Note: Descriptions are shown in the official language in which they were submitted.
20~06~;~
A 1 2 j 1 9 97
A process for the production of transgenic plants
with increased nutritional value via the
expression of modified 2S storage albumins
This invention relates to a process for the production
of plants with increased content of appropriate aminoacids
having high nutritional properties through the modification
of plant genes encoding plant storage proteins, more particu-
larly the 2S albumins.
More particularly, the invention aims at providing
genetically modified plant DNA and plant live material in-
cluding said genetically modified DNA replicable with the
cells of said plant material, which genetically modified
plant DNA contains sequences encoding for a polypeptide
containing said appropriate aminoacids which expression is
under the control of a suitable plant promoter.
A further object of the invention is to take advantage
of the capacity of 2S albumins to be produced in large
amounts in plants.
A further object of the invention is to take advantage
of a hypervariable region of the 2S albumins, which supple-
mentation with a number of said appropriate aminoacid codons
in said hypervariable region of the gene enco~ing said 2S
albumins, do not disturb the correct expression! processing
and transport of said produced modified storage proteins in
the protein bodies of the plants.
Animals and men obtain directly or indirectly their
essential aminoacids by eating plants. These essential
aminoacids include lysine, thryptophane, threonine, methion-
ine, phenylalanine, leucine, valine and isoleucine. For the
easiness of the language these aminoacids are called "appro-
priate aminoacids". Rather recently, agricultural scien-
tists concerned with the world's hungry problem, concentrat-
ed their work on developing plants with high nutritional
yield. These new varieties, obtained through breeding in
OGC, ~~
CA 02000661 1998-06-30
the most cases, were richer in carbohydrates but usually
poorer in essential proteins than the wild type varieties
from which they were derived. Currently, increasing recogni-
tion of the role of plants in supplying essential aminoacids
to the animal world had led to emphasis on the development
of new food plants having a better aminoacid content.
Classical breeding however has limitations for achieving
this goal. Molecular genetics, on the contrary, offers a
possibility to overcome these difficulties. Reference is
lo made to EP 0 208 418 published January 14, 1987 and the
communication of Brown et al., 1986, in which a gene encod-
ing a corn seed storage protein, (the so called zeins) is
modified by the addition of sequences encoding lysine
codons.
Seed storage proteins represent up to 90% of total seed
protein in seeds of many plants. They are used as a source
of nutrition for young seedlings in the period immediately
after germination. The genes encoding them are strictly
regulated, being expressed in a highly tissue specific and
stage specific fashion (Walling et al., 1986; Higgins,
1984). Thus they are expressed almost exclusively in devel-
oping seed, and different classes of seed storage proteins
may be expressed at different stages in the development of
the seed. They are generally restricted in their intercellu-
lar location, being stored in membrane bound organelles
called protein bodies or protein storage vacuoles. These
organelles provide a protease-free environment, and often
also contain protease inhibitors. A related group of pro-
teins, the vegetative storage proteins, have similar ami-
noacid compositions and are also stored in specialized vac-
uoles, but are found in leaves instead of in seeds
(Staswick, 1988). These proteins are degraded upon flower-
ing, and are thought to serve as a nutritive source for
developing seeds.
CA 02000661 1998-06-30
The expression of foreign genes in plants is well estab-
lished (De Blaere et al., 1987). In several cases seed stor-
age protein genes have been transferred to other plants. In
most of these cases it was shown that within its new environ-
ment the transferred seed storage protein gene is expressed
in a tissue specific and developmentally regulated manner
(Beachy et al., 1985; Sengupta-Gopalan et al., 1985; Marris
et al., 1988; Ellis et al., 1988; Higgins et al., 1986, Oka-
muro et al., 1986). It has also been shown in at least two
cases that foreign seed storage proteins are located in the
protein bodies of the host plant (Greenwood and Chrispeels,
1985;
Hoffman et al., 1987). It has further been shown that stable
and functional messenger RNA's can be obtained if a cDNA,
rather than a complete gene including introns, is used as
the basis for the chimeric gene (Chee et al., 1986).
Storage proteins are generally classified on the basis
of solubility and size (more specifically sedimentation
rate, for instance as defined by Svedberg (in Stryer, L.,
Biochemistry, 2nd ed., W.H. Freeman, New York, page 599)). A
particular class of seed storage proteins has been studied,
the 2S seed storage proteins, which are water soluble albu-
mins. They represent a significant proportion of the seed
storage proteins in many plants (Youle and Huang, 1981)
(Table I) and their small size and consequently simpler
structu~e makes t~em an attractive target for modification
(see also EP 0 319 353 published June 7, 1989) . Several 2S
storage proteins have been characterized at either the pro-
tein, cDNA or genomic clone levels (Crouch et al., 1983;
Sharief and Li, 1982; Ampe et al., 1986; Altenbach et al.,
1987; Ericson et al., 1986; De Castro et al., 1987; Scofield
and Crouch, 1987; Josefsson et al., 1987; EP 0 319 353,
Krebbers et al., 1988). 2S albumins are formed in the cell
from two subunits of 6-9 and 3-4 kilodaltons (kd) respective-
ly, which are linked by disulfide bridges.
-
2 ~
4 M A I 2 ~ 1~ 97
The work in the references above showed that 2S albu-
mins are synthesized as complex prepropeptides whose organi-
zation is shared between the 2S albumins of many different
species and are shown diagrammatically for three of these
species in figure 1. Several complete sequences are shown
in figure 2.
As to Fig. 2 relative to protein sequences of 2S albu-
mins, the following observations are made. For B. napus, ~.
excelsia, and A. thaliana both the protein and DNA sequences
have been determined, for R. communis only the protein se-
quence is available (B. napus from Crouch et al., 1983 and
Ericson et al., 1986; B. excelsia from Ampe et al., 1986, De
Castro et al., 1987 and Altenbach et al., 1987, _. communis
from Sharief and Li, 1982). Boxes indicate homologies, and
raised dots the position of the cysteines.
Comparison of the protein sequences at the beginning of
the precursor with standard consensus sequences for signal
peptides reveals that the precursor has not one but two
segments at the amino terminus which are not present in the
mature protein, the first of which is a signal sequence
(Perlman and Halvorson, 1983) and the second of which has
been designated as the amino terminal processed fragment (the
so-called ATPF). Signal sequences serve to ensure the co-
translational transport of the nAsc~nt polypeptide across the
membrane of the endoplasmic reticulum (Blobel, 1980), and are
found in many types of proteins, including all seed storage
proteins examined to date (Herman et al., 1986). This is
crucial for the appropriate compartmentalization of the pro-
tein. The protein is further folded in such a way that cor-
rect disulfide bridges are formed. This process is probably
localized at the luminal site of the endoplasmatic reticulum
membrane, where the enzyme disulfide isomerase is localized
(Roden et al., 1982; Bergman and Kuehl, 1979). After translo-
cation across the endoplasmic reticulum membrane it is
thought that most storage proteins are transported via said
(~iGC, TECI~ISO~IRCE
2 ¦ 19~7
endoplasmic reticulum to the Golgi bodies, and from the lat-
ter in small membrane bound vesicles (~dense vesiclesn) to
the protein bodies (Chrispeels, 1983; Craig and Goodchild,
1984; Lord, 1985). That the signal peptide is removed co-
translationally implies that the signals directing the fur-
ther transport of seed storage proteins to the protein bodies
must reside in the remainder of the protein sequence
present. Zeins and perhaps some other prolaminins deviate
from this pathway; indeed the protein bodies are formed by
budding directly off of the endoplasmic reticulum (Larkins
and Hurkman, 1918). As already of record, 2S albumins contain
sequences at the amino end of the precursor other than the
signal sequence which are not present in the mature polypep-
tide. This is not general to all storage proteins. This amino
terminal processed fragment is labeled ATPF in figure 1.
In addition, as shown in figure 1, several aminoacids
located between the small and large subunits in the precursor
are removed (labeled IPF in the figure, which stands for
internal process~ fragment). Furthermore, several residues
are removed from the carboxyl end of the precursor (labeled
CTPF in the figure which stands for carboxyl terminal pro-
cessed fragment). The cellular location of these latter pro-
cessing steps is uncertain, but is most likely the protein
bodies (Chrispeels et al., 1983; Lord, 1985). As a result of
these processing steps the small subunit and the large sub-
unit remain. These are linked by disulfide bridges, as dis-
cussed below.
When the protein sequences of 2S albumins of different
plants are compared strong structural similarities are ob-
served. This is more particularly illustrated by figure 2
which provides the aminoacid sequences of the small subunit
and large subunit respectively of representative 2S storage
seed albumin proteins of different plants, i.e.,:
R. comm. : Ricinus communis
GC T~c~o~ C~
0 fi ~ ~ ~ A i 2 ¦ 19 ~ 7
A. thali.: Arabidopsis thaliana
B. napus : Brassica napus
B. excel.: Bertholletia excelsia (Brazil nut)
It must be noted that in Fig. 2:
- the aminoacid sequences of said subunits extend on
several lines; the cysteine y~OU~_ of the aminoacid
sequences of the exemplified storage proteins and iden-
tical aminoacids in several of said proteins have been
brought into vertical alignment; the hyphen signs which
appear in some of these sequences represent absent
aminoacids, in other words direct linkages between the
closest aminoacids which surrounded them:
- the aminoacid sequences which in the different proteins
are conserved are framed.
It will be observed that all the sequences contain
eight cysteine residues (the first and second in the small
subunit, the remainder in the large subunit) which could
participate in disulfide bridges as diagrammatically shown
in Fig. 3, which represents a hypothetical model (for the
purpose of the present discussion) rather than a representa-
tion of the true structure of the 2S albumin of Arabidopsis
thaliana.
Said hypothetical model has been inspired by the dis-
ulfide ~ridge mediated loop-formation of animal albumins,
such a~ serum albumins (Brown, 1976), alpha-fetoprotein
(Jagodzinski et al., 1987; Morinaga et al., 1983) and the
vitamine D binding protein where analogous constant C-C
doublets and C-X-C triplets were observed (Yang et al.,
1985).
As can be seen on Fig. 2, the regions which are interca-
lated between the first and second cysteines, between the
fifth and sixth cysteines, and between the seventh and eight
cysteines of the mature protein show a substantial degree of
conservation or similarity. It would thus seem that these
regions are in some way essential for the proper folding
OGC, TECHSol JR~'J~
6 ~ ~, 2¦! t997
and/or stability of the protein when synthesized in the
plants. An exception to this conservation consist in the
distance between the sixth and seventh
cysteine residues. This suggests that these arrangements are
structurally important, but that some variation is permissi-
ble in the large subunit between said sixth and seventh cys-
teines where little conservation of aminoacids is observed.
An analogous suggestion has been made by Slightom and Chee
(1987), where the viciline type seed storage proteins from
peas were compared. These authors indeed suggest that ami-
noacid replacement mutations designed to increase the number
of sulphur containing aminoacids should be placed in regions
which show little or no conservation of aminoacid sequences.
The authors however conclude that the proof that such modifi-
cations can be tolerated will need to be tested in the seeds
of transgenic plants. Moreover, the teaching provided in
their paper on the properties of the through deletion modi-
fied storage protein concerns only the influence on expres-
sion levels and not on processing of said storage proteins.
An embodiment of this invention is the demonstration
that a well chosen region of the 2S albuuin allows variation
without altering the properties and correct processing of
said modified storage protein in plant cells of transgenic
plants.
This region (diagrammatically shown in Fig. 3 by an
enlarged hatched portion) will in the examples hereafter
referred to be termed as the "hypervariable region~. Fig. 3
also shows the respective positions of the other parts of the
precursor sequence, including the "IPF~ section separating
the small subunit and large subunit of the precursor, as well
as the number of aminoacids (aa) in substantially conserved
portions of the protein subunits cysteine residues. The pro-
cessing cleavage sites (as determined by Krebbers et al.,
1988) are shown by symbols.
OGC, T~CHSf~
... ~Q~6~ Y2~;'1997
The seeds of many plants contain albumins of approximate-
ly the same size as the storage proteins discussed above.
However, for ease of language, this document will use the
term "2S albumins" to refer to seed proteins whose genes
encode a peptide precursor with the general organization
shown in figure 1 and which are processe~ to a final form
consisting of two subunits linked by disulfide bridges. The
process of the invention for producing plants with an in-
creased content of appropriate aminoacids comprises :
cultivating plants obtained from regenerated plant cells
or from seeds of plants obtained from said regenerated
plant cells over one or several generations, wherein the
genetic patrimony or information of said plant cells,
replicable within said plants, includes a nucleic acid
sequence, placed under the control of a plant promoter,
which can be transcribed into the mRNA encoding at least
part of the precursor of a 2S albumin including the
signal peptide of said plant, said nucleic acid being
hereafter referred to as the "precursor encoding nucleic
acid"
. wherein said nucleic acid contains a nucleotide se-
quence (hereafter termed the "relevant sequencen) which
relevant sequence comprises a nonessential region modi-
fied by a heterologous nucleic acid insert forming an
open reading frame in reading phase with the non modi-
fied parts surrounding said insert in said relevant
sequence.
. wherein said insert includes a nucleotide segment
encoAing a polypeptide containing appropriate ami-
noacids.
It will be appreciated that under the above mentioned
conditions each and every cell of the cultivated plant will
include the modified nucleic acid. Yet the above defined
recombinant or hybrid sequence will be expressed at high
levels constitutively or only or mostly in certain organs of
QGC TECH.SrJt I~CE
~ ~ ~ Q ~fi ~ ~Y 21 ~997
the cultivated plantc dependent on which plant promoter ha~
been chosen to conduct its expression. In the case of
seed-specific promoters the hybrid storage protein will be
produced mostly in the seeds.
It will be understood that the ~heterologous nucleic
acid insert" defined above consists of an insert which con-
tains nucleotide sequences which at least in part, may be
foreign to the natural nucleic acid encoding the precursor of
the 2S albumins of the plant cells concerned and encode the
appropriate aminoacids. Most generally the segment encoding
polypeptide containing said appropriate aminoacids will it-
self be foreign to the natural nucleic acid enco~;nq the
precursor of said storage protein. Nonetheless, the ter~
"heterologous nucleic acid insertN does also extend to an
insert containing a segment as above-defined normally present
in the genetic patrimony or information of said plant cells,
the "heterologous" character of said insert then addressing
to the different genetic environment ~hich surrounds said
insert.
In the pr~ceAing definition of the process according to
the invention the so-called "nonessential region~ of the
relevant sequence of said nucleic acid encoding the precur-
sor, consists of a region whose nucleotide sequence can be
modified either by insertion into it of the above-defined
insert or by replacement of at least part of said nonessen-
tial region by said insert, yet without disturbing the stabil-
ity and correct processing of said hybrid storage protein a~
well as its transport into the above-said protein bodies.
Sequences consisting of said insert or replacement and repre-
senting the coding region for a polypeptide containing appro-
priate aminoacids can either be put in as synthetic oligomers
or as restriction fragments isolated from other genes, as
thought by Brown, 1986. The total length of the hybrid stor-
age protein may be longer or shorter than the total length of
the non-modified 2S albumin.
~)GC, TECHSOURC~
CA 02000661 1998-06-30
With respect to the choice of the region to be modified,
the present invention is clearly distinguishable from other
work which has been done in this field. Reference is made to
DD-A-240911 patent from the Akademie der Wissenschaften der
DDR where legumin genes from Vicia faba, (glutine and prola-
mine) were modified in vitro with sequences encoding methion-
ine. As place of insert a natural occurring PstI site has
been chosen. At the EMBO workshop "Plant storage protein
genes", (Breisach, FRG, September 1986) the authors presented
their work and informed the audience that plant transforma-
tion experiments were ~ust started with the modified gene.
Mo further results have yet ~eer, published.
Reference is also made to patent application WO 87/07299
published December 3, 1987 and correspon~;ng publication of Radke
et al., 1988. These papers describe the modification of the napin
gene, which encodes the 2S albumin o~ Brassica napus, ~y a
r.ucleotide sequence encoding nine aminoacid residues includ-
ing 5 consecutive methionines. The region of modification is
a naturally occurring SstI site within the region encoding
the mature protein. Such a modification would result in a
insertion directly adjacent to a cysteine residue and more-
over in a region between two cysteines, namely the 4th and
the 5th cysteines of the mature protein which correspond with
the 2nd and 3rd cysteines of the large subunit, whose length
is strongly conserved (see above). We believe such a modifi-
cation is likely to disrupt a normal folding and stability of
the 2S albumin (EP 0 319 353 publiRhed June 7, 1989). Moreover,
above cited reference~ provide no evidence that the desired modi-
fied 2S albumin was successfully synthesized, correctly pro-
cessed or correctly targeted.
In the present invention the precursor-coding nucleic
acid referred to above may of course originate from the same
plant species as that which is cultivated for the purpose of
the invention. It may however originate from another plant
species, in line with the teachings of Beachey et al., 1985
and Okamurc et al., 1986 already of record.
2 0 ~ ~ 6 ~ Y 21-j 19~7
In a similar manner the plant promoter may originate
from the same plant ~pecies or from another, sub~ect in the
last instance to the capability of the host plant's polymeras-
es to reco~nize it. It may act constitutively or in a
tissue-specific manner, such as, but not limited to,
seed-specific promoters.
Regions such as the ones at the end of the small sub-
unit, at the beginning or end of the large subunit, show
differences of such a magnitude that they can be held as
presumably having no substantial impact on the final proper-
ties of the protein. The extreme carboxyl terminus of the
small subunits and the amino terminus of the large subunit
may, however, be involved in the processing of the internal
proceC~s~ fragment. A region which does not seem essential,
consists of the middle position of the region located in the
large subunit, between the sixth and the seventh cysteine of
the nature protein, but not immediately adjacent and at least
3 aminoacids separated from said cysteines. Thus in addition
to the absence of similarity at the level of the aminoacid
residues, there appears a difference in length which makes
that region eligible for substitutions in the longest 2S
albumins and for addition of aminoacids in the shortest 2S
albumins or for elongation of both. The same should be appli-
cable at approximately of the end of the first third part of
the same region between said sixth and seventh cysteine; see
the sequence of R. communis which is much shorter at that
region than the corresponding regions of the other exempli-
fied 2S proteins.
It is of course realized that caution must be exorcised
against hypotheses based on arbitrary choices as concerns the
bringing into line of similar parts of proteins which else-
where exhibit substantial differences. Nevertheless such
comparisons have proven in other domains of genetics to pro-
vide the man skilled in the art with appropriate guidance to
reasonably infer from local structural differences, on the
C)GC, T~CL~S~,~J~E
2 ~ 6 ~ 21 19~7
one hand, and from local similarities, on the othe~ hand, in
similar proteins of different sources, which parts of such
proteins can be modified and which parts cannot, when it is
sought to preserve some basic properties of the non modified
protein in the same protein yet locally modified by a foreign
or heterologous sequence.
The choice of the adequate nonessential regions to be
used in the process of the invention will also depend on the
length of the polypeptide containing the appropriate ami-
noacids. Basically the method of the invention allows the
modification of said 2S albumins by the insertion and/or
partial substitution into the precursor nucleic acid of se-
quences encoding up to 100 aminoacids.
When the complete protein sequence of the region to be
inserted into a 2S albumin has been determined, the nucleo-
tide sequence to encode said protein sequence must be deter-
mined. It will be recognized that while perhaps not absolute-
ly neces~Ary the codon usage of the encoAing nucleic acid
should where possible be similar to that of the gene being
modified.
The person skilled in the art will have access to appropriate
computer analysis tools to determine said codon usage.
Any appropriate genetic engineering tec~nique may be used for
substituting the insert for part of the selected
precursor-coding nucleic acid or for inserting it in the
appropriate region of said precursor-coA i ng nucleic acid. The
general n vitro recombination techniques followed by cloning
in bacteria can be used for making the chimeric genes.
Site-directed mutagenesis can be used for the same pU~ 5
as further exemplified hereafter. DNA recombinants, e.g.
plasmids suitable for the transformation of plant cells can
also be produced according to techniques disclosed in current
technical literature. The same applies finally to the produc-
tion of transformed plant cells in which the hybrid storage
protein encoded by the relevant parts of the selected
O~JC, T~CH.~~,' J~Cf~
6 ~ ~
~Y 21 1997
13
precursor-coding nucleic acid can be expressed. By way of
example, reference can be made to the published European
applications no. 116 718 or to International application WO
84/02913 and, which disclose appropriate te~hniques to that
effect.
When designing the sequences rich in appropriate ami-
noacids, care must be taken that the resulting peptide con-
taining said appropriate aminoacids does not influence the
stability of the modified 2S albumin. Certain insertions may
indeed disrupt the structure of the protein. For example,
long stretches of methionines may result in rod ~hAp~d heli-
ces which would result in instabilities due to disruption of
normal folding patterns. Thus such sequences must occasional-
ly include aminoacids which interrupt the helical structure.
The procedures which have been disclosed hereabove apply
to the adequate modification of the nonessential region of any
of 2S albumins by an heterologous insert containing a DNA
sequence enco~ing a peptide containing appropriate aminoacids
with nutritional properties and then to the transformation of
the relevant plants with the chimeric gene obtained for the
production of a hybrid protein containing the Fe~nce of said
peptide in the cells of the relevant plant. Needless to say
that the person skilled in the art will in all ins~nc~C be
able of selecting which of the existing tec~ique~ would at
best fulfill its needs at the level of each step of the produc-
tion of such modified plants, to achieve the best production
yields of said hybrid storage protein.
For instance the following process can be used in order
to exploit the capacity of a 2S albumin, to be used as a suit-
able vector for the production of plants with increased nutri-
tional value, by inserting in said 2S albumins nucleotide
codons encoding methionine and/or lysine and/or thryptophane
and/or threonine and/or phenylalanine and/or leucine and/or
valine and/or isoleucine when the corresponding
OGC, TEC~Is~! J~rr
6 fi ~ 21 1997
precursor-coding nucleic acid has been sequenced. Such process
then comprises:
1) locating and selecting one of said relevant sequences
of the precursor-coding nucleic acid which comprises a
nonessential region enco~ing a peptide sequence which can
be modified by substituting an insert for part of it or
by inserting of said insert into it, which modification
is compatible with the conservation of the configuration
of said 2S albumins and this preferable by determining
the relative positions of the codons which encode the
successive cysteine residues in the mature protein or
protein subunits of said 2S albumins and identifying the
corresponding successive nucleic acid regions located
upstream of, between, and downstream of said codons with-
in said sub-sequences of the precursor-coding nucleic
acid and identifying in said successive regions those
parts which undergo variability in either aminoacid se-
quence or length or both from one plant species to anoth-
er as compared with those other regions which do exhibit
substantial conservation of aminoacid sequence in said
several plant species, one of said nucleotide regions
being then selected for the insertion therein of the
nucleic acid insert as described hereunder.
An alternative would consist of st,udying any 3-D struc-
tures which may become available in the future.
2)inserting a nucleic acid insert in the selected region
of said precursor nucleic acid in appropriate reading
frame relationship with the non-modified parts of said
relevant sequence, which insert includes a determined
segment enco~;~g a peptide containing all or part of the
above mentioned appropriate aminoacids.
3) inserting the modified precursor-coding nucleic acid
obtained in a plasmid suitable for the transformation of
plant cells which can be regenerated into full
OGC, T~C~ r~
~l 21 ~g97
seed-forming plants, wherein ~aid insertion is brought
under the control of regulation elements, particularly a
plant pro~oter capable of providing for the exp~ ion
of the open reading-frames associated therewith in said
plants;
4) transforming a culture of such plant cells with such
modified plasmid;
5) assaying the expression of the chimeric gene encoding
the hybrid storage protein and, when achieved;
6) regenerating said plants from the transformed plant
cells obtained and growing said plants up to maturity.
In the case the chimeric gene is under the control of a
seed specific promotor, growing up the transformed plants to
seeds must precede step 5)
Hence embodiment as described under 1) of the invention
hereabove provides that in having the hybrid 2S albumins in a
plant, it will pass the plant protein disulfide isomerase
during membrane translocation, thus increasing the chances
that the correct disulfide bridges be formed in the hybrid
precursor as in its normal precursor situation, on the one
hand
The invention further relates to the recombinant nucleic
acids themselves for use in the process of the invention;
particularly to the
- recombinant precursor encoding nucleic acid defined
in the context of said process;
- recombinant nucleic acids containing said modified
precursor encoding nucleic acid under the control of
a plant promoter, whether the latter originates from
the same DNA as that of said precursor coding nucleic
acid or from another DNA of the same plant from which
the precursor encoding nucleic acid is derived, or
from a DNA of another plant, or from a non-plant
organism provided that it is capable of directing
gene expression in plants.
OGC TEC!~ rF
fi ~
~ 9 9 7
16
- vectors, more particularly plant plasmids e.g.,
Ti-derived plasmids modified by any of the preceding
recombinant nucleic acids for use in the transforma-
tion of the above plant cells.
The invention also relates to the regenerable source of
the hybrid 2S albumin, which is formed of in the cells of a
seed-forming-plant, which plant cells are capable of being
regenerated into the full plant or seeds of said seed-forming
plants wherein said plants or seeds have been obtained as a
result of one or several generations of the plants resulting
from the regeneration of said plant cells, wherein further
the DNA supporting the genetic information of said plant
cells or seeds comprises a nucleic acid or part thereof,
including the sequences encoding the signal peptide, which
can be transcribed in the mRNA corresponding to the precursor
of a 2S albumin of said plant, placed under the control of a
plant specific promoter, and
. wherein said nucleic acid sequence contains a relevant
modified sequence encoding the mature 2S storage protein
or one of the several sub-sequences encoding for the
corresponding one or several sub-units of said mature 2S
albumins,
. wherein further the modification of said relevant
sequence takes place in one of its noneccential regions
and consists of a heterologous nucleic acid insert form-
ing an open-reading frame in reading phase with non
modified parts which surround said insert in the rele-
vant sequence,
. wherein said insert consists of a nucleotide segment
e~coAing a peptide containing methionine and/or lysine
and/or thryptophane and/or threonine and/or phenylala-
nine, and/or leucine and/or valine and/or isoleucine.
It is to be considered that although the invention
should not be deemed as being limited thereto, the nucleic
inserts encoding the above mentioned appropriate aminoacids
QGC TEC~Sf',~ F
fi ~ y
2~ ly97
17
will in most instances be man-made synthetic oligonucleotides
or oligonucleotides derived from procaryotic or eucaryotic
genes or of from cDNAs derived of procaryotic or eucaryotic
RNAs, all of which shall normally escape any possibility of
being inserted at the appropriate places of the plant cells
or seeds of this invention through biological proceC~?-~
whatever the nature thereof. In other vords, these inserts
are "non plant variety specific~, specially in that they can
be inserted in different kinds of plants which are genetical-
ly totally unrelated and thus incapable of eY~h~nging any
genetic material by standard biological procesces, including
natural hybridization process~s.
Thus the invention further relates to the seed forming
plants themselves which have been obtained from said trans-
formed plant cells or seeds, which plants are characterized
in that they carry said hybrid precursor-coding nucleic acids
associated with a plant promoter in their cells, said inserts
however being expressed and the corresponding hybrid protein
produced in the cells of said plants.
There follows an outline of a preferred method which can
be used for the modification of a 2S albumin gene and its
expression in the seeds obtained from the transgenic plants.
The outline of the method given here is fol~owed by a specif-
ic example. It will be understood from the person skilled in
the art that the method can be suitably adapted for the modi-
fication of other 2S albumin genes.
1. Replacement or supplementation of the hypervariable
region of the 2S albumin gene by a sequence encoding
peptide containing appropriate aminoacids which possess
nutritional properties.
Either the cDNA or the genomic clone of the 2S albumin
can be used. Comparison of the sequences of the hypervariable
regions of the genes in figure 2 shows that they vary in
length. Therefore if the sequence encoding a peptide contain-
OGC, TrCHSCUF~CE
CA 02000661 1998-06-30
18
ing the appropriate aminoacids is short and a 2S albumin with
a relatively short hypervariable region is used, said se-
quence of interest can be inserted. Otherwise part of the
hypervariable region is removed, to be replaced by the insert
containing a larger segment or sequence encoding the peptide
containing the appropriate aminoacids. In either case the
modified hybrid 2S albumin may be longer than the native
one. In either case two standard techniques can be applied;
convenient restriction sites can be exploited, or mutagenesis
vectors (e.g. Stanssens et al. 1987) can be used. In both
cases, care must be taken to maintain the reading frame of
the message.
The sequence encoding the signal peptide of the precur-
sor of the storage protein used either belongs to this precur-
sor or can be a substitute sequence coding for the signal
peptide or peptides of an heterologous storage protein.
2. The altered 2S albumin coding region is placed under the
control of a plant promoter. Preferred promoters in-
clude the strong constitutive exogeneous plant promoters
such as the promoter from cauliflower mozaic virus di-
recting the 35S transcript (Odell, J.T. et al., 1985),
also called the 35S promoter; the 35S promoter from the
CAMV isolate Cabb-JI (Hull and Howell, 1987), also
called the 35S3 promoter; the bidirectional TR promoter
which drives the expression of both the 1' and the 2'
genes of the T-DNA (Velten et al., 1984).
Alternatively a promoter can be utilized which is not
constitutive but specific for one or more tissues or
organs of the plant. Given by way of example such kind
promoters may be the light inducible promoter of the
ribulose-l, 5-bi-phosphate carboxylase small subunit
gene (EP 0 193 259 published December 3, 1986, if the expres-
sion is desired in tissue with photosynthetic activity,
or may be seed specific promoters.
19 ~ 6 ~ ~a~r 21
A seed ~pecific promoter is used in order to ensure
subsequent expression in the seeds only. This may be of
particular use, since ~eeds constitute an important food or
feed source. Moreover, this specific expression avoids possi-
ble stresses on other parts of the plant. In principle the
promoter of the modified 2S albumin can be used. But this is
not necessAry. Any other promoter serving the same purpose
can be used. The promoter may be chosen according to its
level of efficiency in the plant species to be transformed.
In the examples below the 2S albumin promoter from the 2S
albumin gene from Arabidopsis is used, which constitutes the
natural promotor of the 2S albumin gene which is modified in
said examples. Needless to say that other seed specific promo-
tors may be used, such as the conglycinine promotor from
soybean. If a chimeric gene is so constructed, a signal pep-
tide encoAin~ region must also be included, either from the
modified gene or from the gene whose promotor is being used.
The actual construction of the chimeric gene is done using
stAn~rd molecular biological tech~ques described in Mania-
tis et al., 1982. (see example).
3. The chimeric gene construction is transferred into the
appropriate host plant.
When the chimeric or modified gene construction is com-
plete it is transferred in its entirety to a plant transforma-
tion vector. A wide variety of these, based on disarmed
(non-oncogenic) Ti-plasmids derived from A~robacterium tumefa-
ciens, are available, both of the binary and cointegration
forms (De Blaere et al., 1987). A vector including a
selectable marker for transformation, usually antibiotic
resistance, should be chosen. Similarly, the methods of
plant transformation are also numerous, and are fitted to
the individual plant. Most are based on either protoplast
transformation (Marton et al., 1979) or formation of a small
piece of tissue from the adult plant (Horsch et al., 1985).
In the example below, the vector is a binary disarmed
C)GC TECH~ l lRC~
~3Q~
I'Vl A 1 2 l ~997
Ti-plasmid vector, the marker is kanamycin resistance, and
the leaf disc method of transformation is used.
Calli from the transformation procedure are selected on
the basis of the selectable marker and regenerated to adult
5plants by appropriate hormone induction. This again varie~
with the plant species being used. Regenerated plants are
then used to set up a stable line from vhich seeds can be
harvested.
Further characteristics of the invention will appear in
10the course of the non-limiting disclosure of specific exam-
ples, particularly on the basis of the drawings in which:
- Figs. 1, 2 and 3 refer to overall features of
2S-albumins as already discussed above. The numbers
15refer to the number of aminoacids observed in the
different fragments of the protein precursor.
- Fig. 4 represents the sequence of lkb fragment con-
taining the Arabidopsis thaliana 2S albumin gene and
shows related elements. The NdeI site is underlined.
20- Fig. 5 provides the protein sequence of the large
subunit of the above Arabidopsis 2S protein together
with related oligonucleotide sequences.
- Fig. 6A shows diagrammatically the successive phA~eF
of the construction of a chimeric 2S albumin-Arabidop-
25thaliania gene including the deletion of practi-
cally all parts of the hypervariable region and its
replacement by a AccI site, the insertion of DNA
sequences rich in methionine codons, given by way of
of example in the following disclosure, in the AccI
30site, particularly through site-directed mutagenesis
and the cloning of said chimeric gene in plant vector
suitable for plant transformation.
- Fig. 6B shows diagrammatically t~e protein sequence
of the large subunit of several Arabidopsis 2S albu-
35mins and indicates the region removed from the genes
~G~ TEC~S r!!!~ ~ F
2 (~ ~ n 6 ~ 11 M,~Y 21 1~97
enco~;ng said 2S albumins, and shows diagrammatic~lly
where an AccI site has been created and how oligonu-
cleotides rich in methionine codons are inserted into
said AccI site in such a way that the open re~ng
frame is maintained.
- Fig 7 diagrammatically compares the protein sequenc-
es of the large subunits of the unmodified 2S albu-
min, in which most of the hypervariable region has
been deleted, and those of the modified 2S albu-
mins. The resulting number of methionine residues
are indicated.
- Fig. 8 shows the restriction sites and genetic map
of a plasmid suitable for the performance of the
above site-directed mutagenesis.
- Fig. 9 shows diagrammatically the different steps of
the site-directed mutagenesis procedure of St~nc~Qn~
et al (1987) as generally applicable to the modifica-
tion of nucleic acid at appropriate places.
- Fig. 10 gives the restriction map of pGSC1703A.
Example I :
As a first example of the method described, a prGc~
is given for the production of transgenic plant seeds with
increased nutritional value by having inserted into tbeir
genome a modified 2S albumin protein from Arabidopsis
thaliana having deleted its hypervariable region and re-
placed by way of example by a methionine rich peptide hav-
ing 7 aminoacids with the following sequence :I M M M M R
M. A synthetic oligomer encoAing said peptide is substitut-
ed for essentially the entire part of the hypervariable
region in a genomic clone encoding the 2S albumin of Arabi-
dopsis thaliana. Only a few aminoacids adjacent to the
sixth and seventh cysteine residues remained. This chimer-
ic gene is under the control of its natural promoter and
;3GC, ~:C'~SClJRcE
6 ~ ~Y
~' 21 ~9~7
22
signal peptide. The process and constructions are diagram-
matically illustrated in Fiq. 6A, 6B and 7. The entire
construct is transferred to tobacco, Arabidopsis thalian~
and Brassica napus plants using an Agrobacterium mediated
transformation system. Brassica napus is of particular
interest, since this crop is widely used as protein source
for animal feed.
Plants are regenerated, and after flowering the seeds are
collected and the methionine content compared with untrans-
formed plants.
1. Cloninq of the Arabidopsis thaliana 2S albumin gene.
The Arabidopsis thaliana gene has been cloned accord-
ing to what is described in Krebbers et al., 1988. The
plasmid containing said gene is called pAT2S1. The se-
quence of the region containing the gene, which is called
AT2Sl, is shown in figure 4.
2. Deletion of the hypervariable re~ion of AT2S1 gene and
replacement by an AccI site.
Part of the hypervariable region of AT2S1 is replaced by
the following oligonucleotide:
5'- CCA ACC TTG AAA GGT ATA CAC TTG CCC AAC - 3'
30-mer
P T L K G I H L P N
in which the underlined sequences represent the AccI site
and the surrounding ones sequences complementary to the cod-
ing sequence of the hypervariable region of the Arabidopsis
2S albumin gene to be retained. This results finally in the
aminoacid sequence indicated under the oligonucleotide.
O(~C, TF~-~HSCllR~E
CA 02000661 1998-06-30
The deletion and substitution of part of the sequence encod-
ing the hypervariable region of AT2Sl is done using site
directed mutagenesis with the oligonucleotide as primer. The
system of Stanssens et al. (1987) is used.
The Stanssens et al. method is described in EP 0 319 353 publis-hed
June 7, 1989. It makes use of plasmid pMac5-8 whose restriction
and genetic m~Ap and the positions of the relevant genetic loci are
shown in Fig. 8. The arrows denote their functional orientation.
origin of replication of filamentous phage fl; ORI:
ColE1-type origin of replication; BLA/ApR : region coding
for B-lactamase; CAT/CmR : region coding for chlorampheni-
col acetyl transferase. The positions of the amber mutations
present in pMc5-8 (the bla-am gene does not contain the ScaI
site) and
pMc5-8 (cat-am; the mutation eliminates the unique PvuII
site) are indicated. Suppression of the cat amber mutation in
both suDE and supF hosts results in resistance to at least 25
ug/ml Cm. pMc5-8 confers resistance to +20 ug/ml and 100
ug/ml Ap upon amber-suppression in supE and supF strains
respectively. The EcoRI, BalI and NcoI sites present in the
wild-type cat gene (indicated with an asterisk) have been
removed using mutagenesis techniques.
Essentially the mutagenesis round used for the above men-
tioned substitution is ran as follows. Reference is made to
Fig. 9, in which the amber mutations in the Ap and Cm select-
able markers are shown by closed circles. The symbol
represents the mutagenic oligonucleotide. The mutation itself
is indicated by an arrowhead.
The individual steps of the process are as follows:
- Cloning of the HindIII fragment of pAT2Sl containing the
coding region of the AT2Sl gene into pMaS-8 (I). This
vector carries on amber mutation in the CmR gene and
M ~ 1 21 1997
specifies resistance to ampicillin. The resulting plas-
mid is designated pMacAT2S1 (see figure 6A step 1).
- Preparation of single stranded DNA of this recombinant
(II) from pseudoviral particles.
- Preparation of a HindIII restriction fragment from the
complementary pMc type plasmid (III). pMc-type vectors
contain the wild type CmR gene while an amber mutation
is incorporated in the Ap resistance marker.
- Construction of gap duplex DNA (hereinafter called
gdDNA) gdDNA (IV) by in vitro DNA/DNA hybridization. In
the gdDNA the target sequences are exposed as single
stranded DNA. Preparative purification of the gdDNA from
the other components of the hybridization mixture is not
nececc~ry.
- Annealing of the 30-mer synthetic oligonucleotide to the
gdDNA (V).
- Filling in the remaining single stranded gaps and seal-
ing of the nicks by a simultaneous i~ vitro Klenow DNA
polymerase I / DNA ligase reaction (VI).
- Transformation of a mutS host, i.e., , a strain
deficient in mismatch repair, selecting for Cm resis-
tance. This results in production of a mixed plasmid
progeny (VII).
- Elimination of progeny deriving from the template strand
(pMa-type) by retransformation of a host unable to sup-
press amber mutations (VIII). Selection for Cm resi~-
tance results in enrichment of the progeny derived from
the gapped strand, i.e., , the strand into which
the mutagenic oligonucleotide has been incorporated.
- Screening of the clones resulting from the retransforma-
tion for the presence of the desired mutation. The re-
sulting plasmid containing the deleted hypervariable
region of AT2Sl is called pMacAT2SlC40 (see figure 6A
step 2).
OGC, TECH~OURCF
6 ~ ~
~ 21 1997
3. Insertion of sequences rich in methionine codons into
the AT2Sl gene whose sequences encoding the hypervariable
region have been deleted.
As stated above when the sequences enco~;ng moct of the
hypervariable loop were removed an AccI site was inserted in
its place. The sequences of interest will be inserted into
this AccI site, but a second AccI site is also present in the
HindIII fragment containing the modified gene. Therefore the
NdeI-HindIII fragment containing the modified gene is sub-
cloned into the cloning vector pBR322 (Bolivar, 1977) also
cut with NdeI and HindIII. The position of the NdeI site in
the 2S albumin gene is indicated in figure 4. The resulting
subclone is designated pBRAT2Sl (Figure 6A, step 3).
In principle any insert desired can be inserted into the
AccI site in pBRAT2Sl. In the present example said insert
encodes the following sequence: I.M.M.~.M.R.M. Therefore
complementary oligonucleotides encoding said peptide are
synthesized taking into account the codon usage of AT2Sl and
ensuring the the ends of the two complementary oligonucleo-
tides are complementary to the staggered ends of the AccI
site, as shown here (the oligonucleotides are shown in bold
type) :
5' GT ATA AT& ATG AT& ATG CGC ATG ATAC 3'
3' CA TAT TAC TAC TAC TAC GCG TAC TATG 5'
The details of this insertion, showing how the reading
frame is maintained, are shown in figure 6B. The two oligonu-
cleotides are annealed and ligated with pBRAT2Sl digested
with AccI (figure 6A, step 4). The resulting plasmid is
designated pAD4.
OGC, T~f~,'u.Sf.--;'. ''--,
~iA~ ~1 1997
26
4. Reconstruct~on of the com~lete modified AT2Sl gene with
its natur~l ~romoter.
The complete chimeric gene is reconstructed a~ follows
(see figure 6A): The clone pAT2SlBg contains a 3.6kb BglII
fragment inserted in the cloning vector pJB65 (Botterman et
al., 1987) which encompasses not only the l.Okb HindIII frag-
ment containing the coding region of the gene AT2Sl but suffi-
cient sequences upstream and downstream of this fragment to
contain all neceC~ry regulatory elements for the proper
expression of the gene. This plasmid is cut with HindIII and
the 5.2kb fragment (i.e., that portion of the plasmid not
containing the coding region of AT2Sl) is isolated. The
clone pAT2Sl is cut with HindIII and NdeI and the resulting
320 bp HindIII-NdeI fragment is isolated. This fragment
represents the one removed from the modified 2S albumin in
the construction of pBRAT2Sl (step 3 of figure 6A) in order
to allow the insertion of the oligonucleotides in step 4 of
figure 6A to proceed without the complications of an extra
AccI site. These two isolated fragments are then ligated in
a three way ligation with the NdeI-HindIII fragment from pAD4
(figure 6A, step 5) containing the modified co~3ing sequence.
Individual tranformants can be screened to check for appropri-
ate orientation of the reconstructed HindIII fragment within
the BglII fragment using any of a number of sites. The re-
sulting plasmid, pAD17, consists of a 2S albumin gene modi-
fied only in the hypervariable region, suLLo~"~ed by the same
flanking sequences and thus the same promoter as the unmodi-
fied gene, the entirety contained on a BglII fragment.
5. Transformation of plants.
The BglII fragment containing the chimeric gene is in-
serted into the BglII site of the binary vector pGSC1703A
(Fig. 10) (see also Fig. 6A step 6). The resultant plasmid
is designated pTAD12. Vector pGSC1703A contains functions
OGC, TECHSGURCE
fi ~
~,A~ 21 ~997
27
for selection and stability in both ~. Çli and ~. tumefa-
cien8, as well as a T-DNA fragment for the transfer of for-
eign DNA into plant genomes (Deblaere et al., 1987). It fur-
ther contains the bi-directional TR promotor (Velten et al.,
1984) with the neomycin pho~photransferase protein coding
region (neo) and the 3' end of the ocs gene on one side, and
a hygromycin transferase gene on the other side, ~o that
transformed plants are both kanamycin and hygromycin resis-
tant. This plasmid does not carry an ampicillin resistance
gene, so that carbenicillin as well as claforan can be used
to kill Agrobacterium after the infection step. Using stan-
dard procedures (Deblaere et al., 1987), pTAD12 is trans-
ferred to the Agrobacterium strain C58ClRif carrying the
plasmid pMP90 (Koncz and Schell, 1986). The latter provides
in trans the vir gene functions required for successful trans-
fer of the T-DNA region to the plant genome. This Agrobacteri-
um is then used to transform plants. Tobacco plants of the
strain SRl are transformed using st~n~rd procedures (De-
blaere et al., 1987). Calli are selected on 100 ug/ml kan-
amycin, and resistant calli used to regenerate plants.
The techniques for transformation of Arabidopsis thaliana
and Brassica napus are such that exactly the same construc-
tion, in the same vector, can be used. After mobilization to
Agrobacterium tumefaciens as described hereabove, the proce-
dures of Lloyd et al., (1986) and Rlimaszewska et al. (1985)
are used for transformation of Arabidopsis and Brassica re-
spectively. In each case, as for tobacco, calli can be se-
lected on 100 ug/ml kanamycin, and resistant calli used to
regenerate plants.
In the case of all three ~pecies at an early stage of
regeneration the regenerants are checked for transformation
by inducing callus from leaf on media supplemented with kan-
amycin (see also point 6).
3 6. Screening and analysis of transformed plants.
6 ~ ~
28 I~'IAI 21 1997
In the cace of all three species, regenerated plants are
grown to seed. Since different transformed plants can be
expected to have varying levels of expression (~position
effectsn, Jones et al., 1985), more than one tranformant must
initially be analyzed. This can in principle be done at
either the RNA or protein level; in this case seed RNA was
prepared as described (Beachy et al., 1985) and northern
blots carried out using standard tec-hniques (Thomas et al.,
1980). Since in the case of both Brassica and Arabidopsis
the use of the entire chimeric gene would result in cross
hybridization with endogeneous genes, oligonucleotide probes
complementary to the insertion within the 2S albumin were
used; the same probe as used to make the construction can be
used. For each species, 1 or 2 individual plants were chosen
for further analysis as ~iscllcsed below.
First the copy number of the chimeric gene is determined
by preparing DNA from leaf tissue of the transformed plants
(Dellaporta et al., 1983) and probing with the oligonucleo-
tide used above.
The methionine content of the seeds is analyzed using
known methods (Joseph and Marsden, 1986; Gehrke et al., 1985;
~l~in and Griffith, 1985 (a) and (b)).
Example II
As a second example of the method described, the same
procedure is followed for the production of transgenic plant
seeds with increased nutritional value by having inserted
into their genome a modified 2S albumin protein from ArabidoD-
sis thaliana having deleted its hypervariable region and
replaced by way of example by a methionine rich peptide hav-
ing 24 aminoacids with the following sequence :
C ~C~'
29 M ~1 21 1997
I ~ ~ ~ Q P R G D ~ ~ Q P R G ~ ~ ~
All different steps going from constructs to transformants
as disclosed for example I are executed with the only differ-
ence that in step 3 the following oligonucleotide has been
synthesized and inserted into pBrAT2Sl
(the oligonucleotides are shown in bold type)
5 ' GT ATA ATG ATG ATG CAA CCA AGG GGC GAT ATG ATG ATG ATA
Aq~G ATG ATG
3' CA TAT TAC TAC TAC GTT GGT TCC CCG GTA TAC TAC TAC TAT
TAC TAC TAC
CAA CCA AGG GGC GAT ATG ATG ATG ATA C - 3'
GTT GGT TCC CCG CTA TAC TAC TAC TAT G - 5 '
The relevant plasmids are indicated in figure 6A, details
of the insertion in figure 6B and resulting aminoacid se-
quence of the hybrid subunit shown in figure 7. The relevantplasmids as indicated in figure 6A are pAD3, pAD7 and pTAD10.
The examples have thus given a complete illustration of
how 2S albumin storage proteins can be modified to incorpo-
rate therein an insert enco~ing a methionine rich polypeptidefollowed by the transformation of plant cells such as tobacco
cells, Arabidopsis cells and Brassica napus cells with an
appropriate plasmid cont~in~ng the corresponding modified
precursor nucleic acid, the regeneration of the transformed
plant cells into correspon~ing plants, the culture thereof up
to the seed forming stage, the recovery of the seeds and
finally the analysis of the methionine content of said seeds
compared with the seeds of corresponding non transformed
plants.
CA 02000661 1998-06-30
It goes without saying that the invention is not limited
to the above examples. The person skilled in the art will in
each case be able to choose the desired combination of appro-
priate aminoacids to be inserted into the hypervariable re-
gion of the 2S storage protein, in function of the plant he
wants to improve with regard to its nutritional value and in
function of the desired application of the modified plant.
There follows a list of bibliographic references which
have been referred to in the course of the present disclosure
to the extent when reference has been made to known methods
for achieving some of the process steps referred to herein or
to general knowledge which has been established prior to the
performance of this invention. -
It is further confirmed
- that plasmid pAT2Sl has been deposited with the DSM on
4879 on October 7, 1988
- plasmids pMa5-8 has been deposited with the DSM on 4567
and pMc on 4566 on May 3, 1988
- plasmid pAT2SlBg has been deposited with the DSM on 4878
on October 7, 1988
- plasmid pGSC1703a has been deposited with the DSM on
4880 on October 7, 1988
nowithstanding the fact that they all consist of constructs
that the person skilled in the art can reproduce them from
available genetic material without performing any inventive
work.
3 ~ I MMAY 21 ~7
References :
Altenbach, S.B., Pear~on, K.W., Leung, F.W., Sun, S.S.M
(1987) Plant Mol. Biol. 8, 239-250.
Ampe C., Van Damme, J., de Castro, L.A.B., Sampaio,
M.J.A.M., Van Montagu, M. and Vandekerckhove, J. (1986) Eur.
J. Biochem. 159, 597-604.
Beachy, R.N., Chen, Z.-L., Horsch, R.B., Rogers, S.G., Hoff-
man, N.J. and Fraley, R.T. (1985) EMBO J. 4, 3047-3053.
Bergman, L.W. and Kuehl, W.N. (1979) J. Biol. Chem. 254,
5690-5694.
Blobel, (1980) Proc. Natl. Acad. Sci. 77, 1496-1500.
Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C.,
Heynecker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S.
(1977) Gene 2, 95.
Botterman, J. and Zabeau, M. (1987) DNA 6, 583-591.
Brown, J. (1976) Fed. Proc. Am. Soc. Exp. Biol. 35,
2141-2144.
Brown, J.W.S., Wandelt, Ch., Maier, U., Dietrich, G.,
Schwall, N., and Feix, G. (1986) EMBO workshop "Plant Stor-
age Protein Genes" ~LGy~am and Abstracts page 19, Eds. J.
Brown and G. Feix, University of Freiburg, 1986.
Chee, P.P., Klassy, R.C. and Slightom, J.L. (1986) Gene 41,
47-57.
Chr~peel~, N.J. (1983) Planta 158, 140-152.
Craig, S. and Goodchild, D.J. (1984) Protoplasma 122, 35-44
Crouch, M.L., Tembarge, K.M., Simon, A.E. and Ferl, R.
(1983) J. Mol. Appl. Gen. 2, 273-283.
De Blaere, R., Reynaerts, A., Hofte, H., Hernalsteens,
J.-P., Leemans, J. and Van Montagu, M. (1987) Methods in
Enzymology 153, 277-291
De Castro, L.A.B., Lacerada, Z., Aramayo, R.A., Sampaio,
M.J.A.M. and Gander, E.S. (1987) Mol. Gen. Genet. 206,
338-343.
~C ~C~~~
~1 21 ~997
32
Dellaporta S.L.; J.; Wood, J. and Hicks, B. (1983) Plant
Molecular Biology Reports 1, 19-21.
Ellis, J.R., Shirsat, A.H., Hepher, A., Yarwood, J.N., Gate-
house, J.A., Croy, R.R.D. and Boulter, D. (1988) Plant Molec-
ular Biology 10, 203-214.
Elkin, R.G., and Griffith, J.E. (1985a) J. Assoc. Off. Anal.
Chem. 68, 1028-1032.
Elkin, R.G., and Griffith, J.E. (1985b) J. Assoc. Off. Anal.
Chem. 68, 1117-1127.
Ericson, M.L., Rodin, J., Lenman, M., Glimeliums, K.,
Lars-Goran, J. and Rak, L. (1986) J. Biol. Chem. ~1, 14
576-14 581.
Greenwood, J.S. and Chrispeels, M.J. (1985) Plant Physiol.
79, 65-71.
Gehrke, C.W., Wall, L.L., Absheer, J.S., Kaiser, F.E. and
Zumwalt, R.W. (1985) J. Assoc. Off. Anal. Chem. 68, 811-821.
Herman, E.M., Shannon, L.M. and Chrispeels, M.J. (1986) In
Molecular Biology of Seed Storage Proteins and Lectins, L.M.
Shannon and M.J. Chrispeels Eds., American Society of Plant
Physiologists.
Higgins, T.J.V. (1984) Ann. Rev. Plant Physiol. 35, 191-221.
Higgins, T.J.V., Llewellyn, D., Newbigin, E. and Sr~ncer, D.
(1986) EMBO workshop "Plant Storage Protein Genes~ am
and abstract page 19, Eds. J. Brown and G. Feix, University
of Freiburg, 1986.
Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D.,
Rogers, S.G. and Fraley, R.T. (1985) Science 227, 1229-1231.
Hoffman, L.M., Donaldson, D.D., Bookland, R., R~shkA~ K.,
Heroan, E.M. (1987) EMBO J. 6, 3213-3221.
Hull and Howell (1987) : Virology, 86, 482-493
Jagodinski, L., Sargent, T., Yang, M., Glackin, C., Bonner,
J. (1987) Proc. Natl. Acad. Sci. USA. 78, 3521-3525.
Jones J.D.G.; Dunsmuir, P. and Bedbrook, J. (1985) EMBO J. 4
(10), 2411-2418.
oGC ~C~S~ c~
MAI 21 1997
3 3
Joseph, H. and Marsden, J. (1986) "HPLC of Small Molecules -
A practical approach" in: IRL Press Oxford - WA~hin~ton D.C.
"Amino Acids and Small Peptides~ Ed.: Kim , C.K., 13-27.
Josefsson, L-G.; Lenman, M., Ericson, M.L. and Rask,L.
(1987). J. Biol. Chem. 26~2 (25), 12196-12201.
Klimaszewska, K. and Keller, W.A. (1985) Plant Cell Tissue
Organ Culture, 4, 183-197.
Krebbers, E., Herdies, L., De Clercq, A., Seurinck, J.,
Leemans, J., Vandamme, J., Segura, M., Gheysen, G., Van
Montagu M. and Vandekerckhove, J. (1988) Plant Physiol.
87(4), 859-866.
Koncz, C. and Schell, J. (1986) Mol. Gen. Genet. 204,
383-396.
Larkins B.A. and Hurkman,W.J. (1978) Plant Physiol. 62,
256-263.
Lloyd, A.H., Barnason, A.R., Rogers, S.G., Byrne, M.C.,
Fraley, R.T. and Horsh, R.B. (1986) Science 234, 464-466.
Lord, J.M. (1985). Eur. J. Biochem. 146, 403-409.
Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecu-
lar Cloning. Cold Spring Harbor Laboratory, Cold Spring
Harbor,New York.
Marris, C., Gallois, P., Copley, J. and Kreis, N. (1988)
Plant Molecular Biology 10, 359-366.
Marton, L., Wullems, G.J., Molendijk, L. and Schilperoort,
R.A. (1979) Nature, ~, 129-131.
Morinaga, T., Sakai, N., Wegmann, T., Tanaoki, T. (1983)
Proc. Natl. Acad. Sci. 80. 4604-4606.
Odell, J.T., Nagy, J. and Chua, N.M.(1985) Nature 313,
810-812.
Okamuro, J.X., Jofuku, K.D. and Goldberg, R.B. (1986) Proc.
Natl. Acad. Sci. USA. 83, 8240-8244.
Perlman, D. and Halvorson, H.O. (1983) J. Mol. Biol. 167,
391-409.
~rr ~r~ C)~\~C
M ~ 1 21 i ~0~?~7
- 2 ~ ~ ~ fi 6 ~
Radke, S.E., Andrews, B.M., Moloney, M.M., Crouch, M.L.
Kridl, J.C. and Knauf, V.C. (1988) Theor. Appl. Genet. 75,
685-694.
Roden, L.T., Miflin, B.J., Freedman, R.B. (1982) FEBS Lett.
138, 121-124.
Scofield, S.R. and Crouch, M.L. (1987) J. Biol. Chem. 262
(25), 12202-12208.
Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall,
T.C. and Remp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82,
3320-3324.
Sharief, S.F. and Steven, S.-L. (1982) J. Biol. Chem.
257(24), 14753-14795.
Slightom, J.L. and Chee, P.P. (1987) Biotechn. Adv. ~,
29-45.
Stanssens, P., McKeown, Y., Friedrich, K., and Fritz, H.J.
(1987) Manual EMB0 Laboratory Course; 'Directed mutagensis
and protein engineering' held at Max Planck Institute f r
Biochemie, Martinsried, W-Germany, July 4-18, 1987.
Staswick, P.E. (1988) Plant Physiol. 87, 250-254.
Thomas, P.S. (1980) Proc. Natl. Acad. Sci. 77, 5201.
Velten, J., Velten, L., Hain, R. and Schell, J. (1984) EMB0
J. 3, 2723-2730
Walling, L.; Drews, G.N. and Goldberg, R. (1986) Proc. Natl.
Acad. Sci. 83, 2123-2127.
Yang, F., Luna, V.G., McAnelly, R.D., Noberhaus, K.H., Cup-
ples, R.L., Bowman, B.H. (1985) Nucl. Acids Res. a~,
8007-8017.
Youle, R. and Huang, A.H.C. (1981) American J. Bot. 68,
44-48.
OGC ~
2 ~ 6 ~
M A 1 2 1 ~957
2S Albumin As ~o Of Total Seed Protein
TABLE 1
Family, spccies %
(common name)
Compositae
Helianthus artnuus
(sunflower) 62
Cruciferae
Brassica spp.
(mustard) 62
Llnaceae
Linum ~sit~tissimu~n
(linseed) 42
Legumlnosae
Lupinus polyphyllus
(lupin) 38
Arachis hypogaea
(peanut) 20
Lecythidaceae
BcrthollcRa exçelra
(brazil nut) 30
Liliaceae
Yucca spp.
(yucca) 27
Euphorbiaceae
Ricinus communis <~
(castor bean) 44
(~
From Youle and Huang, 1981
