV). The term col~LiLuLi~re as used herein
does not nPcess~rily in-lir~te tnat a gene is expressed at the same level in all cell types, but
that the gene is expressed in a wide range of cell types, although some variation in
2 0 abun~nre is often detçct~hle.
In providing for Ll~ st;lil,lion and/or e~ s~ion of the sucrose metabolizing enzyme
seqllenrçs, for various reasons one may wish to limit the expression of these enzymes to
plant cells which function as carbon sinks. Towards this end, one can identify useful
transcriptional initiation regions that provide for e~lession preferentially in specific tissue
2 5 types, such as roots, tubers, seeds or fruit. These sequences may be illrntifird from cDNA
libraries using dirrerellLial screening teçh~li~les, for example, or may be derived from
sequrnres known in the literature.
Many tissue specific promoter regions are known, such as the Rubisco small subunit
promoter which plefelelllially is expressed in leaf tissue, the patatin promoter which is
30 ~lefelellLially in potato tubers. Other LlallscliL,Lional initiation regions which ~lefelenlially
provide for ~lallscli~Lion in certain tissues or under certain growth conditions, include
those from napin, seed or leaf ACP, zein, and the like. Fruit specific promoters are also
known, one such promoter is the E8 promoter, described in Deikman et al. (1988) EMBO
J. 2:3315-3320; and DellaPenna et al. (1989) Plant Cell 1:53-63, the te~ching~ of which
3 5 are incorporated herein by reference. An E8-SPS construct (fruit-specific promoter) will
express SPS in a fruit-specific manner, whereby the levels of sucrose produced in the fruit
may be elevated. If coupled with antisense acid invertase, the increase in sucrose would be
16
CA 0223~80l l998-04-24
WO 97/~5678 PCT~US96/17351
m~int~inPci This is a particular issue in tomatoes where acid invertase present in the fruit
drives the production of glucose and fructose from sucrose..
The protein and DNA enro~iing SPS of the subject inveMion is obtainable from anysource cont~ining an endogenous SPS and can be wholly or partially synthetic. Among
5 the pler~l.ed SPSs are those obtainable from corn. By '~obtainable from corn" is meant that
the sequence, wllcLh~,. an amino acid seq~nre or nucleic acid-seq~onre, is related to a corn
SPS, including a SPS recovered through use of nucleic acid probes, antibody preparations.
sequenre colllp~l.isons or derivatives obtained through protein modeling or mutagenesis for
example. Thus, one skilled in the art will readily recognize that antibodies, nucleic acid
10 probes (DNA and RNA) and the like can be ~r~aled and used to screen other plant
sources for SPS and recover it. Typically, a homologously related nucleic acid sequence
will show at least about 60% homology, and more preferably at least about 70% homology
between the corn SPS and the given plant SPS of interest, excluding any deletions which
may be present. Homology is found when there is an identity of base pairs and can be
15 del.,..llhled upon comparison of sequence il~ollllation, nucleic acid or amino acid, or
through hybridization reactions con~urtt?d under relatively ~L~ gclll conditions, e.g., under
conditions where there is a fairly low pe,ccllL~ge of non-specific binding with corn SPS
probes.
Probes can be considerably shorter than the entire sequence, but should be at least
2 0 about 10, preferably at least about lS, more preferably at least 20 nucleotides in length.
Longer oligonucleotides are also useful, up to full length of the gene encoding the
polypeptide of interest. Both DNA and RNA probes can be used. A genomic library
prepared from the plant source of interest can be probed with conserved seq~erlres from
corn SPS to identify homologously related se~-,ellces. Use of the entire corn SPS cDNA
2 5 may be employed if shorter probe sequences are not i(~entifi~ocl Positive clones are then
analyzed by lc~Lliclion enzyme digestion and/or sequencing. In this general manner, one
or more sequences can be ic~ontifir-l providing both the coding region, and the
Llalls.;li~Lional regulatory elements of the SPS gene from such plant source. As an
example, probes derived from corn SPS are used for isolating SPS from corn and sources
3 0 in addition to corn. A probe or a battery of probes lc~ .sel~ g all or segments of the SPS
coding region of corn SPS are preferably used. The corn SPS sequences can be compared
by conventional gene bank searches and the conserved and nollcons~ ed regions used in
the design of additional probes if needed. In addition, the conserved and nonconserved
regions for probe design are i(lentifi~hle through standard hybridization terhniques or, for
3 5 example, by COIll~alillg amino acid and/or nucleic acid sequenres of corn SPS to SPS
seque~res from diverse sources inrlu~ling rice, potato, sugar beet, spinach ,or Arabidopsis
thaliana. which is a flowering plant n,elllber of the mustard family Brasicaceae.
17
CA 0223~801 1998-04-24
W O 97/15678 PCTAUS96/17351
In use, probes are typically labeled in a ~etPct~hle manner (for example with 32p_
labelled or biotinylated nucleotides) and are inrub~tP-l with single-stranded DNA or RNA
from the plant source in which the gene is sought, although llnl~help~ oligonucleotides are
also useful. Hybridization is detect~Pci by means of the label after single-stranded and
double-stranded (hybridized) DNA or DNA/RNA have been separated, typically usingnitrocellulose paper or nylon l,lclllbldnes. Hybridization techniques suitable for use with
oligonucleotides are well known to those skilled in the art.
From the cDNA sequences, one skilled in the art can obtain the corresponding
genomic DNA seql~en~es related thereto to obtain the coding region of the SPS, including
intron sequences, transcription, translation initiation regions and/or Llanscli~t l~""i"~ion
regions of the rt,s~eclive SPS gene. The regulatory regions can be used with or without the
SPS gene in various probes and/or constructs. The complete SPS reading frame can be
assembled using restriction enzyme fragmPnt~ of SPS 90, SPS 61 and SPS 3, ~ Fig. 6.
When expressed in E. coli, the SPS cDNA produces a protein which is recognized
by anti-SPS antisera and has the same electrophoretic mobility as SPS extracted from corn
leaves. We show that this E. coli SPS is as active as plant SPS, i.e. for complete
el-,y~llalic activity in E. coli no other plant factor is needed but the SPS cDNA.
Plants obtained by the method of transformation and cont~ining fusions of SPS
cDNA to tissue specific promoters in order to modify or alter the composition of certain
2 0 plant organs are also included.
A DNA construct of this invention can include lldnscli~lional and translational
initiation regulatory regions homologous or heterologous to the plant host. Of particular
interest are llanscli~lional initiation regions from genes which are present in the plant host
species, for example, the tobacco ribulose biphosphate carboxylase small subunit (SSU)
2 5 transcriptional initiation region; the cauliflower mosaic virus (CaMV) 35S llallscli~LiOnal
initiation region, including a "double" 35S CaMV promoter, the tomato fruit-specific E8
(E8) transcriptional initiation region, and those associated with T-DNA, such as the opine
synthase transcriptional initiation region, e.g., octopine, mannopine, agropine, and the
like.
Any one of number of regulatory sequences may be pl~fe,.l~d in a particular
situation, depending upon whether col~LiLuLi~e or tissue and/or timing int1~lce~1 Lldnscli~lion
is desired, the efficiency of a particular promoter in conjull~;lion with the heterologous SPS,
the ability to join a strong ~rolllotc. with a control region from a different promoter to
provide for inducible transcription, ease of construction and the like. For example, tissue
3 5 specific promoters can be employed to selectively modify or alter the composition of
certain plant organs. Promoters which function in, or are specific by fruit, root and/or leaf
are examples. These regulatory regions find ample prece~1enre in the lill,ldl~
CA 0223~80l l998-04-24
W O 97/~678 PCT~US96/11351
The termination region may be derived from the 3'-region of the gene from which
the initiation region was obtained, from the SPS gene, or from a different gene. Preferably
the termination region will be derived from a plant gene, particularly, the tobacco ribulose
biphosphate carboxylase small subunit ~c~ A~ion region; a gene associated with the Ti-
~ 5 plasmid such as the octopine synthase l~ . Il lil .,.~ ion region or the tml termin~tion region.
In developing the expression cassette, the various fragmPnt.~ co~ "isillg the
~ regulatory regions and open reading frame may be subjected to different processing
conditions. such a ligation, restriction, resection, in vitro mutagenesis, primer repair, use
of linkers and adapters, and the like. Thus, nucleotide transitions, transversions,
insertions, deletions, or the like, nay be pc.rol-lled on the DNA which is employed in the
regulatory regions and/or open reading frame.
During the construction of the expression cassette, the various fragments of theDNA will usually be cloned in an a~,lopliate cloning vector, which allows for
amplification of the DNA, modification of the DNA or manipulation by joining or
removing of the sequences, linkers, or the like. Normally, the vectors will be capable of
replication in at least a relatively high copy number in E. coli. A ~ be. of vectors are
readily available for cloning, inrl~ ing such vectors as pBR322, pUC series, M13 series,
etc. The cloning vector will have one or more lllalh._l~ which provide for selection or
~lal~ru.ll.dll~. The Illdlh~l~ will normally provide for resistance to cytotoxic agents such
2 0 as antibiotics, heavy metals, toxins, or the like. By ~ro~ c restriction of the vector
and cassette, and as a~,o~liate, mollifir~tion of the ends, by chewing back or filling in
overhangs, to provide for blunt ends, by addition of linkers, by tailing, comple...~o.-l;.. y
ends can be provided for ligation and joining of the vector to the ex~,c~sion cassette or
component thereof.
2 5 A~fter each manipulation of the DNA in the development of the cassette, the plasmid
will be cloned and isolated and, as required, the particular cassette col,l~onell~ analyzed as
to its sequence to ensure that the proper seq~lenre has been obtained. Depending upon the
nature of the manipulation, the desired seqllPnre may be excised from the plasmid and
introduced into a dirrc,el,t vector or the plasmid may be restricted and the c~rcssion
cassette component manipulated, as a~,ul,,ialc.
The manner of transformation of E. coli with the various DNA constructs (plasmids
and viruses) for cloning is not critical to this invention. Conjugation, tr~n~d~ctinn,
~ldn~rcction or Ll~,~rollllation, for example, calcium phosphate mrtli~ted ~Idl~rûllllation,
may be employed.
3 5 In addition to the expression cassette, depending upon the manner of introduction of
the expression cassette into the plant cell, other DNA sequences may be required. For
example when using the Ti- or Ri-plasmid for transformation of plant cells, as described
19
CA 0223~801 1998-04-24
W O 97/15678 PCTAJS96/17351
below, at least the right border and frequently both the right and left borders of the T-DNA
of the Ti- or Ri-plasmids will be joined as fl~nking regions to the expression cassette. The
use of T-DNA for transformation of plant cells has received extensive study and is amply
described in Genetic Engineering, Principles and Methods (1984) Vol 6 (Eds. Setlow and
Hollaender) pp. 253-278 (Plenum, NY); A. Hoekema, in: The Binary Plant Vector Sysrem
(1985) Offsetdrukkerij Ranters, 8.V. Alblasserdam.
Alternatively, to enh~nre integration into the plant genome, terminal repeats oftransposons may be used as borders in conju~ ion with a transposase. In this situation,
expression of the transposase should be inducible, so that once the e~ .sion cassette is
inte~ldL~d into the genome, it should be relatively stably integiaL~d and avoid hopping.
The expression cassette will normally be joined to a marker for selection in plant
cells. ConveniellLly, the marker may be reci~ts-nre to a biocide, particularly an antibiotic,
such as Kanamycin, G418, Bleomycin, Hy~ lllychl, Chloramphenicol, or the like. The
particular marker employed will be one which will allow for selection of Llan.rulllled plant
cells as compared to plant cells lacking the DNA which has been introduced.
A variety of techniques are available for the introduction of DNA into a plant cell
host. These t~chni~ues include Lldllsrullllation with Ti-DNA employing A. tumefaciens or
A. rhizogenes as the transforming agent, protoplast fusion, injection, electroporation, DNA
particle bombardlll~ , and the like. For ~ rulllldLion with Agrobacterium, plasmids can
2 0 be ple~aled in E. coli which plasmids contain DNA homologous with the Ti-plasmid,
particularly T-DNA. The plasmid may be capable of replication in Agrobacterium, by
inclusion of a broad ~.~ecLlulll prokaryotic replication system, for example RK290, if it is
desired to retain the expression cassette on a independent plasmid rather than having it
integrated into the Ti-plasmid. By means of a helper plasmid, the expression cassette may
2 5 be transferred to the A. tumefaciens and the resl-lting Llall~rolllled organi~.lll used for
L~a,~rol,lling plant cells. Conveniently, explants may be cultivated with the A. tumefaciens
or A. rhizogenes to allow for transfer of the e~ression cassette to the plant cells, and the
plant cells dispersed in an a~Lopliate selection ",~-li"." The Agrobacterium host will
contain a plasmid having the vir genes ~-~ces~,.,y for ~ldn~.rtl.
After trall.Çollllation, the cell tissue (for example protoplasts, explants or
cotyledons) is ~lal~r~ ,d to a legelleldLion m~ lm~ such as Murashige-Skoog (MS)m-oflil-m for plant tissue and cell culture, for formation of a callus. Cells which have been
transformed may be grown into plants in accordance with conventional ways. See, for
example, McCormick et al., Plant Cell Reports (1986) 5:81-84. The Lldl~.rolllled plants
3 5 may then be analyzed to determine whether the desired gene product is still being produced
in all or a portion of the plant cells. After ~ ression of the desired product has been
CA 0223~801 1998-04-24
WO 97/1~678 PCT~US96/173SI
demo~ al~d in the plant, the plant can be grown, and either pollinated with the same
transformed strain or different strains and the resulting hybrid having the desired
phenotypic characteristic i~ntifi~c~ Two or more ge~ tions may be grown to ensure that
the subject phenotypic characteristic is stably m~int~in~-~ and inherited.
- 5 To identify the desired phenotypic characteristic, transgenic plants which contain
and express a given SPS tr~n.cg,on~ are cc~ led to control plants. Preferably, transgenic
- plants are selected by mea~ul~ ,.ll of SPS activity in leaf, fruit and/or root. The SPS
activity may be periodically measured from various stages of growth through sc-lesccl~e
and compared to that of control plants. Plants or plant parts having increased or decreased
SPS activity colllpaled to controls at one or more periods are selecte~l Transgenic plants
exhibiting SPS activity from about 1 to 12 fold that of cont.ol plants are preferred, with
about 1 to 5 fold being more plcr~ ;d, depending on a desired secondary trait. The
activity can be compared to one or more other traits inrln~ling SPS type, ll~.nscli~ion
initiation type, translation initiation type, termin~ n region type, transgene copy number,
transgene insertion and placement.
When ev,qln~ing a phenotypic characteristic associated with SPS activity, the
transgenic plants and control plants are preferably grown under growth chamber,
greenhouse, open top chamber, and/or field conditions. Tdtontifir~tion of a particular
phenotypic trait and colllpalison to controls is based on routine ~ ir~l analysis and
scoring. Statistical dirr~.e.lces between plants lines can be acslosse(i by colll~alhlg SPS
activity between plant lines within each tissue type ~ essillg SPS. Expression and
activity are colll~aled to growth, development and yield paldlneLCl, which include plant
part morphology, color, llulllbeL, size, ~~im~neione, dry and wet weight, ripening, above-
and below-ground biomass ratios, and timing, rates and duration of various stages of
2 5 growth through senescence, inf In(ling vegetative growth, fruiting, flowering, and soluble
solid content including sucrose, glucose, fructose and starch levels. To identify tr~neg~onic
plants having other traits, the plants can be tested for photosynthetic and metabolic activity,
as well as end-product synthesis. For example, material isolated from tr~negenic plant
cells and plant parts such as leaf, fruit and root are measured for end-products such as
3 0 starch, sucrose, glucose, fructose, sugar alcohols, and glycine and serine from
photorespiratory metabolism following standard protocols. SweeLlless based on sugar
content, particularly fructose, can be tested as well. For some plants, it may be nPces.s~ry
to modify growth conditions to observe the phenotypic effect. As an example, oxygen,
carbon dioxide and light can be controlled and measured in an open gas chamber system,
3 5 and carbon partitioning measured by C'4 labeling of carbon dioxide or other metabolic
substrates. Carbon partitioning also can be deterrnined in extracts from fruit, leaf and/or
root by chromatographic techniques or by Brix using a sugar refractometer. Thesecharacteristic also can be compared against or in-1uce-1 by growth conditions which vary
CA 0223~801 1998-04-24
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gas exchange pala~ Lt:l~, light quality and quantity, L~lllpelatule~ substrate and moisture
content bcLweell lines within each type of growing condition.
The following examples are offered by way of illustration and not by way of
limit~tic)n.
F,XAl~PT ,F.S
Fx~n~le 1
p,.rific~tion of Sucrose Phosph~t~ Synth~ of Corn
1.1 - Mt?thod of det~ll"il~ion of en7,ym~rir ~tivity (SPS)
10 During purification SPS activity is followed in 2 ways:
a) either by means of a colo~ lt;Ll;c test (Kerr et al., Planta., 1987, 170:515-519) called
resorcinol test described below.
Sucrose Phosphate Synthase catalyzes the reaction:
UDPG + Fructose 6-P < = > Sucrose 6-P + UDP
UDPG : Uridine Di-Phospho Glucose
Fructose 6-P or F6P: Fructose 6-Phosphate
Sucrose 6-P : Sucrose 6-Phosphate
The sucrose 6-P formed reacts with the resol~;illol to give a red-colored compound
~I"~"Iiri~hle by spectrophotc)llleLl~/ at 520 nm (nanometer) (Optical Density (O.D.) = 520
nm). In practice, to 45 ~11 (microliter) of enzymatic plepalaLion 25 ~11 of a buffered
solution cont~ining the two substrates is added (UDPG 70 mM, F6P 28 mM, MgCI2 152 5 mM, HEPES 25 mM pH 7.5). After inrub~tion at 37~C, the reaction is stopped by adding
70 ,ul of NaOH in solution and heating at 95~C during 10 min. After cooling, 0.25 ml of a
solution 0.1% resorcinol in ethanol 95% is added; then 0.75 ml of HCI 30% is added. The
OD at 520 mm is read after inr~b~tion for 8 min at 80~C, and cooling.
b) or by means of a coupled enzymatic system (Harbron et al., Anal. Biochem. 1980,
30 107:56-59) being composed in the following way:
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UDPG + F6P < = > Sucrose 6-P + UDP
SPS
UDP + ATP < = > ADP + UTP
Nucleoside Diphosphokin~e NP2K
ADP + PEP < = > Pyruvate + ATP
Pyruvate kinase PK
- Pyruvate + NADH < = > NAD + lactate
Lactate dehydrogenase LDH
The disa~pe~lance of the NADH absorption at 340 mn is monitored: 1 mole of
NAD formed or 1 mole of NADH co~ ...r~l collespol~ds to 1 mole of sucrose 6 P formed.
In practice, in a quartz ~ecLlo~hotometric tun thermostated at 37~C, the following
solution are added.
- 540 ,ul of HEPES buffered 50 mM, MgCI2 10 mM, KCI 20 mM pH=7.5,
- 250 ~11 of a llli~LUle of substrates PEP (1.6 mM NADH 0.6 mM1 ATP 4 mM UDPG 112
mM),
- 60 ~l of an enzyme mixture (LDH 166.7 U/ml PK 333.3 U/ml, NPzK 66.7 U/ml),
- 100 ~11 of F6P 112 mM.
After homogenization, 50 ,ul of the preparation cont~inin~ SPS is added, the
2 0 ~imimltion of optical density at 340 nm is added with a spectrophotometer (WIKON 860,
KONTRON h~Ll-llllcllL~i). The lneasul~ is done with the kinetic of the m~rhin,o.
1.2 Purifie~tion of th.o SPS (prep~r~tion of th~ im m--no~
1.2.1 F~xtraction
The starting material for the ~ul;fi~lion are nature leaves of young corn plants (Zea
m~ys L. cv Pioneer 3184), which have been harvested in late morning, cut up, deveined,
frozen in liquid nitrogen and stored at -70~C.
250 g of leaves are suspended in 1 liter of 50 mM HEPES 10 mM MgCI2, 1 mM
EDTA, S mM DTT, pH=7.5 buffer (extraction buffer) which has observed to it 11 g of
3 0 Polyvinyl-pyrrolidone, nitrogen is bubbled through and the suspension is cooled to 0~C.
The leaves are ground, until a homogeneous liquid is obtained. This ground product is
filtered, and then centrifuged at 14,000 xg for 20 mimltes at 4~C. While the bubbling
through of nitrogen is m~int~inP-l a solution of 50% polyethylene glycol (PEG 8000
"Breox" at 50% w/v of extraction buffer) is added to the ~u~e.llatant until a final
conce~LldLion of PEG of 6% is reached. Then the suspension is cooled at 0~C. After
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WO 97/15678 PCTAJS96/173SI
centrifuging at 14,000 g for 20 minutes tne supernatant has added to it 50% PE~G until a
final conce~ aLion of PEG of 12% is reached. After a repeated centrifugation, the
s--~,el~lat~ is discarded and the residue is solubilized with 60 ml of 50 mM HEPES, 10
mM MgCI., 1 mM EDTA, 5 mM DTT, 10% ethylene glycol (EG), 0.08 M KCI, pH 7.5
buffer (recovery buffer). This solution is clarified by centrifuging at 40,000 g for 10
minutes. The supernatant co~ es the final extract.
1.2.2 T ow plcs~ nion-exch~.~e chrorn~tr,gr~y: f~t-flow nF.~l~ Seph~rose
exch~r~er
The final extract is cl~olllat~graphed on a column 25 mm x 162 m~m of 80 ml of
Fast-Flow DEAE Sepharose (Pharrnacia) equilibrated with recovery buffer. After washing
the column with the same buffer, the l~loLt:hls adsorbed on the support are eluted by means
of a linear gradient with increasing ionic strength between 0.08 M KCI and 0.35 M KCI in
the 50 mM HEPES, 10 mM MgCI2, 1 mM EDTA, 5 mM DTT, 10% EG, pH 7.5 buffer
15 (buffer A). The flow rate applied during this e~ lle.ll is 180 ml/h and chromatography
is executed at 4~C.
The SPS activity is eluted at about 0.17 M KCl.
1.2.3 Chrom~t~r~ny on hPp~rin Seph~rose
The fractions cont~ining the SPS activity are collected and diluted to one fifth in
buffer A, then added to 12 ml of heparin Sepharose previously equilibrated with buffer A.
After one hour of inrnb~tion with gentle agitation at 4~C, the gel is washed witn about 10
volumes of buffer A + 0.05 M KCI, then repacked in a cmulllatugraphy column.
The proteins adsorbed are eluted in an isocratic way by means of a 10 mM CAPS,
25 10 mM MgCI2, 1 mM EDTA, S mM DTT, 10% EG, 0.01% Tween 80, 1 mg/m'l heparin,
1% Fructose, 0.25 M KCI, pH 10 buffer, delivered at 60 ml/h. Cl~olllaLography isexecuted at 4~C. The fractions cont~ining the SPS activity are collected (heparin fraction)
and preserved on ice until the following purification stage. The enzyme at this stage is
stable for a least one week.
The following purification steps are carried out using a system of High Pe.rollllance
Liquid Cnromatography (HPLC); the purification is followed by means of a detector fitted
with a filter enabling absolbcn~;y in tne ultra-violet at 280 nm (A280) to be measured. The
buffers and the fractions recovered are kept at low ~ Idture.
35 1.2.4 High pelroll,-~,n~f ~nion-exch"~.~e chrom~togr~'rly Mono Q
The heparin fraction is diluted by adding one third volume of 20 mM
24
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WO 97/15C78 PCT/US96/17351
Triethanolamine, 10 mM MgCI2, 1 mM EDTA, 10 mM DTT, 3% EG, 0.3% Tween 80,
pH 7.5 buffer (buffer A) and loaded on an FPLC Mono Q HR10/10 column, (10 x 100 mrn
Ph~rm~ri~) previously equilibrated with the same buffer which has added to it NaCl (final
concentration 0.18 M). After the A280 has returned to 0, the proteins adsorbed on the
- 5 chromatography support are eluted by means of a salt-complex gradient with buffer A (~
above) and buffer B (buffer A + NaCI, 1 M) on a Mono Q column as shown below in
- Table 1.
T~hle 1
Salt Gradiellt for Mono Q Colllmn
tim~ (,mim-t~c) % B
0 18
0.1 24
24
19 26
23 26
33 31
38 31
41 100
43 18
The flow rate applied to the Mono Q column is 180 mlth. The SPS activity is eluted
between 0.26 and 0.31 M NaCI. The active fractions are collected together ("Mono Q
fraction").
1.2.5 HPT C on Hydroxyz~tite
The Mono Q fraction is loaded on an HPLC column of hydro~Lya~aLile 4 mrn x 75
mm neutralized with 20 mM KH~PO4/K~HPO4, 3% EG, 0.3% Tween 80, 5 mM DTT, pH
7.5 buffer. After the A280 absorbance has returned to 0, the proteins adsorbed to the
3 0 column are eluted by means of the following phosphate gradient using buffer A (~
above) and buffer B (the same as buffer A ~ itic)n~11y cont~ining but 500 mM Phosphate
of K) as shown below in Table 2.
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T~hle 2
Phosph~te Gr~tlient for E~ydro7cyap~tite Coll-mn
tim-? ~imlt~s) % B
0 2
11
9 13
14 13
29 40
31 100
1 0 32 100
The flow rate applied to the column is 60 ml/h. At this stage, the phosphate will partially
inhibit SPS activity and Lh~lefo~e it is ~limr~llt to c~lru1~te a specific activity and also a
purification factor (~ Table 1) at this stage. The SPS activity is eluted under these
conditions with about 60 mM phnsph~re. The active fractions are collected together and
constitute the HAC fraction.
1.2.6 HPT C on nF~F SPW
2 0 The HAC fraction is loaded on an anion-exchange HPLC column of Di Ethyl
Amino Ethyl type (DEAE-5PW) previously neutralized with a buffer of 20 mM
Triethanolamine, 10 mM MgCI2, 1 mM EDTA, 3% EG, 2.5 mM DTT, 2% betaine, pH
7.5 buffer (buffer A) + 0.15 M NaCI.
After the A280 absorbance h-as re~urned to 0, the ploleills adsorbed to the colurnn
2 5 are eluted by means of the following NaCI gradient using buffer A (~ above) and buffer
B (the same as buffer A but additionally cont~ining 1 M NaCI) as shown below in Table 3.
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W O 97/lS678 PCTAJS9~17351
T~hle 3
Salt Gradie~t for nF~F Colnmn
tim~ s) % B
0 . 15
0.1 20
22 35
27 35
100
31 15
The flow rate applied to the column is 60 rnl/h. The SPS activity is eluted with about
0.3M NaCl.
15 1.2.7 Prep~r~tion of the fin~l prep~r~tion- collrto~ .tion
The final pl~paldLion is concellLldted by HPLC chromatography on a Mono Q
HR5/5 exchanger (5 X 50 rnm, Ph~rm~ri~) and rapid elution. The DEAE 5PW fraction(or the G200 fraction) is diluted to two thirds with buffer A ~ 1.2.6) and loaded on the
column which previously has been neutralized with buffer A + 0.18 M NaCl. The
2 0 following gradient is then applied on the column using buffer A and B ~ 1.2.6) as shown
below in Table 4.
T~hle 4
Gradient for Col-r~ tion
timr (minntrc) % B
0 18
12 100
13 18
The flow rate applied to the column is 60 ml/h. The SPS activity is eluted with about 0.3
M NaCl. The final preparation is stored at -20~C until used.
The results obtained at the various purification stages in terrns of qll~nthirs of
proteins recovered and of SPS activity are sl~mm~rized in Table 5 below.
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W O 97/15678 pcTrus96/l73
T~hle 5
pllrifir~tion of Corrl SPS
Conr~ntr~tion Volnme
of ~lotei~
(~/rnl)
Ground 1 1000 0.05 0 100
product
Final 4 < < 8 60 0.30 6 144
Extract
DEAE FF0.4< <0.8 70 3 60 168
fraction
Heparin0.2 < < 0.425 9 180 90
fraction
Mono Q (0.02)4 30 -5 5 5
fraction
HAC (0-03)4 8 -5 -5 -5
fraction
Final 0.05 2 25 500 5
1 dLio
n
'sA = Specific enzymatic activity: 1 U corresponds to 1 ~lmole of sucrose
formed per minute per mg of protein at 37~C. The meas~n~e.l~,ll of the
quantity of ploleills iS carried out using the Bradford method. As Tween
illL~lr~les enormously with this method, it is not possible to determine the
proteins and then to r~ t~ an sA at the level of the stages cont~ining one.
Furthermore, as phosphate is an inhibitor of SPS activity, the del~lll,hlation
during the HAC stage gives an undc~e~l;...~t~ result.
2pI~= Purification factor
3Y= Yield. The increasing yield during the initial stages of purification can
be explained by the elimin~tion, during purification, of certain inhibitors of
SPS activity.
4( )= approximate value
5-= not deLt:llllhled
An SDS-PAGE profile at various stages of the purification process and the quality
2 0 of the final preparation is given in Figure 1. The 120, 95 and 35 kd ~r~ills are correlated
to the SPS activity. The 35 and 95 kd plo~eills are very likely breakdown products of the
120 kd protein as it can be shown by the nucleotide sequence coding for the SPS protein.
Furthermore, the antibodies directed against the 35 and 95 kd proteins also recognize the
protein 120 kd in imml-nodetection after membrane Lldl~rt:l, which demonstrates an
2 5 antigenic identity between these three pl~tehls (see below). It must be pointed out,
however, that the addition of protease inhibitors in the buffers during purification has not
enabled us to obtain a single 120 kd protein.
28
CA 0223~801 1998-04-24
W O 97/~5678 PCTAUS96/17351
Gel ~ on chiullla~ographies were carried out in order to de~~ hle the
apparent molecular weight of the native SPS protein. Briefly the HAC fraction was
concentrated by HPLC chromatography on a Mono Q H R 5/5 inchanger (~Q 1.2.7). The
active fractions were collected together (about 2 ml) and loaded on an G 200 column
5 previously washed with a buffer co~t~ining 20 mM triethanolamine 10 mM MgCI2 1 mM
EDTA, 3% E.G., 2.5 mM DTT, 2 % betain, 0.3 M NaCl pH 7.5. The SPS activity was
eluted with a major protein peak corresponding to an apparellL mass of 270-280 kda which
is in a~l~e~ with the results obtained by Harbron et al. (Arch. Biochem. Biophys.,
1981, 212:237-246) with the spinach SPS. It can be noted that the chloll,atography on a
1 0 TS lambda 60000 perm~ti~)n column lead to the elution of the SPS activity at a retention
time cullc~olldillg to an a~a~"L mass of 440 kda which is close to the value obtained by
Doehlert and Huber (Plant Physiol., 1983, 73:989-994) witb. the spinach SPS using an
AcA34 permeation column.
The SPS protein seems therefore to be a di or tetrameric protein having as the basic
15 sub-unit a 120 kda protein (homodimeric or homo-te~,dl"~ lic). The results of SDS page
analysis at various stages of purification are shown in Figure 1. The bands of ~roleil,s
visible at about 120 kd (1) 95 kd (2) and 35 kd (3) are correlated during the
chromatography stages, with the a~e~ ce of SPS activity in the ,espe~ re fractions.
2 0 FY~r~le 2
Process for ~hP Pl~ ion of Monoclo~l Anfibo-lies
nirected ~in.~t SPS
2.1 I.l...~ l ion.c
BALB/c mice were ;~ d by subcutaneous injection (pads and paws) according
to the following methodology: Day 0 injection of about 5 micrograms of ~lo~eil~s (or about
0.3 U SPS per mouse): Mono Q pool ern~ ifiell volume for volume with Freund's
Complete Adjuvant (FCA).
Day 14 injection of about 5 micrograms of p,otei"s (or about 0.3 U SPS per
mouse): Mono Q pool emlll~ifiPcl volume for volume with Freund's Incomplete Adjuvant
(FIA).
Day 27 Idem D14
Day 0 + 60 injection of about 20 micrograms of piotei"s: final pool in FIA
Day 0 + 90 injection of about 12 micrograms of ~,uLeh~s: final pool in FIA
Day 0 + 135 injection by intravenous route (IV) in the tail of about 20 micrograrns
of proteins: final pool.
Fusion is achieved 3 days after the IV i.. i~ ion.
29
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W O 97/15678 PCT~US96/173~1
The sera were removed at D34, D61, D98 and D159 in order to measure the
lIP response (~ sclcellillg).
5 2 .1.1 Scl ~ m~th~d
Two methods were used to detect antibodies specific to the SPS used for
immnni7~tions:
- detection method of antibodies inhibiting the SPS activity
- detection method of antibodies directed against the SPS (inhibiting or not).
a) Detection method of antibodies inhibiting the SPS activity
This method of screening allows the detection of antibodies which h,~el~,e with the
active site of the SPS or on a site close to the latter, and therefore prevent the access of
substrates. In practice, 70 ~11 of serum or of ~u~elllaLd~L of hybridoma culture diluted in a
15 suitable way was mixed with 70 ,ul of SPS ple~aldLion (Heparin fraction). After one hour
of inr~lb~tion at ambient ten~ dth,e, the residual SPS activity was deltllllilRd by coupled
enzymatic deLt~ ation (~ 1.1). The results are expressed as a ~lcellLdge of inhibition
as compared to the same SPS prepalation treated in the same way but without antibodies.
2 0 b) Detection method of antibodies directed against SPS (inhibiting or not)
This method is based on the pl~ i~Lion of the antibody-SPS complex by goat
anti-mouse IgG coupled to sepllaluse beads (GAM sepharose). In practice, 60 ~11 of serum
or supernatant of hybridoma culture diluted in any suitable manner were added to 60 ,ul of
SPS preparation (Heparin fraction). After 2 hours of i..~ bd~ion at ambient tt;lll~eldLure,
2 5 the mixture was added to 50 ,ul of 25 % GAM-Sepharose previously washed three times
with a buffer of 50 mM HEPES, 10 mM MgCI2, 1 mM EDTA, 10% EG, 5 mM DTT, pH
7.5. The Illi~l~le was in~-nb~t~tl overnight at 4~C with strong agitation. After centrifuging
the mixture for S .,.i....lt~c at 3000 rpm, the residual SPS activity in the s~ dLdllt was
determined by coupled enzymatic detellllil,dlion (~ 1.1). The results are expressed as a
30 percentage of precipitation (% prec.) as cu",~ared to the same SPS pll,pal~.tion treated in
the same way without antibodies.
2. 1.2 Results
10 mice were ;.. -.. I~i,Pd according to the protocol described previously. The
35 following table gives the results of the precipitation deLel...i.~tion.c carried out with the
h~lcloa~llisera of the 10 mice on D159. The sera are diluted to one two-hundredth.
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W O 97/15678 PCT~US96/173~1
T~hle 6
Percem~e ~lec~ ion of Antibody-SPS Comrlex
Mouse 1 2 3 4 5 6 7 8 9 10
% Prec. 45 22 32 64 36 30 22 16 39 37
Additional dilutions of the serum of mouse 4 give the following results:
T~hle 7
Pel.;cnl~e Plcc~ ion of Seri~l
n;lutionc of MollcP 4 Sen-m
nillltion % Plcc~ tion
1/20067
1/40048
1/60029
1/100020
The spleens of mice 1 and 4 were used for the fusion with myeloma cells.
2.2 Celhll~r fncion
The splenocytes of the mice were fused with myeloma cells of SP2/0-Agl4 mice
according to a ratio of 2:1 in the presence of 45~ polyethylene glycol 1500. The selection
of the hybridomas was effected by adding hypo~r~nthinP and azaserine to the culture
2 5 mP~ lm 24 and 48 hours after fusion.
The hybridomas were cloned and sub-cloned by the method of limited dilution.
2.2.1 Res--ltc of thP screeni~ of h~ybritlc ~n~i clonPC
Results from scle~.~ing of hybrids, clones and sub-clones are shown below in Table
30 8.
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W O 97/15678 PCT~US96/17351
T~hle 8
~Iybrid. Clon~ antl Sub-clon~ Screenir~
~Iybri~lc
Mo-lce 4 (SpA filcion) Monce 1 (SPR filciorl)
2 positive hybrids out of 45 6 positive hybrids out of 52
SPA2: 38 % prec. SPB3: 17 % prec.
SPAl9: 7 % prec. SPB5: 67 % prec.
SPB8: 53 % prec.
SPB13: 68 % prec.
SPB25: 13 % prec.
SPB34: 17 % prec.
1 0 Clonec
SPA fusion SPR fusion
2 clones retained out of 36 7 clones retained out of 46
SPA2-2: 85 % prec. SPB3-2: 19 % prec.
SPA19-7: 8 % prec. SPB5-1: 76 % prec.
SPB5-2: 71 % prec.
SPBS-3: 45 % prec.
SPB54: 24 % prec.
SPB13-1: 79 % prec.
SPB13-2: 53 % prec.
Sub-Clon.oc
SPA fi-cion SPR filcion
sub-clones retained out of 48 sub-clones retained out of 72
SPA2-3 : 60 % prec. SPB3-2-19: 21 % prec.
SPA2-2-33: 33 % prec. SPB5-2-10: 86 % prec.
SPA2-2-25: 92 % prec. SPBS-4-2: 46 % prec.
SPB13-1-7: 87 % prec.
SPB13-2-2: 93 % prec.
2.2.2 Production of anti-SPS monoclon~l ~ntiho-lies
The hydridomas were injected by the intra-~ ,l~al route into female BALB/c
2 0 mice previously treated with p~ e. The monoclonal antibodies were partially purified
from ascites fluids precipitated wit_ 18% sodium s--lrh~te. The ~loLeillsso precipitated
were dissolved then dialyzed against PBS (F18).
2.2.3 Ch~racleli,i.lion of ~nri-SpS nnonoclon~l antibo~ c
2 5 a) Typing
The typing was done using an ELISA test. Anti-IgG rabbit and anti-IgM mouse
CA 02235801 1998-04-24
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antibodies (Zymed) were fixed at the bottom of the wells of a 96-well plate. After one
night at ambient ~ ,.dture the Imoccl-pied sites were saturated with a solution of 3 %
bovine serum albumin in PBS. After one hour of in~ bation at 37~C and several washes,
the various F18's were deposited in the wells. After in-nb~tion and several washes, goat
5 or rabbit antibodies, anti-class and anti-sub class mouse immlln~globulins linked with
peroxidlase, were added. After one hour at 37~C, the antibody type was icl~ntifitod using an
H202/ABTS system. All the anti-SPS monoclonal antibodies were found to be of IgG, type.
b) Inhibition of SPS activity
The delc.lllhlalion of the c~acily of the antibodies to inhibit the SPS activity was
10 carried out by the technique mentioned previously (~Q 2.1.1 a) using F18's. The results
are shown below in Table 9.
T~hle 9
Tnhibition of SPS Activity
Co~ l . d~ ion of
,AntihOdY ~ntiho(li~c (~T~/mV % Inhihition
SPA2-2-3 50 0
SPA2-2-22 50 0
SPA2-2-25 50 0
SPA3-2-19 50 0
SPA5-2-10 50 0
SPA54-2 50 0
SPA13-1-7 50 50
2.5 10
2.1
SPB13-2-2 50 60.1
59.1
33.8
2.5 14.2
8.7
c) Tmmnno-precipitation of the SPS activity
The ~ ion of the ability of the antibodies to immnn-)plecil~i~te the SPS
activity was carried out by the terhniqlle mentioned previously (~ 2.1,1 b) using F18's.
20 The results are shown below in Table 10.
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W O 97/15678 pcTrus96ll7351
T~hle 10
I,,,,,,,~,,o~.ee~l~iLi~tion of SPS A~tivity
AntibodY Con~ lion of % Prec~it~tion
~ntibodies (~/nnl)
SPA2-2-3 50 95
92
2.5 40
SPA2-2-22 50 95.7
51
48.2
2.5 25
10.1
SPA2-2-25 50 91.3
95.3
90.4
2.5 22.8
12.5
SPB3-2-19 50 95
27.8
2.5 17.8
9.3
SPB5-2-10 50 95
81.1
2.5 41.4
22.6
SPB5-4-2 50 95
86.1
2.5 57.2
26.1
SPB13-1-7 50 95
65.4
48.1
2.5 15
SPB13-2-2 50 95
71.8
2.5 43.5
34
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Fx~rr~le 3
Use of the Monnclon~l Antibodies for th~ Ch~rac~ ion
~n-i pllrific~tion of SPS
3.1 Ch~racteri7~tion of Corn SPS
This characterization was carried out with SPB3-2-19 and SPB13-2-2 antibodies bythe technique of immlmo-detection after Lldl~r~. of the ~lOt~ills from an electrophoresis gel
under denaluli.lg conditions (SDS-PAGE) on nitrocellulose ll,~l"b,dne (Western). After
electrophoretic separation in a 12.5% acrylamide gel (Nature 277(1970) 680-685), the
proteins were ~~a~r.,ll~d onto a 0.2211m nitrocellulose lllc;lllblal1e (Schleicher and
Schuell). The buffer was a standard electrophoresis buffer (3.03 g/l. TRIS base, 14.4 g/l.
Glycine, 0.1% SDS, pH 8.3, 20% m~th~nr~
After transfer, the membrane was put in a blocking bath (0.5% Casein in PBS).
After one hour at 37~C under gentle agitation, the membrane was washed 3 to 4 times in a
washing buffer (0.1% Casein, 0.5% Tween 20, in PBS) then inruh~tr-l with a solution of
10 micrograms/ml of the monoclonal antibody to be tested. A part of the membrane was
incubated in parallel with a non-imm-m~ antibody (negative control). After one hour of
inrllbat;on at ambient t~lllpclalule followed by 9 or 10 washes, the lllelllbldne was
2 0 inruhatecl in the plcsellce of an anti-mouse antibody labeled with '25I diluted in a washing
buffer (50,000 cpm per cm2 of llle--lbl~-ne). After one hour of inrnbatit~n at ambient
L~ alul~ followed by 9 or 10 washes, the l-l~,~llblalle was dried, then autoradiographed
(X-OMAT AR Kodak film and Crone XTRA Life Dupont amplifying screen). The resultsof the autoradiography are shown in Figure 2. In the autoradiograph, a strong signal is
2 5 observed at the protein bands 120 kd, 95 kd and 35 kd which correlates with the previous
results (see first part).
3.2 pllrffllr~tion of Sucrose Phosph~t~ Synth~e~ by Immnno~ffinity Chrom~t~r~hy
A mrtho~lology for the purification of corn Sucrose Phosphate Synthase on an
3 o immnnc)affinity support has been pc-rt;~;Led in order to increase the quantity of protein
recovered while reducing the number of L"llirlcaLion stages and to obtain qn~ntitirs
sufficient for protein sequencing.
3.2.1 Prepar~rion of the imm--nr,-adsorbe~t
The F18 (see 2.2.2) corresponding to the SPB13-1-7 antibody or to the SPB13-2-2
~ antibody were mixed with activated CH-Sepharose, (1 mg of antibody per ml of gel).
After inrllhation for 2 hours at ambient temperature, the sites not occupied by the
antibodies were saturated with lM ethanolamine, pH 9. The support was then washed
CA 0223~801 1998-04-24
W O 97/15678 PCT~US96/17351
alternately with 0.1M acetate, 0.5 M NaCl, pH 4 buffer and 0.1 M TRIS, 0.5 M NaCl~
pH 8 buffer. The immllno~fflnity support thus plc~al~,d was preserved at 4~C in a 50 mM
HEPES, 10 mM MgCl2, 1 mM EDTA, 1 mM PMSF, zero 0.01% sodium nitride (azide),
pH 7.5 buffer.
3.2.2 Imml-no~fflnit,y Chrom~t~r~
50% PEG was added to the Heparin fraction of SPS (see 1.2.3.) to give a final
collc~llLldLion of PEG of 20%. After in~lb~tion for 30 ~ s at 4~C with gentle
agitation, the llli.XLUlC was centrifuged at 1600 g for 30 mimlt~s. The protein deposit was
10 taken up in half of the initial volume with the 50 mM HEPES, 10 mM MgCl2, 1 mM
EDTA, 10% ethylene glycol, pH 7.5 buffer. This stage allows the previous buffer, which
is incompatible with the illnlllll,oafflnity clllulllatography~ step to be elimin~tt-d, and the
proteins to be concelllldled. The yield of SPS activity was from 80 to 90%.
The solution obtained was applied with a flow rate of 0.1 ml/min over 1 ml of
15 immnn( ~fflnity support packed in a column and on which had been fixed an antibody not
directed against the SPS (activated CNBr-Sepharose, on which an antineomycin antibody is
fixed). This first stage allows the elimin~ti- n of certain cnl.l;tlllil-~l~l~ which are fixed
nons~,ecirlcally on the chrollldtography support. The effluent of the non-specific column
was in turn applied to the anti-SPS immllno~ffinity support (2 ml in an 11 x 20 mm
2 0 column) with a flow rate of 0.1 ml/min. These two stages were carried out at laboratory
Le.~lpc.alllle. The column was washed with 10 ml of load buffer and then with a washing
buffer (load buffer with the addition of 0.25 M NaCl and 0.3% Tween 20) until absorbency
in ultra-violet at 280 nm was close to base level. The plo~eins adsorbed on the support
were eluted with a solution of 50 mM triethylamine, pH 11. This elution was carried OUt
25 at 4~C and the imml-no~ffinity column was leve.~ed to obtain an O~llilllUlll yield. The
SDS-PAGE profile of the final plepdldlion obtained co~ ollds to that obtained using the
standard protocol (see 1). It must be noted that the elution method of the ploLeills adsorbed
on the immlmn~fflnity support hl.,~.sibly destroys the SPS activity but the recovery yield
of the eluted SPS ploteills is optimal colllpdl~,d to tests carried out in native elution
30 conditions. The eluate of the immlm~ffinity column was dec~lt~d using a Sephadex G25
column, against a 0.14% Glycerûi, 0.07% 2-,llelcd~o-ethanol, 0.04% SDS, O.9 mM TRIS
pH 6.8 buffer (electrophoresis buffer in reducing conditions diluted 70 times). After
des~lin~tinn, the protein pl~Jdlalion was coluellLl~L~d 70 times with a collcelll~dtor under
vacuum and the SPS proteins were purified by SDS-PAGE (~ below).
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WO 97/15678 PCTrUS96/17351
F~n~le 4
Parti~l Sequeîlrir~ of SPS Polypeptides
4.1 pllrifir~tion of SPS Polypepti~lrs for Sequenriru~
Samples of a purified protein preparation obtained as described in Example 3.2.2.
5 were subjected to preparative SDS-PAGE. After electrophoresis, the protein bands were
vi~ li7tod with KCI tre~tm~onr as described by B~ all and Joernvall (Eur. Biochem.
(1978) 169:9-12) and the bands observed at 90kd and 30kd were excised. The ~ eills
from these gel fr~gmPrlt~ were electroeluted using an Electrophoretic CoIlce~ dLol
according to m~nnf~rtllrer's instructions (ISCO; Lincoln, NE) in 4 mM sodium acetate,
1 0 pH8. After electroelution, protein yields were y, ~ by cu..Ipalisoll to a bovine
serum albumin (BSA) standard on a Comassie Blue-stained gel. Approxirnately 30 mg of
the 30 kd protein and 75 ~g of the 90 kd protein were obtained.
4.2 T~yprir nigestion ~n~l Protein Sequenri~ of SPS polypeptides
The ~.oL~;i.. s were coIlce.. Lldled by acetone l~lL-,ipiL~Lion, and resuspended in 50 mM
ammonium carbonate buffer, pH 8. Tryptic digestion and HPLC purification were
p~,.ro--~Ied as described by Sturm and CLis~eels (Biol. Chem. (1987) 262:13392-13403).
Briefly, digestion was ~L.Ço.-lled by addition of trypsin (5% of SPS protein), and
inrllb~tion for two hours at 37~C. The digestion was then repeated. The proteins were
2 0 conceI-Lldted by Iyophiii7~fit)n and resuspended in 50mM sodium phnsph~tt? buffer, pH 2.2.
This mixture was subjected to reverse phase HPEC separation by application to a C18
column in phosphate buffer. Elution was pc.ru.-l.ed using an inc.easillg gradient of
acetonitrile. Eluted material from the phosphate buffer/aceLolliL,ile gradient was monitored
at 214 nm. The fractions corresponding to peaks of absorbance at 214 nm were collected,
25 Iyophilized, ,~ e~ ed in 0.1% trifluoroacetic acid, reapplied to the C18 column
(equilibrated with 0.1 % trifluoroacetic acid), and eluted using an ~c~Lo~ ile gradient.
Eluted material from the trifluoroacc-~ic acid/acetonitrile gradient was monitored at 214 nm.
The fractions corresponding to peaks of abso.l,ance at 214 nm were collected, Iyophilized,
and subjected to standard Edman degradation protein seqllenring on an ~ntom~t~ protein
3 0 sequencer (Applied Biosystems; Foster City, CA). Seq~Pnres of five peptides were
obtained. ~ Fig. 3 (SEQ ID NOS: 1-5).
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W O 97/lS678 PCTrUS96/17351
Fx~ le S
I~ol~tion ~nrl A~.omhly of a Full-T .on~th cnNA for SPS
5.1 RNA I.col~tion from Corn ! P~f
Total RNA was isolated from corn leaves (~ 1.2.1.) according to the method of
5 Turpen and Griffith (Biotechni~ues (1986) 4:11-15). Briefly, 250 gm of material was
homogenized in 4M gn~ni~inP thiocyanate and 2% sarcosyl. The ~ LLull, was then
centrifuged and the cleared ~u~ aLdllL was layered into a 5.7 M CsCI cushion andcentrifuged for 5.5 hours at 50,000 rpm. The RNA pellet was dissolved in water, extracted
with phenol and chloroforrn, and precipitated with ethanol. The resulting pellet was
10 ~e~ )ended in water. The final yield from the RNA isolation step was qn~ntit~t~d by UV
s~e~ ophotometry.
5.2 Poly~A) PNA T~ol~tion
A saturated suspension of cellulose powder/water was added to the RNA/water
15 mixture obtained in 5.1, at 10% of the total volume, to remove residual polysaccharides.
After centrifugation, the ~upellldldlll, co..l;.i.-i..g the RNA, was applied to an oligo(dT)-
cellulose column as described by Maniatis et al. (Molecular Cloning: A Laboratory
Manual, (1982) Cold Spring Harbor, New York). The fraction cont~ining the poly(A)+
RNA was then reapplied to the column. The eluted fraction cont~ining the poly(A)+ RNA
2 0 was extracted with phenol, and the RNA was p,~,cipildted with ethanol. Analysis by gel
electrophoresis showed complete absence of ribosomal RNA.
5.3 Con~t~-rtion of Tot~l Corn T P~f T ihr~ry
cDNA synthesis was performed according to the m~mlf~rtnrer's instructions
2 5 (RiboClone cDNA Synthesis System by P~lllc~d, Madison, WI), using five ,ug of
poly(A)+ RNA as template, except that M-MLV reverse l,a~ Ldse (BRL; Bethl-s~
MD) was substituted for AMV reverse L.alls~ dse. EcoRI linkers were added to theblunt-ended cDNA, and the resulting fr~gmPntc were cloned into an expression vector
(LambdaZAP, Stratagene; La Jolla, CA) according to the m~mlf~l~tnrer's instructions. The
30 resulting library contained approximately 1.5 x 106 llal~rOllllallL~.
5.4 PCR Gen~?r~tion of a p~rri~l SPS cnNA Probe
Using the sequence information from the peptides of Example 4 (SEQ ID NOS: 8-9)
and the polymerase chain reaction (PCR), a 1200 bp SPS cDNA fragment was generated.
35 Total corn leaf cDNA (5.3.) was used as a template, and degel.~"ale oligonucleotides (SEQ
ID NOS: 10-13), designed from two peptide sequences of the 30kd and 90kd SPS
38
CA 0223~801 1998-04-24
W O 97/1'.678 PCTnUS96/1~351
polypeptides, were used as primers. These primer sets were designated as CD3 (SEQ ID
NOS: 10-11) and CD4 (SEQ ID NOS: 12-13). ~ Fig. 4. PCR was carried out, according
to the m~mlf~rtllrer's instructions (GeneAmp DNA Amplifir~tion Reagent Kit and DNA
Thermal Cycler of Perkin Elmer Cetus; Norwalk, CT) except that the reaction was carried
5 out for 30 cycles, and t_e ~nn.o~ling steps were programmed to be at 50~C for 1 minute.
The PCR reactions were analyzed by agarose gel electrophoresis. Use of the correct set of
primers, CD3, resulted in a 1200 bp band being gene,aled by the PCR reaction. PCR using
the other set of l,lhlle~., CD4, gave no specific signals. ~ Fig. 5. Southern analysis (~
Fig. 5) coll~llllled that the PCR band was not an artifact. The probe 4K5 (SEQ ID NO: 14)
10 was used because the cc,..c~ ollding seq~lPnre of the probe was predicted to be within the
1200bp fragment if the fragment corresponded to tne SPS sequenre~ The probe hybridized
to the 1200 bp band generated by PCR using the primer set CD3 but not to PCR products
generated by the primer set CD4. ~ Fig. 5.
15 5.5 Isol~tion of SPS R~rtt~rioph~e T ~mhtl~ cnNA Clonrs
The 1200 bp PCR-~el~.aLt:d fragment was labeled with 32p (as per the Random
Primed DNA Labeling Kit, Bochlillge. Mannheim, T~ polis, IN) and used as a probeto screen approximately 250,000 plaques of the cDNA library (5.3.). The inserts of the
positive clones were analyzed by restriction analysis with EcoRI, and the clones witn the
20 longest inserts, SPS#3 and SPS#18, were selectr~l for furtber analysis. ~Q Fig. 6. A 0.4 kb
HindIII/EcoRI fragment from the S' end of SPS#3 was isolated, then labeled with 32p by
random priming (Random Primed DNA Labeling Kit) and used as a probe to re-screen the
library. Another clone, design~t~cl SPS#61, which extends further u~sLl~ ll than SPS#3,
was isolated. ~ Fig. 6. DNA seqllenring in-lir~tP~i that the 5' end of the SPS reading
2 5 frame was not reached.
To isolate cDNA clones that inrl~lde(l more of the 5' region than SPS#3 or SPS#61,
a new cDNA library was ~lGpalcd, as per Exarnple 5.3., (RiboClone cDNA SynthesisSystem by Promega; Madison, WI) using M-MLV reverse ll~.cli~L~se instead of AMV
reverse transcriptase. However, instead of using oligo (dT) as a primer, a syntnetic 17 bp
3 0 primer, 23B, derived from the 5' sequence of the SPS#61 clone, was used (~ Fig. 6).
This resulted in cDNAs that contain omy regions u~Ll~,dlll of the SPS#61 5' region. The
Iibrary was screened with tne 32P-labeled EcoRI insert from SPS#61, and 16 positive clones
were obtained. The clones witn the longest inserts, SPS#77 and SPS#90, were selected for
further analysis. DNA sequencing of SPS#77 and SPS#90 showed that the region of
3 5 overlap (greater than 100 bp) with SPS#61 was j~lentir~l in all clones, and tnat both
extended further u~Ll.,~ll into tne 5' region. ~ Fig. 6.
39
CA 0223~801 1998-04-24
W O 97/15678 PCTAJS96/17351
PCR was carried out using single-stranded cDNA (from a reverse transcriptase
reaction corn leaf RNA (5.2.) primed with oligo (dT) as described above) as template and
primers selecte(l from the SPS#90 and SPS#3 seq~enres, confirm~o~ that SPS#90 and SPS#3
originate from the same mRNA llallsc~ . The fragment resulting from this PCR reaction
5 was 750 bp in length, Co~ L~ with the size predicted from the DNA sequ~nre. The 750
bp fragment was subcloned into a Bluescript-derived vector as a Sall/HindIII fragment.
Four of the reslllting subclones were partially seq~l~onre~l, and the sequence obtained
m~trhPd the existing DNA seqll.onre.
1 0 5.6 A~semhly of th~ SPS l~ i~ Fr~m~
Both DNA strands of SPS#90, SPS#61, and SPS#3 were sequenced, using the
method of Sanger et al. (PNAS (1977) 74:5463-5467). All three sequences can be combined
to form one contiguous sequence of 3509 bp. ~ Fig. 7 (SEQ ID NO: 6). Primer
extension expe.il~ using corn leaf poly(A) RNA and an ~ .cel~e primer showed that
15 the 5' end of our DNA sequence repl~sellL~ seqllenres form the actual 5' end of the SPS in
RNA. In the SPS reading frame, as defined by the five peptide seqll~onres (SEQ ID. NOS.:
1-5 respectively) (~ Fig. 3), the first methionine codons are located at bp 112 and bp 250.
Fig. 7 (SEQ ID NO: 6). The codon at bp 112 is similar to the consel~us eukaryotic
translational start site (Kozak, Cell (1986) 44:283-292) and is located 54 bp dow~ ealll of
2 0 a TAG stop codon (bp 58). It is proposed that this codon represents the translational start
of the SPS polypeptide in vivo. After a 1068 codon reading frame, translation is stopped
by TGA. The following 193 bp contain the 3' untran.cl~t~(l region including a poly(A)
addition signal, AAATAAA.
The full-length SPS coding region can be assembled by cc,lllbillillg the 529 bp
25 BamHI/HindIII fragment of SPS#90, the 705 bp Hindlll fragment of SPS#61 and the 2162
bp HindlII/ EcoRI fragment from SPS#3 (~ Fig. 6).
F.Y~n~ple 6
n~t~o~tion of SPS Polypepti~l~s by Specific Anti.c.?ra
3 0 6.1 Prep~r~tion of Antibo(1i.o~ to SPS
Samples of purified protein preparations obtained by the method described in 3.2.2.
were subjected to SDS-PAGE eleckophoresis. The ploteills in the gel were fixed and
stained. The bands corresponding to the 90kd and 30kd polypeptides were excised. Using
CA 0223~801 1998-04-24
WO 97/15678 PCT~US96/17351
this material, polyclonal antisera were raised in rabbits by conventional procedures.
Western analysis (as described by Oberfelder, Focl~s (1989) 11(1):1-5) showed that the
antibodies isolated from the rabbit ;.. ,.i,~cl with SPS 30 recognized the bandscorresponding to the SPS#30 and SPS#120 peptides on a SDS PAGE gel, and that theantibodies isolated from the rabbit i.... i~Pcl with SPS#90 recognized the bands
corresponding to the SPS#90 and SPS#120 polypeptides (see Fig.8).
6.2 ~mmnnolo~ir~l T or~1i7~tion of SPS in th~ Corn Pl~nt
Total plUICillS were extracted from leaves of a 30 day-old corn plant, harvested at
10 11:00 am, by boiling in SDS buffer. The protein extracts were loaded on duplicate
SDS-PAGE gels. One gel was stained with Com~ Blue, while the other was subjectedto Western analysis, using a I~ Lul~ of SPS#30 and SPS#90 antisera as probe. ~ Fig. 9.
The ~lol,lille,lL bands appearing on the Comassie Blue-stained gel were iclentifiPd as
phosphoenolpyruvate carboxylase (PEPcase), an enzyme involved in C4 photo~yl~ esis.
15 The Western blot showed the plesel1ce of the SPS band. The SPS protein pattern was very
similar to the PEPcase protein pattern: not present in roots, nor present in the section of
leaf closest to the stem, nor present in very young leaves. This pattern corresponds with
s~ion associated with photo~yllLl.esis, and is the pattern expected for SPS.
2 0 F.-~m~7le 7
(~on.ctru~tion of Fxproc~ion Con~truct Pl~miti~
7.1 Con~trnl~tion of ~h.- fi~ll-len~h SPS r~ fr~mt?
Clone SPS#90 was digested with HindIII and ligated with the 705 bp Hindm
fragment from clone SPS#61 to create a plasmid co..~ the 5' end of the SPS coding
2 5 region. The res-llting plasmid was digested with BamHI and partially digested with
HindIII, resnlting in a 1340 bp BamHI/Hindm fragment cont~ining the 5' end of the coding
region. The 3' end of the SPS coding region was obtained by digestion of SPS#3 with
EcoRI and partial digestion with Hindm, resulting in a 2162 bp HindIIIlEcoRI fragment.
This 2162 bp HindIIIlEcoRI fr~gm~nt, carrying the 3' end, was ligated with the 1340
3 0 BamHI/~coRI fragment carrying the 5' end into a BamHI/EcoRI-digested pUC-derivative
plasmid Bluescript, to create a plasmid carrying the entire 3403 bp SPS coding region and
3' untr~n~l~tec~ Lldnscli~Lion L~lllhl-dLion region.
41
CA 0223~801 1998-04-24
W O 97tl5678 PCTAJS96/17351
7.2 FxI~ression of SPS in ~. coli
When cloning the 3403 bp BamHIlEcoRI SPS fragment into the plasmid Bluescript
SK (Stratagene, La Jolla, CA), a translational fusion between the plasmid coded lacZ
sequence and the SPS reading frame was created. The resulting fusion protein con~in.c 30
N-termin~l amino acids from the l~ tosi~ e and the complete SPS polypeptide. Thefusion protein was expressed in E. coli under the Bluescribe plasmid lacZ promoter.
Preparation of total protein followed by Western analysis using anti-SPS antisera (see 6.1.)
shows a band comigrating with native plant SPS. For the SPS activity test, the E. coli cells
cont~ining the SPS expression construct as described were opened with Iysozyme and
1 0 sonication. Soluble protein was ~ s~lt~ by a Sephadex G-25 column. This protein extract
was assayed for SPS activity analogous to the method described in l.l.a., except that the
reagent anthrone was used instead of resorcinol (Handel, Analytical Biochemistry, (1968)
22:280-283). This test showed that the SPS protein, expressed from the cDNA in E. coli
does have SPS enzyme activity. By colllpdlison to native plant enzyme it seems to have the
1 5 same specific activity.
7.3. Conctru~tion of thP Tobacco Sm~l~ Subnnit (SSU) Prom~ ter-Tr~n~- r~?tion~l F lcion.c
The SPS coding region can be conveniently cloned as a BamHIlEcoRI (bp 106 - bp
3506) fragment 3 ' of a tobacco small subunit promoter. A SSU ~lolllolel for expression of
2 0 the SPS coding region, was ~ al~d as follows. The SSU promoter region from
pCGN627 (described below) was opened by 1~7nI and the 3' ove~lldllg removed. After
Eco~l digestion, the 3403 bp BamHI (filled in) EcoPI SPS cDNA fragment (~, Example
7.1.) was inserted. After the SPS coding region was ligated into the SSU promoter, the
SSU/SPS region was ligated into a binary vector and integrated into a plant genome via
2 5 Agrobacterium tumefaciens-mPrli~tto~ al~ro-llla~ion. (The SPS region carries its own
llanscliL1lion ~ hldtion region in the cDNA seq~lenre). Insertion of the SSU/SPSconstruct into the binary vector pCGN1557 resulted in pCGN3812.
pCGN627
3 0 The 3.4 kb EcoRI fragment of TSSU3-8 (O'Neal et al., Nucleic Acids Res. (1987)
15:9661-8677), cont~ining the small subunit promoter region, was cloned into the EcoR~
site of M13mpl8 (Yanisch-Perron et al, Gene (1985) 53:103-119) to yield an M13 clone 8B.
Single-stranded DNA was used as a template to extend the oligonucleotide primer "Probe
42
CA 02235801 1998-04-24
W O 97~15678 PCTAJS96/173~1
1 " (O'Neal et al., Nucleic Acids Research (1987) 15:8661-8677) using the Klenowfragment of DNA polymerase I. Extension products were treated with mung bean ml~ e
and then digested with HindIII to yield a 1450 bp fragment co..l;.;..;,.g the SSU promoter
region. The fragment was cloned into HindIII-SmaI-digested pUC13 (Yanisch-Perron et
5 al.. Gene (1985) 53:103-119) to yield pCGN625. pCG2J625 was digested with HindTJ~, the
~ ends blunted with Klenow, and the digested plasmid re-digested with EcoRI. The
EcoRI/blunted-HindIII fragment cont~ining the SSU ~umoLel region was ligated with
SmaI/EcoRI-digested pUC18 to yield pCGN627.
10 7.4. con~trllrtion of a C~ V Promot~r--SPS Tl~"~ ;on~l Fllcion
The 35ESS promoter-DNA fragment from cauliflower mosaic virus was fused to the
SPS DNA as follows. The plasmid pCGN639 was opened by BamHI and EcoR~ and the
3403 bp BamHI-EcoRI SPS cDNA fragment (described in Example 7.1) was cloned intothis plasmid. The hybrid gene was removed from this plasmid as a 4.35 kb X~aI-EcoRI
15 fragment and ligated into a binary vector (McBride and .S~ r~lt, Plant Mol. Bio.
(1990) 14:269-276) and integrated into a plant genome via Agrobacterium tumefaciens
m~ t~-d tral~Ço~ ation. Insertion of the CaMV/SPS co~ into the binary vector
pCGN1~57 (McBride and Su~ r~lt supra) results irl pCGN3815.
2 0 7.4.1. Con~tru~ tion of pCGN639
pCGN164 was digested with EcoRV and BamHI to release a EcoRV-BamHI
fragment which cont~in~d a portion of the 35S promoter (bp 7340-7433). pCG8638 was
digested with Hindm and EcoRV to release a HindIII-EcoRV fragment cont~ining a
different portion of the 35S promoter (bp 6493-7340). These two fr~gm~ontc were ligated
2 5 into pCGN986 which had been digested with HindIII and BamHI to remove the
HindIII-BamHI fragment cont~ining the 35S-l~iulllolt:l; this ligation produced pCGN639,
which contains the backbone and tml-3' region from pCGN986 and the two 35S promoter
fragments from pCGN164 and pCGN638.
30 7.4.2. Con~truction of pCGN164
The AluI fragment of CaMV (bp 7144-7735) (Gardner et al., Nucl. Acids Res.
(1981) 9:2871-2888) was obtained by digestion with AluI and cloned into the HincII site of
M13mp7 (Vieira and Messing, Gene (1982) 19:259-268) to create C614. An EcoR~ digest
43
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of C614 produced the EcoFU fragment from C614 cont~ining the 35S promoter which was
cloned into the EcoRl site of pUC8 (Vieira and Messing, supra) to produce pCGN146. To
trim the promoter region, the BgllI site (bp 7670) was treated with BglII and Bal31 and
subsequently a BglII linker was ~tt~rhPd to the Bal31 treated DNA to produce pCGN147.
5 pCGN147 was ~ligested with EcoRllHphI and the reslliting EcoRI-HphI fragment
cont~ining the 35S promoter was ligated into EcoRI-SmaI digested M13mp8 (Vieira and
Messing, supra) to create pCGN164.
7.4.3. Con~tT U~tion of pCGN638
Digestion of CaMV10 (Gardner, et al., Nucl. Acids Res. (1981) 9:2871-2888) with
BglII produced a BglII fragment co,.l~;..i..g a 35S promoter region (bp 6493-7670) which
was ligated into the BamHI site of pUC19 (Norrander et al., Gene (1983) 26:101-106) to
create pCGN638.
15 7.4.4. Con~trurtion of pCGN986
pCGN986 contains a cauliflower xnosaic virus 35S (CaMV35) promoter and a
T-DNA tml-3' region with multiple restriction sites beLweell them. pCGN986 is derived
from another c~csett~, pCGN206, co..l~ i..g a CaMV35S promoter and a different 3'
region, the CaMV region VI 3'-end and pCGN971E, a tml 3' region. pCGN148a
20 cont~ining a promoter region, selectable marker (k~ yeill with 2 ATG's) and 3' region,
was prepared by digesting pCGN528 with Bgm and ins~.Lillg the BamHI-BglII promoter
fragment from pCGN147 (see 7.4.2. above). This fragment was cloned into the BgnI site
of pCGN528 so that the Bgm site was proximal to the kanamycin gene of pCGN528.
The shuttle vector used for this construct pCGN528, is made as follows: pCGN525
25 was made by digesting a plasmid co..l;~;..;..g Tn5, which harbors a k~ ychl gene
(Joigellsell el al., Mol. Gen. Genet. (1979) 177:65), with HindIII-BamHI and inserting the
HindIII-BamHI fragment co..~ g the kanamycin l.~ re gene into the HindIII-BamHI
sites in the tetracycline gene of pACYC184 (Chang and Cohen, J. Bacteriol. (1978)
134: 1141-1156). pCGN526 was made by illS~l~hlg the BamHI fragment 19 of pTiA6
3 0 (Thomashow et al., Cell (1980) 19:729-739) modified with XhoI linKers inserted into the
SmaI site, into the BamHI site of pCGN525. pCGN528 was obtained by cleleting the small
XhoI and religating.
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pCGN149a was made by cloning the BamHI ka~nycin gene fragment from
pMB9KanXXI into tbe BamHI site of pCGN148a. pMB9KanXXI is a pUC4K variant
(Vieira and Messing, Gene (1982) 19:259-268) which has the X71oI site mi.csing but
cont~in~ a functional kal~my~ gene from Tn903 to allow for efficient selection in
5 Agrobacterium.
pCGN149a was ~~ipestrd with HindIII and BamHI and ligated which pUC8 (Vieira
and Messing, supra) digested with HindIII and BamHI to produce pCGE169. This
removes the Tn903 k~l~llychl marker. pCGN565 and pCGN169 were both digested withHindIII and PstI and ligated to form pCGN203, a plasrnid cont~ining the CaMV 35S10 plullloLel and part of the 5'-end of the Tn5 k~~ yci,l gene (up to the PstI site, (Jolgense
etal., A~ol. Gen. Genet. (1979) 177:65). pCGN565 is a cloning vector based on
pUC8-Cm (K. Buckley, Ph.D. Thesis, UC San Diego 1985), but cont~ining the polylinker
from pUC18 (Yanisch-Perron et al., Gene (1985) 53: 103-119). A 3 ' regulatory region was
added to pCGN203 from pCGN204 (an EcoRI fragment of CaMV (bp 408-6105)
15 cont~ining the region VI 3' cloned into pUC18 (Gardner et al., Nucl. Acids Res. (1981)
9:2871-2888) by digestion with Hindm and PstI and ligation. The reslllting cassette,
pCGN206, is the basis for the construction of pCGN986.
The pTiA6 T-DNA trnl 3'-seql~enres were subcloned from the Baml9 T-DNA
fragment (Thom~chnw et al., CeU (1980) 19:729-739) as a BamHI-EcoRI fragment
2 0 (nucleotides 9062 to 12,823, numbering as in Barker et al., Plant Mol. Biol. (1983)
2:335-350) and combined with the pACYC184 (Chang and Cohen, J. Bacteriol. (1978)134: 1141-1156) origin of replication as an EcoRI-HindII fragment and a gellla,llyci"
reSict~nre marker (from plasmid pLB41), (D. Figurski) as a BamHI-HindII r~ag.~ lL to
produce pCGN417. The unique SmaI site of pCGN417 (nucleotide 11,207 of the Baml92 5 fragment) was changed to a SacI site using linkers and the BamHI-SacI fragment was
subcloned into pCGN565 to give pCGN971. The BamHI site of pCGN971 was changed toan EcoRl site using linkers to yield pCGN971E. The reslllting EcoRI-SacI fragment of
pCGN971E, cont~ining the tml 3' regulatory seqnenre is joined to pCGN206 by digestion
with EcoRI and SacI to give pCGN975. The small part of the TnS k~i~nlycil- resict~nre
3 0 gene was deleted from the 3 '-end of the CaMV 35S promoter by digestion with Sall and
BglII, blunting the ends and ligating with SalI linkers. The final expression cassette,
pCGN986, contains the CaMV 35S pro..,oL~, followed by two Sa~ sites, an XbaI site,
BamHI, SmaI, KpnI sites and the tml 3 ' region (nucleotides 11207-9023 of the T-DNA).
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A sr~ ir ~ y of the construction of the various plasmids is shown in
Figures lOA through lOC.
Fx~ le 8
TrAn~enir SPS Tom~to Pl~ntc
8.1 Prodllrtion of Tic~ Specific SPS "Sen~" Tld~-~gel-ic Tom,t-- Pl,ln
Tomato plants were Llal~Ço~ ed with e~ ,ssion c .~settes cont, ining SPS encoding
sequences (pCGN3812, pCGN3815, pCGN3342, and pCGN3343) via Agrobacterium
tumefaciens m~rliAt~ lansrullllalion (Fillatti, et al., Bio/Technology (1987) 5:726-730) and
legelleldl~d. P~ dldtion of pCGN3812, a tobacco SSU/SPS construct, and pCGN3815, a
CaMV 35S/SPS construct are described in Examples 7.3 and 7.4, ~ eclively. The fruit-
specific E8/SPS constructs pCGN3342 and pCGN3343 were ~le~al~,d as described forpCGN3812 with the following mofiifir~ltions. Appro~cim ltPly 2.1 kb of the 5' region
c~ ndillg to the tomato derived E8 fruit-specific promoter replace the SSU promoter
1 5 region in pCGN3812. The E8 promoter is described in Deikm~nn et al. (1988) EMBOJ,
2:3315-3320; and Della Penna et al. (1989) Plant Cell, 1:53-63. The pCGN3342 andpCGN3343 constructs also contain a SPS cDNA sequence llull~al~d at the ApoI site just 3'
of the SPS coding region (at nucleotide 3318), and fused to a 1.2 kb region of the A.
tumefaciens tml 3' t~lll~indtol region from pTiA6 (Barker et al., (1983) Plant Mol. Biol.,
2 0 2:335-350; sequence 11208-10069 of the T-DNA region from A. tumefaciens Ti plasmid
pTilS955). Constructs pCGN3342 and pCGN3343 L~,~reSelll opposite orientations of the
E8-corn SPS-tml insert in the binary vector pCGN1557, which contains the kanamycin
nptII marker gene under the control of the CaMV 35S ~lulllotel region and the tml 3'
L~llllillalul region described above for pCGN3318 (McBride and Sumerfelt, Plant Mol.
Biol. (1990) 14:269-276). Tomato plant lines are design~t~d with a llulllbc~ corresponding
to the construct used for L-dl~Çollllalion. Tomato lines arising from se~aldîe llalL~rol.~lalion
events are signified by a hyphen and a number following the construct/plant designation.
8.2 Immlmoblot Resllltc
Leaves from transformed tomato plants (pCGN3812 and pCGN3815) and control
tomato and corn leaves were tested as described in Example 6.2 for SPS activity using the
SPS #30 and SPS #90 peptide polyclonal antisera of Example 6. No cross l~,aclivily
between the antisera and the control (endogenous) tomato leaves was seen. This inrli
46
CA 0223~801 1998-04-24
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that the corn and tomato SPS are not highly related. As to the transgenic tomato plants,
leaf extracts from tomato plants cont~ining the pCGN3815 or pCGN3818 constructs
showed signals up to levels several times those observed in the ullLldl~r~lmcd corn leaf
extracts.
8.3 SPS Artivity
Leaf extracts also were tested for SPS activity according to the lci-olcillol protocol
described in Example l.l.a. In colllpalison to leaf extracts from control plants, leaves from
transformed tomato plants co~ ;nil~g the SPS gene showed up to 12-fold increases in SPS
10 activity. Higher SPS activity also was observed in some leaf extracts from lldilsge.lic
tomato plants cont~ining the corn SPS gene as co~ ared to control corn leaf extracts.
8.4 St~rch ~nrl Sucrose T ~vel.c
Leaf tissue was analyzed for starch and sucrose levels according to the method of
15 Haissig, et al., Physiol. Plan (1979) 47:151-157. Two controls were used, leaves from an
untrans~ormed plant and leaves from a Llal~rulll~lt which did not show any corn SPS
immnnnblot signal. The starch and sucrose levels of these two plants were essrnti~lly the
same, and had an almost equal pcrc~.lL~ge of starch (mg/lOOmg dry weight) and sucrose
(mg/lOmg dry weight). High-e~ shlg plants cont~ining pCGN3812 (pCGN3812-9 and
2 0 pCGN3812-11) showed both a reduction in leaf starch by 50% and an increase in sucrose
levels by a factor of two. Thus, the extra sucrose ~yllLhesis provided by the exogenous SPS
activity had a profound affect on carbohydrate partitioning. These data indicate that the
presence of high levels of corn SPS activity rrc--lting from a sllfficierlt level of tr2~n~genir
expression of a SPS tr~n.~gellP funrtion~l in tomato leaves cause a mnrlifir~tion of
2 5 carbohydrate partitioning in this tissue.
8.5 Oxy~en Se..~iliviLy
The interaction belvv~ phoLo~y"Ll,esis and the synthesis of end products in
tomatoes e~plessillg corn SPS was evaluated by gas e~rh~nge analysis. Oxygen se.lsilivily
3 0 of plants was inrlllre-l by lowering growth telllpeldLure and then ~2 sel~iLiviLyllleasuled as
the rate of photo~yl~L~esis in low ~2 (Sage and Sharkey (1987) Plant Physiol. 84:658-664).
Photosynthesis of tomato plants e~L~lessillg corn SPS became oxygen insensitive at
14.2~C (measured in 35 Pa CO2), whereas untransformed controls became in~e~ ive at
17.3~C. Change in the growth temperature from 22~C to 30~C during the day did not
47
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W O 97/15678 PCTrUS96/17351
affect this pattern. Furthermore, the transformed plants did not ~cclim~tr following growth
at high CO. (Worrell et al. (1991). The Plant CeU 3:1121-1131). These data show that
the SPS e"~ ,shlg plants have a reduced ceiling imposed on photosynthesis by end product
synthesis at lower te~ aLLIr,_s. The data also show that the temperature at which
5 photosynthesis becomes oxygen h~sel~iLi~re can be modulated by SPS activity through its
effect on chloroplasts, photosynthetic capacity and end product synthesis and sink
transport/conversion.
8.6 Te~ ,lallllc Fffect on P~rtitionir~
l O The effect of temperature on starch and sucrose partitioning was evaluated in tomato
plants transformed with pCGN3812 (~_ 7.3). The Lldl~rollllcd tomato plants were
colll~ared to control UC82B plants. The rate of starch plus sucrose synthesis as a function
of IC111~CIdLU1~, was assayed by feeding a pulse of 14 C02 to leaves at a normal partial
~,essu~c then chasing with llnl~beled CO2 for a long enough period of time to permit
15 incorporation of the labeled carbon into starch, sucrose, r.u.;Lose, glucose or another end
product but for a short enough period of time so that very little of the carbon was CA~O1 Lcd
from the leaf source tissue. Analysis of end product synthesis showed that sucrose
synthesis appeared more sensitive to Lclll~c.dlule than did starch synthesis. ~or example,
plants e,L~-cssh~g about 5-fold more SPS activity colll~aled to controls did not partition
2 0 more carbon to sucrose at the lowest tcl~ lalule. This in-lirates that the control
cocrrciclll for SPS approaches zero as metabolic activity of the plant is reduced with
lcll-~.dture under these conditions. The additional SPS activity also changed the oxygen
sensitivity in this same te-..~c,.dLu.c range. The above results show that partitioning
between starch and sucrose, end-product synthesis/sink transport and conversion can be
25 modulated as a function of telll~cldlulc. (~ Fig. 11).
8.7 ~
Manipulation of yield by mo~lifir~tion of end-product ~yllllle~is is related to growth
conditions and reproductive/vegetative sink. The effect of growth conditions on tomato
3 0 yield was evaluated in homozygous SSU/SPS (Rubisco small subunit promoter-SPS),
35/SPS (CaMV 355 promoter-SPS) and E8/SPS (E8 fruit-specific promoter-SPS) tomato
plant lines grown under growth chaullber~ open-top chdlllber and field conditions following
standard methods in the art.
When compared to ull~ldn~r~rmed tomato plants~ variation in yield increase was
48
CA 0223~801 1998-04-24
WO 97/15678 PCTAJS96/1~351
observed in the growth ch~mher, open-top ch~mher and field trials. Dirr.,..,nces observed
in fruit yield may be due to earlier flowering and the number of fruits set and filled for
plants grown in growth ~h~mhers and pots co~ dled to those grown in the field. Also,
tomatoes eA~l.,ssi,lg SPS behind the CaMV 35S p~ lolef grew better than tomatoes5 e~lessing the gene behind a Rubisco small subunit promoter under growth chamber
conditions. These data indicate a promoter effect. Additionally, studies in ~ tldlUlC
controlled growth rooms show that there was more yield penalty in the SPS tomatoes at
low ~ eld~ules than at high ~ cldLul~. These data are in accolddllce with the
partitioning data showing a reduction in mo~ tion of sucrose levels at low ~ c;ldthle in
1 0 tomato plants.
8.7.1 Soluble Soli-l~ Tn T2 SSU/SPS Tom~t~- Pl~nt~ Grown Un~lPr Growth Ch~mher ~n-l
Greenhouse Con-lition~
Leaf-specific SSU/SPS tomato lines 3812-9 and 3812-11 were evaluated for solublesolid content. Extracts of fruit from these tomato lines and controls were grown and
harvested in a Biotron growth ch~ hel or under standard greenhouse conditions and served
as the tissue source. T2 plants from the 3812-9 and 3812-11 lines were segle~dlillg as the
original lines were shown to contain at least two SSU-SPS insertions. For growth chamber
20 conditions, T2 plants were ill~ r~l by metal halide lamps at peak level of 500 ~mol
photons/m/s (pot level), at a ~e~ dLulc; of 26~C for the 16h day and 18~C at night, and a
relative hllmi-lity of 60%. Plants were watered daily with half-strength Hoagland's
solution (Hoagland and Arnon, Calif. Argicult. Exp. Sta. Cir. (1938) 357:1-39). Soluble
solids were evaluated as Brix units per unit weight fruit tissue llleasured for the average of
2 5 three fruits per plant. Transgenic SSU/SPS plants grown under growth ch~lll)el conditions
exhibited substantial increases in soluble solids compared to controls. The soluble solids
measured in a segregating T2 population of 3812-11 plants grown under greenhouseconditions showed the same effect, but overall hlcl~ases were reduced colllpa,ed to
SSU/SPS plants in growth chamber tests.
8.7.2 Soluble Solic1~ In T4 SSU/SPS ~n~ 35S/SPS Tonn~tr~ Pl~nt~ Grown Un-il?r
Greenhon~ Con-lition~
Homozygous SSU/SPS tomato lines were generated from original SSU/SPS 3812-9
3 5 transfol,lldllL~ in UC82-B tomatoes following standard products. Two homozygous lines
49
CA 0223~801 1998-04-24
W O 97/lS678 PCTnJS96/17351
desi~n~lPd A and B were grown under greenhouse conditions and fruit evaluated for
soluble solid content using Brix analysis measured per unit weight fruit tissue. Soluble
solids were measured as an average of three plants per line and three fruit per plant. The
average soluble solid content for the SSU/SPS 3812-9 lines was increased si~ ly
5 compared to the UC82-B controls. The data was shown to be signifîc~nt at a 0.01 % level
(99%), according to least .signifi~nt dirrclcilce (LSD) st~tistir~l analysis.
Homozygous lines of tomato plants lldl~rol,-led with the 35S/SPS construct of
pCGN3815 were geneldlcd to cGlllpdle the homozygous leaf-specific SPS construct results
to homozygous col~LiLuLive e,~lc~sion construct. In one line, desi_n~t~d 3815-13-2, a
1 0 subst~nti~l increase in fruit yield was observed, as measured for both fruit size and fruit
number, compared to non-Llol~rol.lled controls and, surprisingly, compared against the
SSUtSPS leaf-specific homozygous line controls. The 3815-13-2 plants also produced a
second flush of fruit.
15 8.7.3 Soluble Solitls In Field Grown T4 SSU/SPS Tom~to Pl~nts
Tomato plants holllo~golls for the SSU/SPS construct were ge~ dlrcl from T4
crosses of original 3812-9 lldl~iÇolllldllL~ as described in Example 8.1. Tomato lines
rl~si_n~tPd A and B, which arose from separate l;lO~Si.lg events, were grown under field
CondiLiolls following standard field trial protocols. Soluble solids were obtained from fruit
2 0 extracts of replicate plants as described for growth cl~u~-bcl and greenhouse studies. The
soluble solids were evaluated by .1~l. . ., I;ll;lllp, the average refractive index (Rl) and specific
sugar content per unit weight fruit tissue using high ~.CS~ulc liquid chromatography
(HPLC). The R~ easu.cll.ents p.~ lr(l analysis of overall sugar and acid content and
the HPLC analysis for collL.ibuLions by individual sugars. Both mPthorls of analysis were
2 5 con~1nctPd following standard protocols. The results are reported in Table 11 below.
CA 0223~801 1998-04-24
W O 97115678 PCTAJS96/17351
T~hle 11
Soluble Soliclc ~n-l Su~r Co~t~ t in T ~f-Specific
SSU/SPS Tolr ~to Pl~ntc
Toln~to T,inP 1~ Sl~r Col-rf~ oll ~%)
Snrrose Glucose Fructose
Control 3.9 0.08 1.33 1.62 3.03
Control 4.2 0.11 1.51 1.75 3.37
(A) SSU/SPS-A-75-5 4.9 0.19 1.58 2.58 4.35
(A) SSU/SPS-A-91-4 4.9 0.19 1.61 2.55 4.35
(B) SSU/SPS-B-87-2 4.6 0.22 1.59 2.37 4.18
Average increase due to 0.75 0.10 0.17 0.81 1.09
SPS
The transgenic tomato lines A and B cnncictently showed higher sugar and acid
content compared to the controls. Sucrose, glucose and fructose levels were in;lea~ed
substantially in tomato fruit of the A and B lines, cc,ll~L)al~d to the controls. Surprisingly,
the contribution of glucose and fructose to the overall increase in soluble solids was
10 prononnred colllpal~d to sucrose, in~ ting a net partitioning and conversion of
photoacsimil~ to the fruit sink tissue.
8.7.4 Soluble Soli~lc In Frllit-Specific F.8/SPS Tom~to Pl~ntc Grown Un-l~r Greenhonc~
Contlition~
The soluble solids in fruit from tomato plant lines 3342 and 3343 e~ressillg thefruit-specific E8-SPS constructs were evaluated as follows. Tomato plant lines arising
from separate lldl~rollllation events with pCGN3342 and pCGN3343 were grown under
standard Greenhouse conditions. Soluble solids from replicate lines and trials were
2 0 measured using RI, SPS specific activity and HLPC analyses. As a control, untransformed
tomato plants and leaf-specific SSU/SPS tomato line were e~min~d in parallel for each
trial. Repl~,sellLdtive data for soluble solid content and di~Llibulioll are reported in Tables
12-14 below.
CA 02235801 1998-04-24
W O 97/15678 PCT~US96/17351
.8
~ P P- P ~
,_ ~ o c~ 00 o ~ ~ n O
O ~ ~ , d~
o
o
U'
C~: U7
C ~
00
a
-- d ~ O ~ C~ x ~ ~ , ~ O -- ~?
,~ d t~X ~ ~
'; o .~
C~
U~
U~
52
CA 02235801 1998-04-24
WO 97/15678 PCTAJS96/17351
Cl~ ~ O C~ ~ U~
c ~ , r-- ~ ~ ~ t-- oo ~I
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l C~ ~ ~ ~ ~ ~ l--
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~ 5c ~ u CO~ g OO CO~ o~. g ~ oO~ oO.
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a~ ~ 1~ oo x ~ oo
c
c o ~
F o ~ ~ ~ ~ ~ d- c~ oo
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CA 02235801 1998-04-24
W O 97/15678 PCTrUS96/17351
~ .
oo ~o~ ~
~Z ooo oZZ~~. ooZ
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_ ' X oo o~ t-- ~ ~ ~-- ~ _ ~ oo
H ~ x ~ ~ ~ c~ c~ ~ ~ ~
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C .C C ~ t oo ~ ~ o o o--Cl~
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3 I.r v~ ~ ~ ~ ~ ~ ~ ~t ~ ~ ~ ~
c~ c~
C ~r C b b b b b b ~D b b b b
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-
c~ x ~
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E ~ ~ ~ S 't oo ~ ~ ~1 S ~t
CA 0223~801 1998-04-24
WO 97/15678 PCTrUS96/17351
Tomato plant lines e~ ;.shlg the fruit-specific E8/SPS constructs consistently showed
an increase in soluble solids reflected by overall sugar content, acid content and distribution.
To assess the correlation between SPS activity and altered soluble solid content, SPS activity
was measured in fruit from control tomato plants and compared to that in fruit from E8/SPS
5 tomato lines 3343-6 and 3342-11. Control fruit from tomato line FL7060 was assayed with a
SPS activity rate of 17.8 ~4mols sucrose/gram fresh weight/hour. Activity was much higher in
the transgenic lines, with the 3343-6 event having a rate of 67.5 ~Lmols sucrose/grown fresh
weight/hour and the 3342-11 event measured at 36.6 ~mols sucrose/gram fresh weight/hour.
These results show that the illcl~ase of fruit-specific activity of the SPS correlates to the
1 0 increase in sugar content of fruit.
Flr~m~r~le 9
T~ ~el-ic SPS Pot~to Pl~n
9.1 Produrtion of SPS pot~t~ Pl~nt~
Potato plants were L~al~rolllled with e~lession c~se~ s cont~ining SPS coding
seqnenreS (pCGN3812) via Agrobacterium tumefaciens me~ te(l LlalL~7follllàLion (Fillatti et al.,
supra) and regenerated. Pl~alation of pCGN3812, a tobacco SSU/SPS construct, is described
in Example 4.3.
20 9.2 OXygen Sen.~itivi~y
Potato is adapted to cool weâtlll . and has a large vegetative sink, whereas thegenetically similar tomato has a large reproductive sink. To evaluate whether potato has a
relatively higher capacity for starch plus sucrose ~yllLhesis, allowing it to avoid oxygen
hlsell~.iLi~ity in the range of 120C to 200C, oxygen sensitivity was ~x;....i..~d in potatoes
2 5 ex~ sillg the corn SPS gene. Potatoes e~L~les~ the corn SPS exhibited a higher capacity
for photosynthesis in elevated CO2 when the plants were three weeks old co~ al~,1 to controls.
When the potato corn SPS e~ ssillg plants were six to seven weeks old with developing
tubers, they showed the acclim~tion to elevated C02 found in many plants and the controls
(Fig. 12). These data show that les~.ollsi~eness of plant growth to elevated CO2 in plants
3 0 having diverse physiological systems can be mo~ ted by manipulating sucrose synthesis
through an SPS which functions in plants.
CA 0223~801 1998-04-24
W O 97/15678 PCT~US96/17351
9.3 T--h~r Yield
Tld.~..lled potatoes expressing corn SPS exhibited greater tuber yield when grown in
both large chambers and in open top chambers out-of-doors (Fig. 13). Rec~nc~ yield in potato
is tuber mass and not fruit, the effect in potato appear different from the effect seen in tomato.
5 Collectively, the tomato and potato yield data indicate that morlifir~tion of SPS activity
through expression of an exogenous t.,.ncg~ encoding SPS directly effects net sucrose
synthesis and mass action in a similar manner in diverse plant systems, even though sucrose
metabolism and its systemic effects may differ, which can be used to manipulate yield.
The above results demonstrate that transgenic plants can be constructed which have
10 altered carbon partitioning through expression of a gene required for sucrose synthesis. Plants
L~dl sr~,.ll.ed with a DNA expression construct capable of controlling the expression of an SPS
gene exhibited mo-lifi~tion of starch and sucrose levels, CO2 and/or ~2 sensitivity,
dlule dependent growth responsiveness, and overall m- ~iifi~tion of carbon partitioning
b- Iween source tissue such as leaf and sink tissue such as fruit or root. The data also show
15 that the plant growth and yield were affected by altered carbon partitioning, as illustrated in
two different plants of the ni~htch~ family Sol~n~ P, potato and tomato. The data also
show that control of carbohydrate parthioning through morlifir~tion of end-product synthesis,
for example, sucrose synthesis and conversion to other sugars in sink tissue, such as glucose
and fructose provide means for altering plant growth and yield of specific plant tissues, plant
2 0 parts and/or whole plant systems. In particular, increased SPS activity and tissue-specific SPS
activity was demonstrated to produce a net increase in overall soluble solids in sink tissue such
as fruit. Increases in the sugars sucrose, glucose and fructose ~ se~ d soluble sugars
analyzed in the soluble solids, with contributions by glucose and fructose being higher than
sucrose. The SPS activity and sugar content data inrli~ ~t~ that the endogenous acid invertase
2 5 found in ripening tomato fruit colllribul~d to the observed i..~ ,ases in glucose and fructose.
Acid levels in the fruit-specific E8/SPS constructs also were observed, correlating acid content
to an increase in sugar content. These data collectively show that SPS can be used to alter the
overall content and ratio of soluble solids in a plant sink tissue, resl-lting in a del.lol~l~ble
phenotype in plants, such as fruit having mo-lifi~ wtx;t..css. Also, tomatoes eA~-~sshlg SPS
3 0 behind the CaMV 35S promoter grew better than tolll~loes e~ ,shlg the gene behind a
Rubisco small subunit promoter under growth l~h~mher conditions. These data indicate a
promoter effect which can be manipulated to control SPS activity in particular plant cells, plant
56
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parts and throughout the plant. In general, the results show that plant growth and yield can be
enh~nred through transgenic e~,l,ssion of SPS, even though its effect on photosynthesis may
be small.
FY~n~le 10
Soluble Solirl~ in T2 SSU-SPS Pl~nt~
Investigation of the soluble solids in the fruits of the SSU-SPS lines was initially done
on extracts from fruit of 3812-9 and 3812-11 lines grown in a Biotron inrllhator. T2 plants
were illll",il~ (l by metal halide lamps at a peak level of 500 ,umol ~hol~ns/m/s (pot level),
26 C for the 16 h day and 18 C at night, and a relative hllmiAity of 60%. Plants were watered
daily with half-strength Ho~gl~n~l's solution (Hoagland and Arnon, Calif. Agricult. Exp. Sta.
Cir. (1938) 357: 1-39). These lines were segregating as the original lines contained at least 2
ilLS~lliol~.
lBrix analysis (soluble solids) on extracts from these plants revealed lines with Brix
readings as much as 40% higher than the controls. The extracts lllea~.u,.,d were the average of
3 fruit from one plant.
Measu,c,l,el,L~. were also taken for fruit from a seg,e~ aling T2 population of 3812-11
plants in the greenhouce. The controls averaged a Brix reading of 3.5 while the transgenics
averaged 4.0, an increase of 14%.
F~ rle 11
Hom-)zy~oll~ Pl~ntc
T4 homozygous lines were ~en.,lated from original 3812-9 llalL~ ll"a.,~. in UC82-B
tomatoes. The original line segl~,galcd 15:1 forKan r ~ nre, i"rlir~ g that ithad two
25 insertion sites. Two homozygous lines were genelalcd and verified to be different by Southern
border analysis. These lines were ~ ign~te-l A and B.
Individual homozygote (T4) lines were grown in the greenhouse, with three fruit taken
from each plant and 3 plants analyzed from each line. The Brix of the UC82B controls was
3.35 while the Brix on the 3812-9 lines ranged from 3.7 to 4.1. This is an increase from 12%
3 0 to 24% . Statistics (LSD) on all the lines in which fruit from 3 plants were analyzed showed
these results to be signifir~nt at a .01 % level (99%).
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Measu.~ll.ellL~ were also made on homozygous lines of tomato plants llàl~rullllcd with
the 35S CaMV promoter-SPS construct pCGN3815. In one line, 3815-13-2 there was asl-hst~nti~l i..-,.-,ase in yield of tomatoes, in terms of an increase in both fruit size and in fruit
number, as llleasùled against non-llalLsro.ll-ed control plants and as against SSU-SPS
5 homozygous line controls. The 3815-13-2 plants also produced a second flush of fruit. A
second Llansgellic line cont~inin~ the pCGN3815 construct did not produce these dramatic
yield increases.
Fx~n~ple 12
l~rix An~lysi~ of Field Tri~l SSU-SPS Frllit
Field trial results of RI mea~ulell~llL~ are provided in Table 15. The RJI (lerlacLi~e
index) was measured several times on the fruit of these plants (Table 15). R/I is a measure of
soluble solids and is indicative of sugars and acids. The transgenic A and B lines consistently
had a higher R/I than the control UC82-B.
Table 15
~llmm~ry of Refr~etive In~lex M~
L~ I Rto~ Re~ 2 I R.o~-li~ 3 ¦ Re~1ir~ 4 1 Over~ll A-~
Trial 1
UC82-B 3.3 3.9 3.6 3.0 3.5
A Lines (X2) 4.0 4.4 4.6 3.8 4.2
Trial 2
UC82-B 4.1 4.1 3.9 4.0
A Lines (X3) 4.8 4.3 4.2 4.4
B Lines (X2) 4.4 4.4 4.2 4.3
Trial 3
UC82B 3.2
A Lines (X1) 4.2
B Lines (X1)
CA 0223~801 1998-04-24
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F.~ ple 13
HPT C An~lysi~ on SSU-SPS Frl-it Sn~rs
Fruit from the SPS plants described in Example 12 were further analyzed by HPLC to
5 determine collLlibuLions of individual sugars to the increased soluble solids content. As seen in
Table 16, sucrose did not increase as much as might be expected based on the fact that sucrose
is the sugar transported by the plant into the fruit. Glucose was not increased as much as
fructose, which increased nearly 50%.
It is evident from the above results, that plant cells and plants can be produced which
10 have improved p[Op~l Lies or may produce a desired phenotype. In accordance with the subject
invention, it is now seen that SPS seq~rnrçs may be introduced into a plant host cell and be
used to express the enzyme to increase soluble solids content in fruit. Moreover, it is seen that
the SPS may be used to alter the overall content and ratio of soluble solids in plant sink tissue,
reslllting in a demonstrable phenotype in planta, such as altered fruit sweetness. In this
15 manner, fruits, such as tomato fruit, having modified s~L less may be obtained.
Fx~n~le 14
Frl-it Specific F~pressiorl of SPS
E8-SPS constructs deci~n~tr-l as pCGN3342 and pCGN3343 contain the tomato E8
20 promoter C~Jlll~lisillg the approximately 2.1 kb 5' region of the E8 promoter. A description of
this promoter region can be found in Deikrnan el al., supra, and in Deikman et al. (Plant
Physiol. (1992) 100:2013-2017).
This E8 promoter is fused to the same SPS encoding seqll~nre used for pCGN3812 and
pCGN3815, only the SPS seq~-Pnre used in these CO1~L1UL;L~ has been truncated at the ApoI site
2 5 just 3 ' of the SPS encoding sequenre (at nucleotide 3318), and fused to a 1.2 kb region of the
tml 3' region from pTiA6 (Barker et al., (1983) Plant Mol. Biol. 2:335-350; sequence 11207-
10069 of the T-DNA region from the Agrobacterium tumefaciens Ti plasmid pTil5955).
COl~.L~u~L~. pCGN3342 and pCGN3343 are the opposite orientations of this E8-SPS-tml
construct in the 35S kan binary, pCGN1557 (McBride and Summerfelt, supra). Tomato lines
3 0 arising l~rom separate tran~7f~ aLion events using pCGN3342 and pCGN3343 are signified by
the construct number followed by a hyphen and an event number.
Table 17 provides data from RI llleasul~ s of soluble solids in tomatoes from
59
CA 0223~801 1998-04-24
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greenhouse studies of T1 plants. The RI was measured several times on the fruit of these
plants.
Assays were made for the SPS activity in control and transgenic fruit from the 3343-6
and 3342-11 events. The control 7060 fruit was assayed with a SPS activity rate of 17.8
5 ,umols sucrose/g/hr. This d~ lo~ ates that the increase sugar conce~ alion of fruit in
al~cg~ ic tomatoes over the control correlates to an increase of SPS activity in the fruit.
Tables 18 and 19 provide an analysis of individual sugars as measured by HPLC from
two separate trials, to ~letPrmin~ co..~ iorl.C of each sugar to the increased soluble solids
content observed in transgenic E8-SPS fruit. The data of Table 18 and 19 demonstrate that
10 increased SPS activity from ll,.~ genic e~lei,sion in fruit by a fruit specific promoter can
produce an overall net increase in sugars in the fruit. Due to the endogenous acid invertase
found in ripening tomato fruit, i...;leases in sugar are found in glucose and fructose.
It also appears that there is a correlating increase in acid levels with an increase in
sugar content in fruit tral~.llled with E8-SPS.
CA 02235801 1998-04-24
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~D
.~ ~ ~ ~ ~ ~
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C ~ o ~ ~ ~ ~
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c ~ ~ ~
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oo o o o o ~
61
CA 02235801 1998-04-24
W O 97/15678 PCTAJS96/17351
C
C O ~ u~ ~ 00 00 0 ~ O
c
C ~ ~ ~ cr~ D ~ ~ O -I
¢ a ~ ~ T ~ V T~
62
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CA 02235801 1998-04-24
W O 97/15678 PCTrUS96/17351
~ ~ ~ cr~
C , ~ r~ ~ -- oo ~ Cl~
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CA 02235801 1998-04-24
W O 97/15678 PCTAJS96/17351
n
o co ~ ~ ~ _ c~ _
c oJ , o -- cr~ ~ -- ~ ~ ~ _ ~ co
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64
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All publications and patent applications mentioned in the specification are indicative of
the level of skill of those skilled in the art to which this invention pertains. All referenced
publications and patent applications are herein incorporated by ,~rel~,ncc to the same extent as
if each individual publication or patent application was specifically and individually intiir~tr
to be incorporated by Ic:r~.e.lce.
The invention now been fully described, it would be al,~a,~.lL to one of ordinary skill in
the art that many changes and mo~lifir~tit~ns can be made thereto without departing from the
spirit or scope of the appended claims.
CA 0223~80l l998-04-24
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S~UU~N~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Van Assche, C.
Lando, D.
Bruneau, J. M.
Voelker, T.
Gervais, M.
(iiJ TITLE OF lNv~NllON: MODIFICATION OF SUCROSE PHOSPHATE
SYN~rHASE IN PLANTS
(iii) NUMBER OF SEQUENCES: 14
(iv) CORRESPON~N~ ~nD~ s
(A) ADDRESSEE: Law Offices of Barbara Rae-Venter
(B) STREET: 260 Sheridan Avenue, Suite 440
(C) CITY: Palo Alto
(D) STATE: Cali~ornia
(E) COUN1~: USA
(F) ZIP: 94306
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) ~uKR~Nl APPLICATION DATA:
(A) APPLICATION NUMBER: NOT YET ASSIGNED
(B) FILING DATE: NOT YET ASSIGNED
(C) CLASSIFICATION: NOT YET ASSIGNED
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/175,471
(B) FILING DATE: 27-DEC-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Barbara Rae-Venter
(B) REGISTRATION NUMBER: 32,750
(C) REFERENCE/DOCKET NU~3ER: CGNE.072.02US
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415)328-4400
(B) TELEFAX: (415)328-4477
66
CA 02235801 1998-04-24
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(2) INFORMATION FOR SEQ ID NO:l:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) ST~I~NI )14:1 ~NI.:.CS
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) s~Qu~N-~ DESCRIPTION: SEQ ID NO:l:
Thr Trp Ile Lys
(2) INFORMATION FOR SEQ ID NO:2:
(i) ~QU~NC~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRPNI~ N~ S:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:2:
Tyr Val Val Glu Leu Ala Arg
l 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) ~QU~N-~ CHARACTERISTICS:
(A) LENGTH: ll amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
67
CA 02235801 1998-04-24
WO 97/15678 PCTrUS96/1735
(xi) s~UU~N~ DESCRIPTION: SEQ ID NO:3:
Ser Met Pro Pro Ile Trp Ala Glu Val Met Arg
l 5 l0
(2) INFORMATION FOR SEQ ID NO:4:
(i) S~Q~N~ CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAN~N~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) S~UU~N~: DESCRIPTION: SEQ ID NO:4:
Leu Arg Pro Asp Gln Asp Tyr Leu Met His Ile Ser His Arg
l 5 lO
(2) INFORMATION FOR SEQ ID NO:5:
(i) ~uU~N~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
( C ) S TRA N ~ "'N l~: ~ S:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:5:
Trp Ser His Asp Gly Ala Arg
68
CA 0223~80l l998-04-24
W O 97/lS678 PCT~US96/17351
(2) INFORMATION FOR SEQ ID NO:6:
(i) S~u~ CHARACTERISTICS:
~ (A) LENGTH: 3509 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~R.~S: double
(D) TOPOLOGY: Iinear
(ii) MOLECULE TYPE: cDNA to mRNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 112..3315
(xi) ~uU~N~: DESCRIPTION: SEQ ID NO:6:
GAATTCCGGC GTGGGCGCTG GGCTAGTGCT CCCGCAGCGA GCGATCTGAG AGAACGGTAG 60
A~ilCCGGCC GGGCGCGCGG GAGAGGAGGA GG~lCGGGCG GGGAGGATCC G ATG GCC 117
Met Ala
GGG AAC GAG TGG ATC AAT GGG TAC CTG GAG GCG ATC CTC GAC AGC CAC 165
Gly Asn Glu Trp Ile Asn Gly Tyr Leu Glu Ala Ile Leu Asp Ser His
5 10 15
ACC TCG TCG CGG GGT GCC GGC GGC GGC GGC GGC GGG GGG GAC CCC AGG 213
Thr Ser Ser Arg Gly Ala Gly Gly Gly Gly Gly Gly Gly Asp Pro Arg
20 25 30
TCG CCG ACG AAG GCG GCG AGC CCC CGC GGC GCG CAC ATG AAC TTC AAC 261
Ser Pro Thr Lys Ala Ala Ser Pro Arg Gly Ala His Met Asn Phe Asn
35 40 45 50
CCC TCG CAC TAC TTC GTC GAG GAG GTG GTC AAG GGC GTC GAC GAG AGC 309
Pro Ser His Tyr Phe Val Glu Glu Val Val Lys Gly Val Asp Glu Ser
55 60 65
GAC CTC CAC CGG ACG TGG ATC AAG GTC GTC GCC ACC CGC AAC GCC CGC 357
Asp Leu His Arg Thr Trp Ile Lys Val Val Ala Thr Arg Asn Ala Arg
70 75 80
69
CA 0223~801 1998-04-24
W O 97/15678 PCT~US96/17351
GAG CGC AGC ACC AGG CTC GAG AAC ATG TGC TGG CGG ATC TGG CAC CTC 405
Glu Arg Ser Thr Arg Leu Glu Asn Met Cys Trp Arg Ile Trp His Leu
85 90 95
GCG CGC AAG AAG AAG CAG CTG GAG CTG GAG GGC ATC CAG AGA ATC TCG 453
Ala Arg Lys Lys Lys Gln Leu Glu Leu Glu Gly Ile Gln Arg Ile Ser
100 105 110
GCA AGA AGG AAG GAA CAG GAG CAG GTG CGT CGT GAG GCG ACG GAG GAC 501
Ala Arg Arg Lys Glu Gln Glu Gln Val Arg Arg Glu Ala Thr Glu Asp
115 120 125 130
CTG GCC GAG GAT CTG TCA GAA GGC GAG AAG GGA GAC ACC ATC GGC GAG 549
Leu Ala Glu Asp Leu Ser Glu Gly Glu Lys Gly Asp Thr Ile Gly Glu
135 140 145
CTT GCG CCG GTT GAG ACG ACC AAG AAG AAG TTC CAG AGG AAC TTC TCT 597
Leu Ala Pro Val Glu Thr Thr Lys Lys Lys Phe Gln Arg Asn Phe Ser
150 155 160
GAC CTT ACC GTC TGG TCT GAC GAC AAT AAG GAG AAG AAG CTT TAC ATT 645
Asp Leu Thr Val Trp Ser Asp Asp Asn Lys Glu Lys Lys Leu Tyr Ile
165 170 175
GTG CTC ATC AGC GTG CAT GGT CTT GTT CGT GGA GAA AAC ATG GAA CTA 693
Val Leu Ile Ser Val His Gly Leu Val Arg Gly Glu Asn Met Glu Leu
180 185 190
GGT CGT GAT TCT GAT ACA GGT GGC CAG GTG A~A TAT GTG GTC GAA CTT 741
Gly Arg Asp Ser Asp Thr Gly Gly Gln Val Lys Tyr Val Val Glu Leu
195 200 205 210
GCA AGA GCG ATG TCA ATG ATG CCT GGA GTG TAC AGG GTG GAC CTC TTC 789
Ala Arg Ala Met Ser Met Met Pro Gly Val Tyr Arg Val Asp Leu Phe
215 220 225
ACT CGT CAA GTG TCA TCT CCT GAC GTG GAC TGG AGC TAC GGT GAG CCA 837
Thr Arg Gln Val Ser Ser Pro Asp Val Asp Trp Ser Tyr Gly Glu Pro
230 235 240
ACC GAG ATG TTA TGC GCC GGT TCC AAT GAT GGA GAG GGG ATG GGT GAG 885
Thr Glu Met Leu Cys Ala Gly Ser Asn Asp Gly Glu Gly Met Gly Glu
245 250 255
CA 0223~801 1998-04-24
O 97/~5678 PCTAJS96/17351
AGT GGC GGA GCC TAC ATT GTG CGC ATA CCG TGT GGG CCG CGG GAT AAA 933
Ser Gly Gly Ala Tyr Ile Val Arg Ile Pro Cys Gly Pro Arg Asp Lys
260 265 270
TAC CTC AAG AAG GAA GCG TTG TGG CCT TAC CTC CAA GAG TTT GTC GAT 981
Tyr Leu LYR Lys Glu Ala Leu Trp Pro Tyr Leu Gln Glu Phe Val Asp
275 280 285 290
GGA GCC CTT GCG CAT ATC CTG AAC ATG TCC AAG GCT CTG GGA GAG CAG 1029
Gly Ala Leu Ala His Ile Leu Asn Met Ser Lys Ala Leu Gly Glu Gln
295 300 305
GTT GGA AAT GGG AGG CCA GTA CTG CCT TAC GTG ATA CAT GGG CAC TAT 1077
Val Gly Asn Gly Arg Pro Val Leu Pro Tyr Val Ile His Gly His Tyr
310 315 320
GCC GAT GCT GGA GAT GTT GCT GCT CTC CTT TCT GGT GCG CTG AAT GTG 1125
Ala Asp Ala Gly Asp Val Ala Ala Leu Leu Ser Gly Ala Leu Asn Val
325 330 335
CCA ATG GTG CTC ACT GGC CAC TCA CTT GGG AGG AAC AAG CTG GAA CAA 1173
Pro Met Val Leu Thr Gly His Ser Leu Gly Arg Asn Lys Leu Glu Gln
340 345 350
CTG CTG AAG CAA GGG CGC ATG TCC AAG GAG GAG ATC GAT TCG ACA TAC 1221
Leu Leu Lys Gln Gly Arg Met Ser Lys Glu Glu Ile Asp Ser Thr Tyr
355 360 365 370
AAG ATC ATG AGG CGT ATC GAG GGT GAG GAG CTG GCC CTG GAT GCG TCA 1269
Lys Ile Met Arg Arg Ile Glu Gly Glu Glu Leu Ala Leu Asp Ala Ser
375 380 385
GAG CTT GTA ATC ACG AGC ACA AGG CAG GAG ATT GAT GAG CAG TGG GGA 1317
Glu Leu Val Ile Thr Ser Thr Arg Gln Glu Ile Asp Glu Gln Trp Gly
390 395 400
TTG TAC GAT GGA TTT GAT GTC AAG CTT GAG AAA GTG CTG AGG GCA CGG 1365
Leu Tyr Asp Gly Phe Asp Val Lys Leu Glu Lys Val Leu Arg Ala Arg
405 410 415
GCG AGG CGC GGG GTT AGC TGC CAT GGT CGT TAC ATG CCT AGG ATG GTG 1413
Ala Arg Arg Gly Val Ser Cys His Gly Arg Tyr Met Pro Arg Met Val
420 425 430
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WO 97/15678 PCT~US96/17351
GTG ATT CCT CCG GGA ATG GAT TTC AGC AAT GTT GTA GTT CAT GAA GAC 1461Val Ile Pro Pro Gly Met Asp Phe Ser Asn Val Val Val His Glu Asp
435 440 445 450
ATT GAT GGG GAT GGT GAC GTC AAA GAT GAT ATC GTT GGT TTG GAG GGT lS09
Ile Asp Gly Asp Gly Asp Val Lys Asp Asp Ile Val Gly Leu Glu Gly
455 460 465
GCC TCA CCC AAG TCA ATG CCC CCA ATT TGG GCC GAA GTG ATG CGG TTC 1557
Ala Ser Pro Lys Ser Met Pro Pro Ile Trp Ala Glu Val Met Arg Phe
470 475 480
CTG ACC AAC CCT CAC AAG CCG ATG ATC CTG GCG TTA TCA AGA CCA GAC 1605
Leu Thr Asn Pro His Lys Pro Met Ile Leu Ala Leu Ser Arg Pro Asp
485 490 495
CCG AAG AAG AAC ATC ACT ACC CTC GTC AAA GCC TTT GGA GAG TGT CGT 1653
Pro Lys Lys Asn Ile Thr Thr Leu Val Lys Ala Phe Gly Glu Cys Arg
500 505 510
CCA CTC AGG GAA CTT GCA AAC CTT ACT CTG ATC ATG GGT AAC AGA GAT 1701
Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu Ile Met Gly Asn Arg Asp
515 520 525 530
GAC ATC GAC GAC ATG TCT GCT GGC AAT GCC AGT GTC CTC ACC ACA GTT 1749
Asp Ile Asp Asp Met Ser Ala Gly Asn Ala Ser Val Leu Thr Thr Val
535 540 545
CTG AAG CTG ATT GAC AAG TAT GAT CTG TAC GGA AGC GTG GCG TTC CCT 1797
Leu Lys Leu Ile Asp Lys Tyr Asp Leu Tyr Gly Ser Val Ala Phe Pro
550 555 560
AAG CAT CAC AAT CAG GCT GAC GTC CCG GAG ATC TAT CGC CTC GCG GCC 1845
Lys His His Asn Gln Ala Asp Val Pro Glu Ile Tyr Arg Leu Ala Ala
565 570 575
AAA ATG AAG GGC GTC TTC ATC AAC CCT GCT CTC GTT GAG CCG TTT GGT 1893
Lys Met Lys Gly Val Phe Ile Asn Pro Ala Leu Val Glu Pro Phe Gly
580 585 590
CTC ACC CTG ATC GAG GCT GCG GCA CAC GGA CTC CCG ATA GTC GCT ACC 1941
Leu Thr Leu Ile Glu Ala Ala Ala His Gly Leu Pro Ile Val Ala Thr
595 600 605 610
72
CA 0223~80l l998-04-24
WO 97/lS678 PCT~US96/17351
AAG A~T GGT GGT CCG GTC GAC ATT ACA AAT GCA TTA AAC AAC GGA CTG 1989
Lys Asn Gly Gly Pro Val Asp Ile Thr Asn Ala Leu Asn Asn Gly Leu
615 620 625
CTC GTT GAC CCA CAC GAC CAG AAC GCC ATC GCT GAT GCA CTG CTG AAG 2037
Leu Val Asp Pro His Asp Gln Asn Ala Ile Ala Asp Ala Leu Leu Lys
630 635 640
CTT GTG GCA GAC AAG AAC CTG TGG CAG GAA TGC CGG AGA AAC GGG CTG 2085
Leu Val Ala Asp Lys Asn Leu Trp Gln Glu Cys Arg Arg Asn Gly Leu
645 650 655
CGC AAC ATC CAC CTC TAC TCA TGG CCG GAG CAC TGC CGC ACT TAC CTC 2133
Arg Asn Ile His Leu Tyr Ser Trp Pro Glu His Cys Arg Thr Tyr Leu
660 665 670
ACC AGG GTG GCC GGG TGC CGG TTA AGG AAC CCG AGG TGG CTG AAG GAC 2181
Thr Arg Val Ala Gly Cys Arg Leu Arg Asn Pro Arg Trp Leu Lys Asp
675 680 685 690
ACA CCA GCA GAT GCC GGA GCC GAT GAG GAG GAG TTC CTG GAG GAT TCC 2229
Thr Pro Ala Asp Ala Gly Ala Asp Glu Glu Glu Phe Leu Glu Asp Ser
695 700 705
ATG GAC GCT CAG GAC CTG TCA CTC CGT CTG TCC ATC GAC GGT GAG AAG 2277
Met Asp Ala Gln Asp Leu Ser Leu Arg Leu Ser Ile Asp Gly Glu Lys
710 715 720
AGC TCG CTG AAC ACT AAC GAT CCA CTG TGG TTC GAC CCC CAG GAT CAA 2325
Ser Ser Leu Asn Thr Asn Asp Pro Leu Trp Phe Asp Pro Gln Asp Gln
725 730 735
GTG CAG AAG ATC ATG AAC AAC ATC AAG CAG TCG TCA GCG CTT CCT CCG 2373
Val Gln Lys Ile Met Asn Asn Ile Lys Gln Ser Ser Ala Leu Pro Pro
740 745 750
TCC ATG TCC TCA GTC GCA GCC GAG GGC ACA GGC AGC ACC ATG AAC AAA 2421
Ser Met Ser Ser Val Ala Ala Glu Gly Thr Gly Ser Thr Met Asn Lys
755 760 765 770
TAC CCA CTC CTG CGC CGG CGC CGG CGC TTG TTC GTC ATA GCT GTG GAC 2469
Tyr Pro Leu Leu Arg Arg Arg Arg Arg Leu Phe Val Ile Ala Val Asp
775 780 785
-
CA 0223~801 1998-04-24
W O 97/15678 PCT~US96/173~1
TGC TAC CAG GAC GAT GGC CGT GCT AGC AAG AAG ATG CTG CAG GTG ATC 2517
Cy8 Tyr Gln Asp Asp Gly Arg Ala Ser Lys Lys Met Leu Gln Val Ile
790 795 800
CAG GAA GTT TTC AGA GCA GTC CGA TCG GAC TCC CAG ATG TTC AAG ATC 2565
Gln Glu Val Phe Arg Ala Val Arg Ser Asp Ser Gln Met Phe Lys Ile
805 810 815
TCA GGG TTC ACG CTG TCG ACT GCC ATG CCG TTG TCC GAG ACA CTC CAG 2613
Ser Gly Phe Thr Leu Ser Thr Ala Met Pro Leu Ser Glu Thr Leu Gln
820 825 830
CTT CTG CAG CTC GGC AAG ATC CCA GCG ACC GAC TTC GAC GCC CTC ATC 2661
Leu Leu Gln Leu Gly Lys Ile Pro Ala Thr Asp Phe Asp Ala Leu Ile
835 840 845 850
TGT GGC AGC GGC AGC GAG GTG TAC TAT CCT GGC ACG GCG AAC TGC ATG 2709
Cys Gly Ser Gly Ser Glu Val Tyr Tyr Pro Gly Thr Ala Asn Cys Met
855 860 865
GAC GCT GAA GGA AAG CTG CGC CCA GAT CAG GAC TAT CTG ATG CAC ATC 2757
Asp Ala Glu Gly Lys Leu Arg Pro Asp Gln Asp Tyr Leu Met His Ile
870 875 880
AGC CAC CGC TGG TCC CAT GAC GGC GCG AGG CAG ACC ATA GCG AAG CTC 2805
Ser His Arg Trp Ser His Asp Gly Ala Arg Gln Thr Ile Ala Lys Leu
885 890 895
ATG GGC GCT CAG GAC GGT TCA GGC GAC GCT GTC GAG CAG GAC GTG GCG 2853
Met Gly Ala Gln Asp Gly Ser Gly Asp Ala Val Glu Gln Asp Val Ala
900 905 910
TCC AGT AAT GCA CAC TGT GTC GCG TTC CTC ATC AAA GAC CCC CAA AAG 2901
Ser Ser Asn Ala His Cy8 Val Ala Phe Leu Ile Lys Asp Pro Gln Lys
915 920 925 930
GTG AAA ACG GTC GAT GAG ATG AGG GAG CGG CTG AGG ATG CGT GGT CTC 2949
Val Lys Thr Val Asp Glu Met Arg Glu Arg Leu Arg Met Arg Gly Leu
935 940 945
CGC TGC CAC ATC ATG TAC TGC AGG AAC TCG ACA AGG CTT CAG GTT GTC 2997
Arg Cys His Ile Met Tyr Cys Arg Asn Ser Thr Arg Leu Gln Val Val
950 955 960
CA 0223~80l l998-04-24
W O 97/15678 PCT~US96/17351
CCT CTG CTA GCA TCA AGG TCA CAG GCA CTC AGG TAT CTT TCC GTG CGC 3045Pro Leu Leu Ala Ser Arg Ser Gln Ala Leu Arg Tyr Leu Ser Val Arg
965 970 975
TGG GGC GTA TCT GTG GGG AAC ATG TAT CTG ATC ACC GGG GAA CAT GGC 3093
Trp Gly Val Ser Val Gly Asn Met Tyr Leu Ile Thr Gly Glu His Gly
980 985 990
GAC ACC GAT CTA GAG GAG ATG CTA TCC GGG CTA CAC AAG ACC GTG ATC 3141
Asp Thr Asp Leu Glu Glu Met Leu Ser Gly Leu His Lys Thr Val Ile
995 1000 1005 1010
GTC CGT GGC GTC ACC GAG AAG GGT TCG GAA GCA CTG GTG AGG AGC CCA 3189
Val Arg Gly Val Thr Glu Lys Gly Ser Glu Ala Leu Val Arg Ser Pro
1015 1020 1025
GGA AGC TAC AAG AGG GAC GAT GTC GTC CCG TCT GAG ACC CCC TTG GCT 3237
Gly Ser Tyr Lys Arg Asp Asp Val Val Pro Ser Glu Thr Pro Leu Ala
1030 1035 1040
GCG TAC ACG ACT GGT GAG CTG AAG GCC GAC GAG ATC ATG CGG GCT CTG 3285
Ala Tyr Thr Thr Gly Glu Leu Lys Ala Asp Glu Ile Met Arg Ala Leu
1045 1050 1055
AAG CAA GTC TCC AAG ACT TCC AGC GGC ATG TGAATTTGAT G~l~ L L 1 lA 3335
Lys Gln Val Ser Lys Thr Ser Ser Gly Met
1060 1065
CALll L~ L'CC 'l''L'l''l ~Ll~AC TGCTATATAA AATAAGTTGT GAACAGTACC GCGGGTGTGT 3395
ATATATATAT TGCAGTGACA AATA~AACAG GACACTGCTA ACTATACTGG TGAATATACG 3455
ACTGTCAAGA TTGTATGCTA AGTACTCCAT TTCTCAATGT ATCAATCGGA ATTC 3509
(2) INFORMATION FOR SEQ ID NO:7:
( i ) S~yU~N~ CHARACTERISTICS:
(A) LENGTH: 1068 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
CA 0223~80l l998-04-24
WO 97/15678 PCTrUs96/1735
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Ala Gly Asn Glu Trp Ile Asn Gly Tyr Leu Glu Ala Ile Leu Asp
l 5 10 15
~er His Thr Ser Ser Arg Gly Ala Gly Gly Gly Gly Gly Gly Gly Asp
Pro Arg Ser Pro Thr Lys Ala Ala Ser Pro Arg Gly Ala His Met Asn
Phe Asn Pro Ser His Tyr Phe Val Glu Glu Val Val Lys Gly Val Asp
Glu Ser Asp Leu His Arg Thr Trp Ile Lys Val Val Ala Thr Arg Asn
~la Arg Glu Arg Ser Thr Arg Leu Glu Asn Met Cys Trp Arg Ile Trp
~is Leu Ala Arg Lys Lys Lys Gln Leu Glu Leu Glu Gly Ile Gln Arg
100 105 110
Ile Ser Ala Arg Arg Lys Glu Gln Glu Gln Val Arg Arg Glu Ala Thr
115 120 125
Glu Asp Leu Ala Glu Asp Leu Ser Glu Gly Glu Lys Gly Asp Thr Ile
130 135 140
Gly Glu Leu Ala Pro Val Glu Thr Thr Lys Lys Lys Phe Gln Arg Asn
145 150 155 160
~he Ser Asp Leu Thr Val Trp Ser Asp Asp Asn Lys Glu Lys Lys Leu
165 170 175
~yr Ile Val Leu Ile Ser Val Hi8 Gly Leu Val Arg Gly Glu Asn Met
180 185 190
Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly Gln Val Lys Tyr Val Val
195 200 205
Glu Leu Ala Arg Ala Met Ser Met Met Pro Gly Val Tyr Arg Val Asp
210 215 220
76
-
CA 0223~801 1998-04-24
WO 97/15678 PCT~US96/173~1
Leu Phe Thr Arg Gln Val Ser Ser Pro Asp Val Asp Trp Ser Tyr Gly
225 230 235 240
~lu Pro Thr Glu Met Leu Cys Ala Gly Ser Asn Asp Gly Glu Gly Met
245 250 255
~ly Glu Ser Gly Gly Ala Tyr Ile Val Arg Ile Pro Cy~3 Gly Pro Arg
260 265 270
Asp Lys Tyr Leu Lys Lys Glu Ala Leu Trp Pro Tyr Leu Gln Glu Phe
275 280 285
Val Asp Gly Ala Leu Ala His Ile Leu Asn Met Ser Lys Ala Leu Gly
290 295 300
Glu Gln Val Gly Asn Gly Arg Pro Val Leu Pro Tyr Val Ile His Gly
305 310 315 320
~is Tyr Ala Asp Ala Gly Asp Val Ala Ala Leu Leu Ser Gly Ala Leu
325 330 335
~sn Val Pro Met Val Leu Thr Gly His Ser Leu Gly Arg Asn Lys Leu
340 345 350
Glu Gln Leu Leu Lys Gln Gly Arg Met Ser Lys Glu Glu Ile Asp Ser
355 360 365
Thr Tyr Lys Ile Met Arg Arg Ile Glu Gly Glu Glu Leu Ala Leu Asp
370 375 380
Ala Ser Glu Leu Val Ile Thr Ser Thr Arg Gln G1U Ile Asp Glu Gln
385 390 395 400
~rp Gly Leu Tyr Asp Gly Phe Asp Val Lys Leu Glu Lys Val Leu Arg
405 410 415
~la Arg Ala Arg Arg Gly Val Ser Cys His Gly Arg Tyr Met Pro Arg
420 425 430
~et Val Val Ile Pro Pro Gly Met Asp Phe Ser Asn Val Val Val His
435 440 445
A
Glu Asp Ile Asp Gly Asp Gly Asp Val Lys Asp Asp Ile Val Gly Leu
450 455 460
77
CA 0223~801 1998-04-24
WO 97/15678 PCTrUS96/17351
Glu Gly Ala Ser Pro Lys Ser Met Pro Pro Ile Trp Ala Glu Val Met
465 470 475 480
~rg Phe Leu Thr Asn Pro His Lys Pro Met Ile Leu Ala Leu Ser Arg
485 490 495
~ro Asp Pro Lys Lys Asn Ile Thr Thr Leu Val Lys Ala Phe Gly Glu
500 505 510
Cy8 Arg Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu Ile Met Gly Asn
515 520 525
Arg Asp Asp Ile Asp Asp Met Ser Ala Gly Asn Ala Ser Val Leu Thr
530 535 540
Thr Val Leu Lys Leu Ile Asp Lys Tyr Asp Leu Tyr Gly Ser Val Ala
545 550 555 560
~he Pro Lys His His Asn Gln Ala Asp Val Pro Glu Ile Tyr Arg Leu
565 570 575
~la Ala Lys Met Lys Gly Val Phe Ile Asn Pro Ala Leu Val Glu Pro
580 585 590
Phe Gly Leu Thr Leu Ile Glu Ala Ala Ala His Gly Leu Pro Ile Val
595 600 605
Ala Thr Lys Asn Gly Gly Pro Val Asp Ile Thr A8n Ala Leu Asn Asn
610 615 620
Gly Leu Leu Val Asp Pro His Asp Gln Asn Ala Ile Ala Asp Ala Leu
625 630 635 640
~eu Lys Leu Val Ala Asp Lys Asn Leu Trp Gln Glu Cys Arg Arg Asn
645 650 655
~ly Leu Arg Asn Ile His Leu Tyr Ser Trp Pro Glu Hi8 Cys Arg Thr
660 665 670
Tyr Leu Thr Arg Val Ala Gly Cys Arg Leu Arg Asn Pro Arg Trp Leu
675 680 685
Lys Asp Thr Pro Ala Asp Ala Gly Ala Asp Glu Glu Glu Phe Leu Glu
690 695 700
78
-
CA 0223~80l l998-04-24
W O 97/15678 PCTrUS96/17351
Asp Ser Met Asp Ala Gln Asp Leu Ser Leu Arg Leu Ser Ile Asp Gly
705 710 715 720
Glu Lys Ser Ser Leu Asn Thr Asn Asp Pro Leu Trp Phe Asp Pro Gln
- 725 730 735
Asp Gln Val Gln Lys Ile Met Asn Asn Ile Lys Gln Ser Ser Ala Leu
740 745 750
Pro Pro Ser Met Ser Ser Val Ala Ala Glu Gly Thr Gly Ser Thr Met
755 760 765
Asn Lys Tyr Pro Leu Leu Arg Arg Arg Arg Arg Leu Phe Val Ile Ala
770 775 780
Val Asp Cys Tyr Gln Asp Asp Gly Arg Ala Ser Lys Lys Met Leu Gln
785 790 795 800
Val Ile Gln Glu Val Phe Ary Ala Val Arg Ser Asp Ser Gln Met Phe
805 810 815
Lys Ile Ser Gly Phe Thr Leu Ser Thr Ala Met Pro Leu Ser Glu Thr
820 825 830
Leu Gln Leu Leu Gln Leu Gly Lys Ile Pro Ala Thr Asp Phe Asp Ala
835 840 845
Leu Ile Cys Gly Ser Gly Ser Glu Val Tyr Tyr Pro Gly Thr Ala Asn
850 855 860
Cys Met Asp Ala Glu Gly Lys Leu Arg Pro A5p Gln Asp Tyr Leu Met
865 870 875 880
His Ile Ser His Arg Trp Ser His Asp Gly Ala Arg Gln Thr Ile Ala
885 890 895
Lys Leu Met Gly Ala Gln Asp Gly Ser Gly Asp Ala Val Glu Gln Asp
900 905 910
Val Ala Ser Ser Asn Ala His Cys Val Ala Phe Leu Ile Lys Asp Pro
915 920 925
Gln Lys Val Lys Thr Val Asp Glu Met Arg Glu Arg Leu Arg Met Arg
930 935 940
79
CA 0223~801 1998-04-24
WO 97/l5678 PCTrUS96/1735
Gly Leu Arg Cys His Ile Met Tyr Cys Arg A~n Ser Thr Arg Leu Gln
945 950 955 960
Val Val Pro Leu Leu Ala Ser Arg Ser Gln Ala Leu Arg Tyr Leu Ser
965 970 975
Val Arg Trp Gly Val Ser Val Gly Asn Met Tyr Leu Ile Thr Gly Glu
980 985 99o
His Gly Asp Thr Asp Leu Glu Glu Met Leu Ser Gly Leu His Lys Thr
995 1000 1005
Val Ile Val Arg Gly Val Thr Glu Lys Gly Ser Glu Ala Leu Val Arg
1010 1015 1020
Ser Pro Gly Ser Tyr Lys Arg Asp Asp Val Val Pro Ser Glu Thr Pro
1025 1030 1035 1040
Leu Ala Ala Tyr Thr Thr Gly Glu Leu Lyg Ala Asp Glu Ile Met Arg
1045 1050 1055
Ala Leu Lys Gln Val Ser Lys Thr Ser Ser Gly Met
1060 1065
(2) INFORMATION FOR SEQ ID NO:8:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = 'IPossible peptide ~nco~;ng
sequences"
(iii) HYPOTHETICAL: YES
(Xi) ~U~'N~'~ DESCRIPTION: SEQ ID NO:8:
WSNATGCCNC CNATHTGGGC NGARGTNATG MGN 33
CA 02235801 1998-04-24
W O 97/15678 PCTnUS96/1735
(2) INFORMATION FOR SEQ ID NO:9:
( i ) g~yU~N~'~ CH~RACTERISTICS:
(A) LENGTH: 42 base pairs
- (B) TYPE: nucleic acid
(C) STR~Nn~nNR-~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Possible peptide ~nco~
se~uences"
(iii) ~Y~~ CAL: YES
(Xi ) ~U~N~ DESCRIPTION: SEQ ID NO:9:
YTNM~NC'~N~ AYCARGAYTA YYTNATGCAY A~w~N~AYM GN 42
(2) INFORMATION FOR SEQ ID NO:lO:
( i ) g~yu~N~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANn~n~-~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic oligonucleotide
mixture"
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:l0:
A1GC~N~NA THTGGGCNGA 20
(2) INFORMATION FOR SEQ ID NO:ll:
( i ) ~QUh~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRAN~ N~.cS: single
(D) TOPOLOGY: linear
CA 02235801 1998-04-24
WO 97/lS678 PCTrUs96/l735
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic oligonucleotide
mixture"
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:ll:
TGCATNAGRT ARl~Yl~RlC 20
(2) INFORMATION FOR SEQ ID NO:12:
(i) ~yU~N~' CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /de8c = "Synthetic oligonucleotide
mixture"
(Xi ) ~'yUhN-C'~ DESCRIPTION: SEQ ID NO:12:
TCNGCCCADA lNG~N-GG-AT 20
(2) INFORMATION FOR SEQ ID NO:13:
( i ) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STR~NnT~n~s single
(D) TOPOLOGY: linear
( ii ) M~T~CUT~T' TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic oligonucleotide
mixture"
82
CA 02235801 1998-04-24
W o 97n5678 PCTAUS96/1735l
(xi) 8~Uu~ DESCRIPTION: SEQ ID NO:13:
GAYCARGAYT AYCTNATGCA 20
- (2) INFORMATION FOR SEQ ID NO:14:
( i ) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) ST}~NI )I~ NNl~:~s: 8ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: tdesc = "Synthetic oligonucleotide
mixture"
(Xi) S~UU~N~ DESCRIPTION: SEQ ID NO:14:
1 ~l ~N~NC KNAR 14
83
