Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: PLANTS GENETICAI.LY ENHANCED FOR DISEASE
RESISTANCE
INVENTORS: Jesse M. Jaynes and Kenneth S. Derrick
FIELD OF THE INVENTION
The present invention relates to genetically enhanced
plants, and particularly to gymnosperms, monocots and
dicots genetically altered to express antifungal and
antibacterial peptides and/or to express polypeptides
which have high proportions of limiting essential amino
acids.
~ACKGROUND OF ~ INVENTION
Many attempts have been made to obtain improved
plants for cultivation through breeding programs. A
conventional plant breeding program requires as much as
ten years to develop a new variety. In addition to the ~-
initial hybridization step, several years are typically
spent replanting successive generations in order to obtain
homozygous plants. An alternative to a conventional plant
breeding program is anther culturing in which anthers from
one plant are used to pollinate the ovaries of another
plant. However, many traits cannot be successfully
introduced via such hybridization techniques since the
genes for such traits are not found in breeds available
for hybridization.
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An alternative to hybridization is somaclonal
variation. This technique involves the use of vegetative
plant parts, such as callus tissue, as explant material.
For example, callus that develops from vegetative explants
of rice frequently regenerates plants which have genetic
characteristics not found in the variety from which the
explant was originally obtained. These somatic mutants
occur at high frequencies, and the percentage of
regenerated plants which differ from the starting variety
exceeds, for example, 75 percent in rice. This technique
is therefore useful for producing genetic variability.
Again, however, there are limits to the extent of
variation which can be obtained.
Development of plant genetic engineering began in the
early 1940s when experiments were being carried out to
determine the biological principle causing formation of
crown gall tumors. The tumor-inducing principle was shown
to be a bacterial plasmid from the infective organism
Agrobacterium tumefaciens. This plasmid has since been
characterized in much detail utilizing the currently
available techniques of recombinant DNA technology. The
bacterium elicits its respon~e by inserting a small
fragment of bacterial plasmid into the plant nucleus where
it becomes incorporated and functions as a plant gene.
This discovery opened the door to using Agrobacterium and
its plasmids as vehicles to carry foreign DNA to the plant
nucleus. There are, however, limitations to the
application of this technique which include: (1)
susceptibility to infection with the Agrobacterium plasmid
and (2) available tissue culture technology for
regeneration of the transformed plants. Thus, there are
no successful reports on genetic engineering of monocots
such as cereals with Agrobacterium plasmid vectors because
of the general inability of Agrobacterium to infect
monocots.
More recently, other techniques have been used to
genetically transform monocots. For example,
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electroporation of protoplasts of rice, wheat and sorghum
to obtain expression of a foreign gene was reported in
Ou-Lee et al, Botany, vol. 8:3, pp. 6815-6819 (1986).
Similar plant protoplast electroporation and
electroinjection through cell walls and membranes have
also been reported for other monocots, and dicots as well.
See, Fromm et al, Proc. Natl. Acad Sci USA, vol. 82,
pp. 5824-5828 (1985); Hibi et al, J. Gen. Virol., vol. 67,
pp. 2037-2042 ~1986); Langridge et al, Plant Cell Reports,
vol. 4, pp. 355-359 (1985); Fromm et al, Nature, vol. 319,
pp 791-793; Shillito et al, Bio/Technology, vol. 3,
pp. 109~-1103 (1985~; and Okada et al, Plant Cell
Physiol., vol. 27, pp. 619-626 (1986). Similarly, direct
and chemical-induced introduction of DNA into monocot and
dicot cells has been disclosed. See, Lorz et al, Mol.
Gen. Genet., vol. 199, pp. 178-182 (1985); Potrykus et al,
Mol. Gen. Genet., vol. 199, pp 183-188 (1986); Uchimaya et
al, Mol. Gen. Genet., vol. 204, pp 204-207 (1986); Freeman
et al, Plant & Cell Physiol., vol. 25, no. 8,
pp. 1353-1365 (1984); and Krens et al, Nature, vol. 296,
pp. 72-74 (1986). Another technique of interest is the
injection of DNA into young floral tillers of rye plants
reported in de la Pena et al, Nature, vol. 325,
pp. 274-276 (1987).
The agricultural production of major crops has long
been significantly affected by insects and plant
pathogens. For example, blight and blast are major
diseases of rice plants which can decimate a crop. Some
plants cannot be cultivated in certain parts of the world
because of the presence of diseases in such locations to
which the plants are susceptible. For example, the main
diseases in potato are bacterial soft rot and bacterial
wilt caused by Erwinia carotovora and Pseudomonas
solanacearum, respectively. These diseases are primarily
responsible for limiting the growth of potatoes in many
areas of Asia, Africa, South and Central America.
Moreover, pesticides are becoming increasingly difficult
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to use in an effective, and yet environmentally acceptable
manner. Therefore, it would be desirable to have
available for cultivation plants which are resistant to
insects and other pathogens.
It is well known that the pupae of Hyalophora ~a type
of silk moth) respond to bacterial infection by the
synthesis of mRNAs which culminate in the production of
about 15 to 20 new proteins. Lysozyme, the antibacterial
protein found in egg white and human tears, and two other
classes of antibacterial peptides, called cecropins and
attacins, have been purified from hyalophora humor. These
proteins have a rather broad spectrum of activity in that
they are effective on many different types of bacteria.
Thus, the insects have evolved a rather successful and
novel means to fight bacterial infections. Although a
traditional immunologist would think this system lacks
specificity, the insect has a rather potent arsenal of at
least three different antibacterial proteins which may
work in different ways to destroy bacterial pathogens.
Thus, the invading bacteria is presented with a formidable
challenge which is very difficult to circumvent. While a
bacterial pathogen may be naturally resistant to one, it
is highly improbable that it would be resistant to all
three toxins. Although the exact mode of action of the
protein toxins is not fully understood, they are generally
procaryote specific and appear to be benign to eucaryotic
insect cells.
As mentioned above, the property of certain peptides
to induce lysis of procaryotic microorganisms such as
bacteria is well known. For example, U.S. patents
4,355,104 ancl 4,520,016 to Hultmark et al describe the
bacteriolytic properties of some cecropins against
Gram-negative bacteria. Quite interestingly, the
cecropins described in the Hultmark et al patents were not
universally effective against all Gram-negative bacteria.
For example, the cecropins described therein lysed
Serratia marcescens D61108, but not Serratia marcescens
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D611. Moreover, cecropins have generally been reported to
have no lytic activity towards eucaryotic cells such as
insect cells, liver cells and sheep erythrocytes, as
reported in the Hultmark patents; International Patent
Publication WO/8604356; Andreu et al, Biochemistry, vol.
24, pp. 1683-88 (1985)i Boman et al, Developmental and
Comparative Immunoloqy, vol. 9, pp. 551-5S8 (1985)i and
Steiner et al, Nature, vol. 292, pp. 246-248 (1981).
Other lytic peptides heretofore known include, for
example, the sarcotoxins and lepidopterans. Such peptides
generally occur naturally in the immune system of
Sarcophaga peregrina and the silkworm, lepidopteran,
respectively, as reported in Nakajima et al, The Journal
of Biological Chemistry, vol. 262, pp. 1665-1669 (1987)
and Nakai et al, Chem. Abst. 106:214351w (1987).
A number of the antibacterial polypeptides have been
found to be useful when the genes encoding therefor are
incorporated into various ani~als. Such transformation of
animals with genes encoding therefor are described
in International Publication WO 88/00976 published
on February 11, 1988 by Jesse M. Jaynes,
Frederick M. Enright and
Kenneth F. White.
Polynucleotide molecules expressible in E. coli and
having the sequence araB promoter operably linked to a
gene which is heterologous to such host are also known.
The heterologous gene codes for a biologically active
polypeptide. A genetic construct of a first genetic
sequence coding for cecropin operably linked to a second
genetic se~uence coding for a polypeptide which is capable
of suppressing the biological effect of the resulting
fusion protein towards an otherwise cecropin-sensitive
bacterium is also described in International Publication
W086/04356, July 31, 1986.
The Hultmark et al patents mentioned above also
mention that there are no known antibodies to cecropin,
indicating a wide acceptability for human and veterinary
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applications, including one apparently useful application
for surface infections because of the high activity
against pseudomonas. Similarly, ~PO publication 182,278
(1986) mentions that sarcotoxins may be expected to be
effective in pharmaceutical preparations and as foodstuff
additives, and that antibacterial activity of sarcotoxin
can be recognized in the presence of serum. Shiba, Chem.
Abstr. 104: 230430K (1985) also mentions preparation of an
injection containing 500 mg lepidopteran, 250 mg glucose
and injection water to 5 ml.
Several analogs of naturally-occurring cecropins,
sarcotoxins and lepidopterans have been reported. For
example, it is reported in Andreu et al, Proc. Natl. Acad.
Sci. USA, vol. 80, pp. 6475-6479 (1983) that changes in
either end of the amino acid sequence of cecropin
generally result in losses in bactericidal activity in
varying degrees against different bacteria. It is
reported in Andreu et al (1985) mentioned above that Trp2
is clearly important for bactericidal activity of
cecropin, and that other changes in the 4, 6 or 8 position
have different effects on different bacteria. From the
data given in Table II at page 1687 of Andreu et al
(1985), it appears that almost any change from natural
cecropin generally adversely affects its bactericidal .
activity. Cecropin is defined in International
Publication W086/04356 to include bactericidally active
polypeptides from any insect species and analogs,
homologs, mutants, isomers and derivatives thereof having
bactericidal activity from 1% of the naturally-occurring
polypeptides up to 100 times or higher activity of the
naturally-occurring cecropin. Other references generally
discuss the effects of the ~-helix conformation and the
amphiphilic nature of cecropin and other lytic peptides.
It is known that lysozyme and attacins also occur in
insect homolymph. For example, it is reported in Okada et
al, Biochem. J., vol. 229, pp. 453-458 (1985) that
lysozyme participates with sarcotoxin against bacteria,
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but that the bactericidal actions are diverse. Steiner et
al mentioned above suggests that lysozyme plays no role in
the antibacterial activity of insect hemolymph other than
to remove debris following lysis of bacteria by cecropin.
Merrifield et al, Biochemistry, vol. 21, pp. 50~0-5031
(1982~ and Andreu et al (1983) mentioned above state that
cecropin purified from insect hemolymph may be
contaminated with lysozyme, but demonstrate that the
synthetically prepared cecropin is as bactericidally
active as purified cecropin from insect hemolymph.
The treatment of eucaryotic pathogens and other cells
with lytic peptides, and novel lytic peptides, is the
subject matter of International Publication WO 89/00194
published on January 12, 1989 by Jaynes, Enright, Whi~e
and Jeffers.
Approximately 70% of the world's human population
lives in underdeveloped countries, and have diets
nutritionally inadequate in proteins, fats and calories.
Malnutrition in these countries is wide-spread and
persistent. Protein malnutrition can usually be
attributed to a deficiency in the diet of one or more of
the essential amino acids. The major food staples of many
underdeveloped countries, cereals and tubers, are
deficient in most limiting essential amino acids. When a
major portion of the diet consists of such staples, the
result is limiting essential amino acid deficiency. In
children, this condition is particularly debilitating,
because of the large requirement for high quality
polypeptide needed for normal growth and development.
Protein malnutrition of this type could be alleviated
or eliminated by adding supplements to the diet, which
supplements contained polypeptides high in these limiting
essential am:ino acids. Such polypeptides would have to be
susceptible to digestion by normal human or animal
proteases. Further, such polypeptides would be
manufactured by cloning and expression of synthetic DNA.
13211~7
Synthetic DNA of a desired sequence can now be
constructed using modern chemical techniques, and the DNA
can then be cloned into various microorganisms using
recombinant DNA technology. It is known, for example,
that a synthetic DNA which codes for
poly(1-aspartyl-1-phenylalanine) can be cloned and
expressed in E. coli. Doel et al, Nucleic Acids Research,
Vol. 8, No. 20, pp. 4575-4592 (1980). Also Tangus et al,
A~plied a Environmental Microbiology, Vol. 43, No. 3,
pp. 629-635 (March, 1982), have obtained expression of a
cloned homopolymeric synthetic DNA sequence coding for
poly-l-proline.
A number of United States patents have issued
concerning the synthesis of polypeptides by conventional
polypeptide sequencing, or by using DNA technology. Such
patents include 3,796,631 to Choay et al, 3,850,749 to
Kaufman et al, 3,299,043 to Schramm et al, 3,594,278 to
Naylor, and 3,300,469 to Bernardi et al. U.S. Patent
4,338,397, issued July 1982 to ~ilbert et al, discloses a
method for synthesizing within a bacterial host, and
secreting through the membrane of the host, a selected `
mature protein or polypeptide using these DNA techniques.
The polypeptide can be any selected polypeptide such as
proinsulin, serum albumin, and the like.
Summary of the Invention
The present invention involves the genetic
transformation of plants, including gymnosperms, dicots
and monocots, to express foreign genes to enhance one or
more characteristics of the plants, such as disease or
pest resistance, and/or nutritional quality.
In one aspect the invention provides a plant having a
heterologous gene encoding ~or an antimicrobial agent. The
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gene is preferably expressible, and the plant may be, for
example, either a gymnosperm~ a monocotyledon or a
dicotyledon. The antimicrobia:L agent may be, for example,
a lytic peptide such as attacin, lysozyme or cecropin, or
an antiviral agent such as mic~WA.
In another aspect, the invention provides a plant
having a heterologous gene encoding for a polypeptide or
protein comprising at least 25-60 weight percent of
limiting amino acids selected from lysine, tryptophan,
methionine, threonine, and isoleucine. The gene is
preferably expressible, and the plant may be a monocot or
a dicot. In one embodiment, the polypeptide is preferably
the product of genes constructed by random synthesis of a
mixture of codon pairs for the limiting essential amino
acids. In another preferred embodiment, the protein is
designed for stability and digestability, as well as a
high proportion of limiting essential amino acids.
In another aspect of the invention, there is provided
a plant having at least a first heterologous gene encoding
for an antimicrobial agent, and a second heterologous gene
encoding for a polypeptide or protein comprising at least
25-60 weight percent of limiting essential amino acids
selected from lysine, tryptophan, methionine, threonine,
and isoleucine. The plant may be a monocot or a dicot,
and the genes are preferably expressible.
Another aspect of the invention is the provision of
methods for genetically transforming plants with a
heterologous gene coding for (a) antimicrobial agents such
as, for example, cecropin and micRNA, or (b) polypeptides
comprising at least 25-~0 weight percent of limiting
essential amino acids selected from lysine, tryptophan,
methionine, threonine, and isoleucine. One such method
includes the steps of incubating plant protoplasts in the
presence of a vector containing the gene under conditions
effective to induce uptake of the gene by the protoplasts,
selecting and cloning the incubated protoplasts expressing
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132.~157
the gene, and regenerating a whole plant from the clones
to obtain a plant homozygous for expression of the gene.
Another such method includes the steps of
electroporating plant protoplasts in the presence of a
vector containing the gene, selecting and cloning the
electroporated protoplasts expressing the gene, and
regenerating a whole plant from the clones to obtain a
plant homozygous for expression of the gene.
A ~urther such method includes the steps of
introducing a vector containing the gene into a plant
during development of its reproductive structures,
harvesting seeds produced by the plant, generating plants
from the seeds, and selecting and reproducing the
seed-generated plants to obtain a plant homozygous for
expression of the gene.
In yet another aspect of the invention, there is
provided a novel lytic peptide having the amino acid
sequence a~ or ~ wherein ~ represents the amino acid
se~uence LXXLLXLLXXLLXL, ~ represents the amino acid
sequence LLLLLLLLLLLSLS or the mirror image thereof so
that the "SLS" end thereof is near a terminus of the
peptide, and X represents K or R.
According to a further aspect of the invention there
is provided a process for modifying a plant to confer
disease resistance to that plant, said process comprising
inserting into the genome of the plant an expressible
heterologous gene encoding a lytic peptide, wherein said
lytic peptide comprises from about 30 to about ~0 amino
acids, at least some of which are arranged in an
amphiphilic alpha helical confirmation having a
substantially hydrophilic head with a positive charge
density and a substantially hydrophobic tail.
According to yet a further aspect of the invention
there is provided a process for modifying a plant to
confer disease resistance, said process comprising into
the genome of the plant an expressible heterologous gene
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encoding a lytic peptide selected fro~ the group consist-
ing of: cecropins, attacins, and synthetic amphiphilic
homologs thereof.
According to yet a further aspect of the invention
there is provided a process for modifying a gymnosperm,
monocot, or dicot to confer disease resistance, said
process comprising inserting into the genome o~ the
gymnosperm, monocot, or dicot a heterologous gene which
includes a promoter operably linked to a sequence coding
for a lytic peptide, wherein said lytic peptide is
selected from the group consisting of cecropins, attacins,
and amphiphilic homologs thereof.
Description of the Invention
1. Introduction
The plant of the present invention has a heterologous
gene coding for an antimicrobial polypeptide and/or a
polypeptide relatively high in limiting essential amino
acids. As used herein, the term "plant" generally
includes trichophyta, and particularly pteropsida such as
gymnosperms, monocots and dicots. Representative examples
of monocots include rice, whe~t, barley, rye, corn, and
other grasses, ~ananas, palms, lilies, orchids, sedges,
and the like. Representative dicots include potatoes,
carrots, sweet potatoes, willows, oaks, elm, maples,
apples, roses, buttercups, petunias, phlox, violets,
sunflowers, and the like. Generally, the antimicrobial
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gene is desirable in ornamental plant species as well as
species cultivated for food, fiber, wood products, tanning
materials, dyes, pi~ments, gums, resins, latex products,
fats, oils, drugs, beverages and the like. On -the other
hand, the gene for enhanced nutritional quality is
desirably expressed in plants cultivated for food, such
as, for example, grains, legumes, nuts, vegetables,
fruits, spices and the like, but there is generally no
advantage to having this gene expressed in non-food
plants.
The plants should express the heterologous gene, and
are preferably homozygous for expression thereof.
Generally, the gene will be operably linked to a promoter
inducible i~ the cells of the particular plant. The
expression should be at a level such that the
characteristic desired from the gene is obtained. For
example, the expression of the gene for antimicrobial
resistance should confer some measurable enhancement of
pathogen resistance to the plant relative to the same
species without the gene. Similarly, the expression of
the gene for enhanced nutritional guality should result in
a plant having a relatively higher content of one or more
limiting essential amino acids compared to that of the
same species without the gene. On the other hand, it will
generally be desired to limit the excessive expression of
the gene in order to avoid significantly adversely
affecting the normal physiology of the plant, i.e. to the
extent that cultivation thereof becomes difficult.
Promotors such as CaMV l9s, CaMV 35s and the like are
contemplated as being suitable in most plants.
2. Antimicrobial Agents
The antimicrobial genes in the plants of the present
invention generally encode for antibacterial and/or
antifungal polypeptides, and/or antiviral agents such as
micRNA, not normally found in the particular plant
species. Suitable antimicrobial polypeptides are, for
example, derived from insect hemolymph, such as attacin.
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A preferred class of antimicrobial polypeptides include
the lytic peptides. Exemplary lytic peptides include
lysozymes, cecropins, attacins, melittins, magainins,
bombinins, xenopsins, caeruleins, the polypeptide from
gene 13 of phage P22, S protein from lambda phage, E
protein from phage PhiX174, and the like. However, lytic
peptides such as the melittins, bombinins, and magainins
are generally relatively high in lytic activity, and are
therefore less preferred since host plant cells may be
adversely affected thereby.
As used herein, the term "lytic peptide" includes any
polypeptide which lyses the membrane of a cell in an ln
vivo or ln vitro system in which such activity can be
measured. Suitable lytic peptides used in the present
invention have lytic activity toward one or more plant
pathogens such as fungi and bacteria. Exemplary fungal
pathogens include species of Helminthosporium (e.g. late
blight), Trichophyton, Colletotricium, Ceratocystis (e.g.
Dutch elm disease), Fusarium, Phytophthorax, Rhigoctoria
and the like. Representative examples of bacterial
pathogens include species of Pseudomonas, Erwinia (e.g.
fire blight), Xanthomonas, Clavibacter and the like.
Preferred lytic peptides have from about 30 to about
40 amino acids, at least a portion of which are arranged
in an amphiphilic ~-helical conformation having a
substantially hydrophilic head with a positive charge
density, a substantially hydrophobic tail, and a pair of
opposed faces along the length of the helical
conformation, one such face being predominantly
hydrophilic and the other being predominantly hydrophobic.
The head of this conformation may be taken as either the
amine terminus end or the carboxy terminus end, but is
preferably the amine terminus end.
Suitable lytic peptides generally include cecropins
such as cecropin A, cecropin B, cecropin D, and
lepidopteran; sarcotoxins such as sarcotoxin IA,
sarcotoxin IB, and sarcotoxin IC; and other polypeptides
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such as attacin and lysozyme obtainable from the hemolymph
of any insect species which have lytic activity against
bacteria and fungi similar to that of the cecropins and
sarcotoxins. It is also contemplated that lytic peptides
may be obtained as the lytically active portion of larger
peptides such as certain phage proteins such as S protein
of A phage, E protein of Phix174 phage and P13 protein of
P22 phage; and C9 protein of human complement. As used
herein, classes of lytically active peptides such as, for
example, "cecropins," "attacins" and "phage proteins," and
specific peptides within such classes, are meant to
include the lytically active analogues, homologues,
fragments, precursors, mutants or isomers thereof unless
otherwise indicated by context. Of these exemplary lytic
peptides, those having fewer than about 30 amino acids
such as the melittins are generally less preferr~d in the
present invention because of their lack of specificity for
bacteria and fungi as indicated by their hemolytic
potential, whereas those with more than about 40 amino
acids such as attacins and lysozymes may not be
sufficiently lytic when used alone to provide a broad
spectrum of microbial resistance. On the other hand, those
having between about 30 and about 40 amino acids, such as
cecropins and sarcotoxins are generally more preferred
because of their specificity for lysing pathogens over
host cells.
Hydrophilic amino acids generally include and
generally have the respective relative degree of
hydrophobicity (at pH 7.0; kcal/mol) as follows: aspartic
acid (D), -7.4; glutamic acid (E) -9.9; asparagine (N),
-0.2; glutamine (Q), -0.3; lysine (K), -4.2; arginine (R),
-11.2; serine (S), -0.3; and cysteine (C), -2.8.
Hydrophobic amino acids generally include and generally
have the respective relative degree of hydrophobicity as
follows: histidine (H), 0.5; threonine (T), 0.4; tyrosine
(Y), 2.3; tryptophan (W), 3.4; phenylalanine (F), 2.5;
leucine (L), 1.8; isoleucine (I), 2.5; methionine (M),
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1.3; valine (V), 1.5; and alanine (A), 0.5. Glycine has a
relative degree of hydrophobicity of 0 and may be
considered to be hydrophilic or hydrophobic.
The amino acid homology of peptides can be readily
determined by contrasting the amino acid sequences thereof
as is known in the art. Similarly, the amphiphilic
homology of peptides can be determined by contrasting the
hydrophilicity and hydrophobicity of the amino acid
sequences. The amino acid sequences of some preferred
lytic peptides are compared to cecropin B by tabulation
and construction of Edmundson helical wheels in the
aforementioned International Publication wo 89/001~4.
Cecropin B is a potent bacteriolytic peptide which
occurs naturally and can be obtained from insects as
described in the Hultmark et al patents mentioned above,
by direct peptide synthesis, or from genetically
transformed host cells as described in the aforementioned
Publication W0/086/04356. As determined by construction
of a helical wheel, ~ourteen of the sixteen amino acids on
the hydrophilic ~ace of cecropin B are hydrophilic, while
eleven of the twenty amino acids on the hydrophilic face
are hydrophilic, for a total of eleven "imperfections".
It is contemplated that the removal or replacement of
Gly2 3 and pro2 4 would result in a more lytic peptide with
only six imperfections in the amphiphilic helical
conformation. In addition, proline is known to disrupt
the helical conformation, and its removal may permit a
more helical conformation and, hence, more lytic activity.
Note that the helical wheel constructs for cecropin SB-37,
cecropin D and Shiva 1 are typically constructed assuming
that the end region prolines would disrupt the ~-helical
conformation there and this can be indicated by placing
proline and the preceding amino acids outside the
corresponding wheel construct.
Cecropin SB-37 is an analogue which was prepared
using a peptide synthesizer and is about as lytically
active as cecropin B. It has 94% homology therewith in
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its amino acid sequence and its amphiphilicity. As seen
from a helical wheel construct thereof, two of the
seventeen amino acids are hydrophilic on the hydrophobic
face, while eight of twenty are hydrophobic on the
hydrophilic face. It is similarly contemplated that if
Gly2 3 and pro2 4 were removed or replaced, it would have
only five imperfections in the amphiphilic conformation,
and thus be more lytically active.
Similarly, the other naturally occurring lytic
peptides cecropin A, cecropin D, lepidopteran, sarcotoxin
lA, sarcotoxin lB and sarcotoxin lC approximate the
amphiphilic helical conformation of cecropin B to varying
degrees. With the exception of cecropin A which is about
as lytically active as cecropin B and SB-37, these
lS peptides are generally less lytically active against
bacteria than cecropin B. However, it is likewise
contemplated that the lytic activity thereof may be
improved by removing amino acids from the sequence
thereof, for example, Val19 and Ile20 from cecropin D or
20 Gly2 3 and pro2 4 from lepidopteran or cecropin A.
Another peptide designated herein as "Shiva 1" was
prepared using a peptide synthesizer and has the amino
acid seguence MPRWRLFRRIDRVGKQIKQGILRAGPAIALVGDARAVG.
While this peptide has only a 46% amino acid homology with
cecropin B, its amphiphilic homology therewith is 100%.
Quite surprisingly, however, Shiva 1 is generally much
more lytically active than cecropin B, and it is
contemplated that its lytic activity may be further
enhanced by removal or replacement of Gly23 and Pro24.
A cecropin SB-37 homologue designated herein as
"*cecropin SB-37" is identical thereto except for the
substitution of glutamic acid in the fourth position (for
threonine; corresponding to the second position of
cecropin B) and lysine in the eighth position (for
leucine; corresponding to the sixth position in cecropin
B). Substitul:ions in these positions may reduce the lytic
activity of the cecropin SB-37 against procaryotes by as
:. , -, ~ ; . - ................. . :
,
- ~ . , ;. ~ .
.: : .. , , .- . . : ,
-16~ 7
much as 90% as reported in Andreu et al (1985) mentioned
above. Quite surprisingly, however, it has been found
that the lytic activity of *cecropin SB-37 against many
pathogens is generally comparable to cecropin SB-37.
Another preferred class of lytic peptides
contemplated as being suitable for expresison in the
plants of the present invent:ion include polypeptides
having the sequence ~ , and ~, wherein ~ has the amino
acid seguence LXXLLXLLXXLLXL, ~ has the amino acid
sequence LLLLLLLLLLLSLS or the mirror image thereof so
that the l'SLS" end thereof is near or at a terminus of the
peptide, preferably the amino terminus, and each X
independently represents K or R. The following
polypeptides are designated herein as indicated:
DES I GNATI ON SEQUENCE X
Shiva 2 ~ K
Shiva 3 ~ K
Shiva 4 ~ R
Shiva 5 ~ K
Shiva 6 ~ R
Shiva 7 ~ R
Of these, Shiva 3, Shiva 4 and Shiva 7 may be too
lytically active to be used in plants at high expression
levels, and might need to be employed at relatively low
expression levels. However, all of these Shiva
polypeptides are contemplated as having general utility as
lytic peptides separate and apart from expression in the
plants of this invention, e.g. in inducing lysis of cells
in vitro.
The amino acid seguences of the foregoing exemplary
lytic peptides as well as other ly-tic peptides
contemplated for use in the plant transformants in the
present invention are listed in Table I.
~ , - . . ~ ~ .-
:- . : .. : . .
- ~
.. . ..
.. . .. .. ... .
:~
.. , . . ~
, . . ~ . .
-17- ~32~
TABLE I
Peptide Amino Acid Sequence
CECROPIN A KWKLFKKIEKVGQNI~)GIIKAGPAVAWGQATQIAK
CECROPIN B KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKALG
CECROPIN D WNPFKELEKVGQRVRDAVISAGPAVATVANATALAK
LEPIDOPTERAN RWKIFKKIEKMGRNIRDGIVKAGPAIEVLGSAKALG
SARCOTOXIN IA GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR
SARCOTOXIN IB GWLKKIGKKIERVGQHTRDATIQVIGVAQQAANVAATAR
SARCOTOXIN IC GWLRKIGKKIERVGQHTRDATIQVLGIAQQAANVAATAR
SB-37 MP KWKVFKKIEKVGRNIRNGIVKAGPAIAVLGEAKALG
MOD.SB-37 MP KEKVFLKIEKMGRNIRNGIVKAGPAIAVLGEAKALG
SHIVA-l MP R~RLERRIDRVGKQIKQGILRAGPAIAVLGDARAVG
MOD.SHIVA- MP RERLFLRIDRVGKQIKQGILRAGPAIALVGDARAVG
SHIVA-2 LKKLLKLLKKILKLLLLLLLLLLLLSLS
SHIVA-3 SLSLLLLLLLLLLLLKKLLKLLKKLLKL
SHIVA-4 LRRLLRLLRRLLRLLRRLLRLLRRLLRL
SHIVA-5 LKKLLKLLKKLLKLLKKLLKLLKKLLKL
SHIVA-6 LRRLLRLLRRLLRLLLLLLLLLLLLSLS
SHIVA-7 SLSLLLLLLLLLLLLRRLLRLLRRLLRL
PIELITTIN GIGAVLKVLTTGLPALISWIKRKRQQ
MI ~ELITTIN QQRKRKIWSILAPLGTTLVKLVAGIG
ART. MELITTIN LLQSLLSLLQSLLSLLLQWLKRKRQQ
MAGAININ 1 (PGS) GIGKFLHSAGKFGKAFVGEIMKS
PGS GIGKFL}ISAKKFG~AFV OEIMNS
25 MAGAININ 2 ~PGS) GIGKFLHSAKKFGKAFV OEIMNS
BOMBININ GIGALSAKGALKGLAKGLAZHFAD
XENOPSIN PF GWASKIGQTLGKIAKVGLKELIGPK
CAERU~EIN PF GFGSFLGKALKAALKIGANALGGSPQQ
PGL GMASKAGAISGKIAKVALKAL
DEGRADO ACT. LKKLLKLLKKLLKILKKILKLLKKLLKL
C9 INC. KMKNA~LKKQNLERAIEDYINEFSVRR
AS PROTEIN INC. kDKMPEKHDLLAAILAAXEQGIGAILAFAMAYLRGR
P22 13 PRO. INC. MKKMPEKHDLLTAM~ EQGIGAILAFAMAYLRGR
~X174 INC. ~rVRWTLWDTLAFLLLSLLLPSLLIMFIPSFKRPVS
The DNA sequences coding for the foregoing peptides
can be readily determined and synthesized according to
established principles and techniques in the art.
Also included within the scope of the tenm
"antimicrobial agents" as used herein are antiviral
agents, inclusive of micRNA or antisense RNA for viruses
and viroids replicatable in plants. Exemplary viruses
infectious in plants include tobacco mosaic virus, tobacco
ringspot virus, potato leaf-roll virus, potato viruses X
and Y and the like. Representative examples of viroids
replicatable in plants include potato spindle tuber
viroid, cadang-cadang viroid, avocado sun blotch viroid,
and the like. Antisense RNA, or micRNA as it is sometimes
called, generally inhibits or blocks translation of
- ~ ,- . - .:
: . .. .
- ~ ~ "~
-18- ~321~7
viruses and viroids so that replication thereof in
infected plant cells is inhibited or prevented. Such
micRNA is preferably complementary to the micRNA (or DNA
in the case of RNA viruses) of the virus replication gene,
and such complementarity and concomitant hybridization
prevents translation or transcription thereof. In the
case of viroids, a single mic~NA appears to be sufficient
for protection against all known viroids because of a
common DNA sequence in their central conserved regions.
For prophylaxis against viruses, on the other hand,
several micRNA's may be required to block a wide spectrum
of viruses.
An exemplary genetic sequence for a micRNA
complementary to the tobacco mosaic virus is as follows:
5' GATCTCCACGGTTGTGGCCATATAATCATCGTGTTTTTCAA 3'
Exemplary contemplated genetic sequences for micRNA's
complementary to the African cassava mosaic virus are as
follows:
ACMVl: 5' AGGGGCCAACCGTA 3'
ACMC2: 5' GCCCCGGTTGGCAT 3'.
Viroid antisense RNA's are coded by the following
exemplary DNA sequences:
PSTVl: 5' GATCTAGGGATCCCCGGGGAAACCT 3'
PSTV2: 5' GATCTAGGTTTCCCCGGGGATCCCT 3'.
3. Polypeptides High in Limiting Essential Amino Acids
A predominant limiting factor in animal and human
nutrition is protein quality. Certain proteins, such as
egg protein, are considered to be of very high quality
because they contain disproportionately high levels of
limiting essential amino acids. Limiting essential amino
acids are those which are found in very restricted
quantities in most proteins, and which, when ingested by
an organism, limit the amount of protein which that
organism can produce. The five most limiting essential
amino acids are lysine, methionine, tryptophan, threonine
and isoleucine. Of the total protein in egg, 22.8%
consists of these limiting amino acids. The average
cereal grain protein (ric~, corn, wheat, barley) is
~: : ;. ,
, : , , .: .
., , . : .; : , . :
. : : - :: . -~ :
-19- ~ 3~
composed of about 13% o~ these five most limiting
essential amino acids. The following Tables list these
five limiting amino acids in the cereal grains and in
roots and tubers.
TABLE II
AMINO ACID RANK FOR A CHILD'S DIET OF A
CALORIC AMOUNT OF CEREAL GRAIN
Cereal Amino Acid
Isoleucine Lysine Methionine Threonine Tryptophan
10 Barley 1 1 2
Corn 1 3 2
Rice 1 1 4 3 2
Wheat 2 1 3
TABLE III
AMINO ACID RANK FOR A CHILD'S DIET OF A
CALORI~ AMOUNT OF ROOT OR TUBER
Amino Acid
Isoleucine Lysine Methionine Threonine Tryptophan
. . _
Cassava 3 4 l 2 2
20 Potato 4 5 2 3
Sweet
Potato 3 3 2 3
It will be seen that generally lysine is the first
limiting amino acid in a cereal and tryptophan is the
first limiting amino acid in a root or tuber diet for a
human child. However, even if the lysine or tyrptophan
requirement is met, then the other amino acids become
limiting depending upon which of these foods is being
consumed. In the case of legumes, the first limiting
amino acid would be methionine.
The present embodiment provides an ideal polypeptide
for use in supplementing the diets of animals and humans
to meet their daily limiting essential amino acid
requirement. The polypeptide of the present embodiment is
particularly suitable for supplementing diets which
consist largely of cereals, roots, tubers and legumes.
Thus, the present invention provides a polypeptide which
has elevated levels as compared to proteins known in the
art, of lysine, methionine, tryptophan, threonine and
,
- ~ ;
,
., " , : : , . -
, , , .~ ~.................. ..
~ , ; . . .
-20- ~ 321~7
isoleucine. In this broadest embodiment, the polypeptides
of this invention will contain from 25-60 weight percent
of these limiting essential am:ino acids. The polypeptide
preferably contains at least two of these limiting
essential amino acids, more preferably at least three, and
especially four or all five.
For purposes of this embodiment of the invention, a
protein will be defined as an amino acid chain containing
more than 50 amino acids. A polypeptide will be an amino
acid chain having less than 50 amino acids.
According to this embodiment, the cloning and
expression of synthetic DNA containing repeated selected
codons for limiting essential amino acids are used as a
means for improving the amino acid profiles of single cell
proteins. Thus, using these techniques, single cell
production of polypeptides composed largely of limiting
essential amino acids are used to supplement cereal, root,
tuber and/or legume diets and feed grain rations and other
foodstuffs. Such supplemented foodstuffs would include
any suitable foodstuff, particularly legumes, roots,
tubers and cereal grains. Thus, in this embodiment is
provided the synthesis, cloning and expression of genes
which code for polypeptides specifically designed for
their digestibility by animal proteases. These genes code
for polypeptides high in limiting essential amino acids,
particularly for the five most limiting amino acids of the
root, tuber and cereal diet: lysine, methionine,
tryptophan, threonine and isoleucine.
To determine the efficacy of some of the genes of
this embodiment, they were tested with positive results,
for their functional activity in a microbe such as E.
coli. The test method used generally involves isolating a
plasmid from a microbe such as E. coli. The plasmid may
be isolated by any desired procedure, but preferably a
cleared lysate procedure is employed. Next, the plasmid is
digested with restriction enzyme such as, for example,
EcoRl to yield a linear molecule with an interrupted
. :
;: .,
. . .~ . :
-21- ~32~
chloramphenicol resistance gene at the 72nd amino acid of
the chloramphenicol-acetyl transferase (CAT) protein. In
this manner, when a DNA segment is cloned into the site on
the promoter, the plasmid no longer produces an active CAT
protein and any recombinant clone is rendered
chloramphenicol sensitive. These genes, when fused to an
active bacterial plasmid prolmoter region, such as
chloramphenicol-acetyl transferase (CAT), incorporated 14C
lysine at a rate of two to seven times that of a control
during the polypeptide production cycle of the bacteria.
Thus, in the present embodiment, a series of genes that
encode for high quality polypeptides are designed and
synthesized and the genes are expressed in a microbe.
Synthesis of the genes may be by random ligation of
mixtures of small oligodeoxynucleotides. Thus, the
products of ligation are heterogenous with respect to
molecular weight. However, the addition of a dodecomeric
linker, which contains the enzyme recognition sequence for
the restriction endonuclease such as EcoR1 is at a
concentration which should yield, upon digestion with
EcoR1, fragments of a mean size of about 400 base pairs
(bp). Suitable recombinant E. coli. strains are then
selected for further characterization and designated. In
the process of this invention, the designations given were
SP44 and SP47. These designated strains contain
nucleotide fragments cleaved by EcoR1 of approximately 100
bp and 300 bp, respectively.
Polypeptide synthesis is determined by the relative
uptake of labeled amino acids by the recombinant E. coli.
strains as compared to controls. The polypeptide
sequences produced can be derived from the known
nucleotide sequences of the DNA.
In the following Table IV, the c~mino acid profiles
obtained in the synthetic polypeptides just described are
compared with those in lactalbumin and zein.
,
: - . . ~ .
-
.
- ,
-22- ~.32~ 7
TABLE IV
Sp44(1)Sp44(2) Sp47~L) Sp47(2)Lact6alb2umin 4ein
Lys 13.3 20.0 23.0 16.7 9.0 0
Met 6.6 9.9 11.5 8.3 2.3 0
Thr 13.3 0 6.3 3.1 5.2 2.4
Trp 6.6 9.9 11.5 8.3 2.2 0
Total53.1 39.~ 58.6 3~.5 24.9 6.6
Lactalbumin is a high quality milk protein, and
zein is a poor quality major protein in maize. It will be
seen from Table IV that proper supplementation of zein
with SP44(1) or (2) or SP47(1) or (2), would markedly
improve the balance of the five most limiting amino acids.
The method of gene synthesis, according to this
embodiment, is flexible enough to produce amino acid
profiles which would be specifically designed to
supplement the material to be enhanced, in this case,
maize. It should be pointed out that the insertion of
lysine (or arginine) at frequent intervals in these
polypeptides, preferably on the average of every fifth to
tenth amino acid, will provide numerous sites for
proteolytic attack by trypsin.
It will be seen, therefore, that the present
embodiment provides polypeptides which contain appropriate
amino acids to provide a high q~lality product and also to
be in a form which will be used to supplement foodstuffs
having low quality proteins. In the broadest embodiment,
the polypeptides may be described as being composed
largely of the amino acids lysine, methionine, tryptophan,
threonine, and isoleucine, which amino acids constitute
25-60 weight percent of the polypeptide.
In a further embodiment, a protein of the present
invention has the following amino acid seguence:
GDRKK~MDRHPFLHPFLTIPFLKKW~KKWM
TIHPFLHPFLHPFLTIKKWMKKWMKKWMHPF
LKKWMKKWMKKWMTIDRKKWMTIHPFLTIP
In a still further embodiment of the invention, a
smaller polypeptide which has somewhat lower quality than
the foregoing large protein has the following amino acid
sequence:
.
~ ,, "
.
-23- 132~
GTITIHPFLKKWMTIHPFLKKWMTIHPFLP
The importance of the use of the genes of the
present invention may be undexstood from the following
Table V, which sets forth the percentage of limiting
essential amino acids in a number of food proteins, and
compares those percentages with the supplementary
polypeptide of this invention.
TABLE V
Isoleucine Lysine Methionine Threonine Tryptophan Total
Casein 6.55 8.01 3.08 4.23 1.34 23.21
Lactal-
bumin 6.21 9.06 2.25 5.24 2.20 24.96
Whole Egg 6.64 6.40 3.13 4.98 1.65 22.80
Beef 5.23 8.73 2.48 4.41 1.17 22.03
Soybean 5.89 6.92 1.47 4.31 1.51 20.09
Barley 4.26 3.38 1.44 3.38 1.25 13.71
Corn 4.62 2.88 1.86 3.98 .61 13.95
Rice 4.40 3.20 1.80 3.50 .10 13.00
Wheat 4.34 2.74 1.53 2.88 1.24 12.72
Synthetic*
Protein 8.10 21.60 16.20 10.80 10.80 67.50
These numbers represent the calculated theoretical
average based on the experimental protocol used.
It may be seen from the above table, that the total
of these limiting essential amino acids in the synthetic
protein is 67.5 as compared to the other food proteins
which range from 12.72 to 24.96. This is a remarkable
increase in the amount of these limiting essential amino
acids in a polypeptide since the foodstuffs with which it
is compared are generally of high nutritional value.
A particularly preferred embodiment is a protein
which is relatively more stabl~ due to alternating charges
in the amino acid side chains which are adjacent to each
other in ~-helical conformations of the protein, and which
contains few, and preferably no ~-helix disrupters, such
as proline. Such proteins are contemplated as having a
greater utility since they would be more stable, i.e.,
less subject to proteolytic attack by the plant enzymes,
and expressible in a wider variety of plants. An
exemplary protein with such characteristics, and also with
. ~ . .., . , ~ . .
.... . .
-: ~: , ,- .
, ~
-24- ~ 7
approximately 20 percent of its amino acids being randomly
inserted lysine, has the amino acid sequence as follows:
MFKWMKEIWKVLKDMIDKMKTFIDTLFEM
ITKLFTEVEKWMKEIWKVLKDMID~KTFVDTLFEM
LEMITKWFTEVEKWMKEIWKVLKDMIDKM
The DNA seguences coding for the foregoing peptides
can also be readily determinedl and synthesized according
to established principles and t~chniques in the art, e.g.
synthesis of the oligonucleotide, or portions thereof
followed by ligation.
4. Transformation of Plants
The plants of the present invention may be obtained
by any of several methods. Such methods generally include
direct gene transfer, chemically-induced gene transfer,
electroporation, microinjection, Agrobacterium-mediated
gene transfer, and the like. Some methods, such as
Agrobacterium-mediated gene transfer, are generally only
suitable for certain types of plants, e.g. dicots, while
the other methods can generally be used to transform
monocots and gymnosperms as well.
One method for obtaining the present plants is direct
gene transfer in which plant cells are cultured or
otherwise grown in the presence of DNA oligonucleotides
containing the gene desired to be introduced into the
plant. The donor DNA source is typically a plasmid or
other suitable vector containing the desired gene. For
convenience, reference is made below to plasmids, with the
understanding that other suitable vectors containing the
desired gene are also contemplated.
Any suitable plant tissue which takes up the plasmid
may be treated by direct gene transfer. Such plant tissue
includes, for example, reproductive structures at an early
stage of development, particularly prior to meiosis, and
especially 1-2 weeks pre-meiosis. ~enerally, the
pre-meiotic reproductive organs are bathed in plasmid
solution, such as, for example, by injecting plasmid
.
~ ~ .
-25- - ~ 3~
solution directly into the plant at or near the
reproductive oryans. The plants are then sel~-pollinated,
or cross-pollinated with pollen from another plant treated
in the same manner. The plasmid solution typically
contains about 10-50 ~g DNA in about 0.1-10 ml per floral
structure, but more or less than this may be used
depending on the size of the particular floral structure.
The solvent is typically sterile water, saline, or
buffered saline, or a conventional plant medium. If
desired, the plasmid solution may also contain agents to
chemically induce or enhance plasmid uptake, such as, for
example, PEG, Ca2 or the like.
Following exposure of the reproductive organs to the
plasmid, the floral structure is grown -to maturity and the
seeds are harvested. Depending on the plasmid marker,
selection of the transformed plants with the marker gene
is made by germination or growth of the plants in a
marker-sensitive, or preferably a marker-resistant medium.
For example, seeds obtained from plants treated with
2~ plasmids having the kanamycin resistance gene can be
identified to have taken up the plasmid by germinating the
seeds in kanamycin. Plants expressing the kanamycin
resistance gene will remain green, whereas those without
this marker gene are albino. Presence of the desired gene
transcription of mRNA therefrom and expression of the
peptide can further be demonstrated by conventional
Southern, northern, and western blotting technic~es.
In another method, plant protoplasts are treated to
induce uptake of the plasmid. Protoplast preparation is
well-known in the art and typically involves digestion of
plant cells with cellulase and other enzymes for a
sufficient period of time to remove the cell wall.
Typically, the protoplasts are separated from the
digestion misture by seiving and washing. The protoplasts
are then suspended in an appropriate medium, such as, for
example, medium F, CC medium, etc., typically at 104 - 107
cells/ml. To this suspension is then added the plasmid
, . . ~
-26- 13211~7
solution described above and an inducer such as
polyethylene glycol, Ca2 , Sendai virus or the like.
Alternatively, the plasmids may be encapsulated in
liposomes. The solution of plasmids and protoplasts are
then incubated for a suitable period of time, typically
about 1 hour at about 25~C. In some instances, it may be
desirable to heat shock the mixture by briefly heating to
about 45~C, e.g. for 2-5 minutes, and rapidly cooling to
the incubation temperature. The treated protoplasts are
then cloned and selected for expression of the desired
gene, e.g. by expression of the marker gene and conventional
blotting techniques. Whole plants are then regenerated
from the clones in a conventional manner.
The electroporation techni~ue is similar except that
electrical current is typically applied to the mixture of
naked plasmids and protoplasts, in an electroporation
chamber in the absence or presence of polyethylene glycol,
Ca2+ or the like. Typical electroporation includes 1-10
pulses of 40-10,000 DC volts for a duration of 1-2000 ~s
with typically 0.2 second intervals between pulses.
Alternating current pulses of similar severity can also be
used. More typically, a charged capacitor is discharged
across the electroporation chamber containing the plasmid
protoplast suspension.
Another method suitable for transforming dicots
involves the use of Agrobacterium. In this method,
Agrobacterium containing the plasmid with the desired gene
is used to infect plant cells and insert the plasmid into
the genome of the cells. The cells expressing the desired
gene are then selected and cloned as described above. For
example, one method for introduction of a foreign gene
into a dicot, e.g., a tuber, root or legume, by means of a
plasmid, e.g. an Ri plasmid and an Agrogacterium, e.g. A.
rhizogenes or A. tumefaciens, is to utilize a small
recombinant plasmid suitable for cloning in Escherichia
coli, into which a fragment of T-DNA has been spliced.
This recombinant plasmid is cleaved open at a site within
::
,
.
~: -
~ . . .
-27- ~2~ ~7
the T-DNA. A piece of "passenger" DNA is spliced into
this opening. The passenger DNA consists of the gene of
this invention which is to be incorporated into the plant
DNA as well as a selectable marker, e.g., a gene for
resistance to an antibiotic. This plasmid is then
recloned into a larger plasmid and then introduced into an
Agrobacterium strain carrying an unmodified Ri plasmid.
During growth of the bacteria, a rare double-recombination
will sometimes take place resulting in bacteria whose
T-DNA harbors an insert: the passenger DNA. Such bacteria
are identified and selected by their survival on media
containing the antibiotic. These bacteria are used to
insert their T-DNA (modified with passenger DNA) into a
plant genome. This procedure utilizing A. rhizogenes or
A. tumefaciens gives rise to transformed plant cells that
can be regenerated into intact healthy, fertile plants.
The invention is illustrated by way of the examples
which follow.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a restriction map of pUCP521 and also
illustrates a DNA sequence with which a BanII fragment of
pUCP521 can be ligated.
Fig. 2 is a restriction map of pTP369 of Example 2
containing the gene for protein 13 of phage 22.
Fig. 3 is a partial restruction map of pMONP22Ly of
Example 3 containing the lysozyme gene of phage P22.
Fig. 4 is a partial restriction map of pMONT4Ly of
Example 4 containing the lysozyme gene of phage T4.
Fig. 5 is restriction maps of pLYS1023, p76-311,
p20-211 and pCHLY.
Fig. 6 is a restriction map of PMON530.
Fig. 7 is a restriction map of pCA2.
Fig. 8 is a partial restriction map of pIBI76.
Fig. 9 is a partial restriction map of pBI121.
Fig. 10 is a partial restriction map of pCAMV2X.
.
-28-
~ 3 2 ~ 7
Fig. 11 is a graph illustrating the enhancement of
essential amino acid content of the lotononis
transformants of Example 16.
Exampl _
A plasmid containing an attacin cDNA se~uence, the
CaMV 35s promoter and kanamycin/streptomycin/spectinomycin
resistance genes was constructed, and is designated herein
as pMONAATT. The plasmid pCP521 was obtained from K.
Xanthopoulous, and it has a 723 bp insert in the Pstl site
of pBR322 containing the acidic attacin cDNA se~uence
without methionine. pCP521 was treated with BanII and
PstI to remove the attacin gene. The attacin gene was
subcloned into the PstI site of pUCl9 by filling in the 3'
end of the attacin with T4 DNA polymerase and ligating
with T4 DNA ligase. The resulting plasmid was selected
for clockwise orientation and designated pUCP521. A
restriction map of pUCP521 is seen in Fig. 1. This was
digested with BanII. The larger fragments were isolated
by gel permeation and ligated with the DNA sequence:
5' CAGATGTAACAATGGACGCGCACGGAGCC 3'
3' TCGAGTCTACATTGTTACCTGCGCGTGCC 5'
A 657 bp fragment was rescued by digestion with BglII
and EcoRV. This fragment was then ligated between the
BglII and SmaI sites of pMON530 and the resulting plasmid
was designated pMONATT. A restriction map of unmodified
pMON530 is illustrated in Fig. 6.
The plasmid pMONAATT was then placed in competent E.
coli cells. Into 100 ~1 of cells at 10fi cells/ml, was
added 1 ~g of pMONAATT in 10 ~1 sterile water and 100 ~1
0.01M CaCl2 buffer. This mixture was incubated at 4C for
45 minutes, heat shocked at 42C for two minutes, placed
in 1.5 ml Luria broth at 37C for two hours and plated on
LB-Agar containing 50 ~g/ml kanamycin sulfate. After
incubation at 37C overnight, colonies were selected,
grown and screened for kanamycin resistance and the
correct attacin gene. Suitable clones in mid to late-log
phase were suspended at 106/ml and 0.1 ml was placed on
. : : , :
.. "
. . :.,.:- ., , , .
: . , ,~ ~
-29- ~32~57
LB-agar plates with a like amount of disarmed vector A.
tumefaciens. After about 6 hours at room temperature, the
plates were scraped, the cells were serially diluted
several times at 1:10 dilution and replated on LB-agar
containing 25 ~g/ml chloramphenicol, 50 ~g/ml kanamycin
sulfate, and 100 ~g/ml each of streptomycin and
spectinomycin. Surviving A. tumefaciens were diluted 1:10
in PBS and N. tabacum leaf discs were briefly dipped in
this solution and subsequently plated. After two days of
incubation, cefotoxin and kanamycin were added to the
media. All cells died, indicating that no transformants
were obtained.
The pMONAATT clones are digested with EcoRV and
HindIII and the attacin fragment is rescued and purified
from agarose gel. This fragment is subcloned into pIBI76
(Fig. 8) between the EcoRV and HindIII cleavage sites
thereof. The resulting plasmid is then digested with
EcoRV and KpnI and the fragment containing the attacin
gene is recovered. This fragment is then subcloned into
pCA2 (Fig. 12; Science, vol. 236, pp. 1299-1301 (1987))
between the EcoRV and KpnI sites. This plasmid is then
digested with HindIII and PstI to rescue a fragment
containing the attacin gene and duplicated CaMV 35s
promoters. This fragment is then ligated into pBI121
(Fig. 9; obtained commercially from Clontech Laboratories,
Inc.) between the HindIII and PstI sites to obtain the
vector illustrated in Fig. 10 and designated pCAMV2X.
This vector has the attacin gene under the control of the
double CaMV 35s promoters, as well as a ~-glucoronidase
("GUS") gene. This vector is then cloned in E. coli as
described above for pMONATT. The transformed E. coli are
conjugated with disarmed vector A. tumefaciens which are
subsequently used to infect the tobacco l~af discs as
described above. After about 2 weeks, callus tissue
develops. P]ants are regenerated from the callus and
expression of attacin is demonstrable by blotting
techniques. The plants are exposed to Pseudomonas
:~ . ' , ' , ' ,:
. . .
-30- ~32~
syringae but are not infected, in contrast to
untransformed plants of the same species identically
exposed to the P. syringae under the same condi~ions.
ExampLe 2
A plasmid containing P13 protein of phage P22, the
CaMV 35s promoter and kanamycin/streptomycin/spectinomycin
resistance genes was constructed, and is designated herein
as pMONP22P13. The plasmid pI'P369 (Fig. 2) was obtained
from A. Poteete. pTP369 was digested with EcoRI and NaeI
to obtain a fragment of about 1100 bp containing the P13
protein codons. This fragment was ligated into pUC19
between the EcoRI and the HincII cleavage sites thereof to
obtain a plasmid designated pTPEN1041. pTPEN1041 was
digested with EcoRI and PstI, and a fragment of about 1100
bp was isolated. This was then digested with MaeII and
yielded fragments of 437, 348 and 259 bp. The 437 bp
fragment ends were filled in using Klenow enz~me and
purified from agarose gel. This was then ligated into
p~ION530 between the SmaI and LiH sites, and the
orientation was checked with HpaI. The resulting plasmid
was designated pMONP22P13. The plasmid pMONP22P13 was
cloned in E. coli and conjugated with A. tumefaciens which
was then cultured with tobacco leaf discs as described in
Example 1, but no transformed cells were obtained.
The P22 protein gene from the pMONP22P13 clones are
then inserted in the HindIII site of pBI121 as described
in Example 1, and then used in an A. tumefaciens vector to
transform tobacco leaf discs. Callus eventually
develops, and plants regenerated therefrom express the0 phage protein and are resistant to P. syringae.
Example 3
A plasmid corresponding to the partial restriction
map shown in Fig. 3 was constructed and designated
pMONP22Ly. The plasmid pDR116 was obtained from A.
Poteete and contained a fragment of about 590 bp between
HindI and Tac~I sites [as a BamHI-ClaI insert in a pBR322
derivative containing a BamHI in the PvuI cleavage site.]
: ,. . -
, ! . .~. . , ': ~
~ 3 2 ~ 7
pDR116 was digested with BamHI and ClaI and the ends of
the fragment containing the phage lysozyme gene was filled
in using Klenow enzyme and isolated from agarose gel.
This approximately 590 bp fragment was ligated into
pMON530 at the SmaI/LiH site and the orientation was
checked by digestion with EcoRI and DraI. The resulting
plasmid was designated pMONP22Ly, cloned in E. coli and
conjugated with A. tumefaciens which was then cultured
with tobacco leaf discs as described in Example 1. Again,
no tobacco transformants resistant to kanamycin were
obtained.
The P22 lysozyme is then rescued from pMONP22Ly and
inserted into pBI121 which is then used to transform E.
~oli, which in turn is conjugated with A. tumefaciens and
used to infect tobacco leaf discs as described in Example
1. Callus eventually develops, and plants regenerated
therefrom express the phage lyso~yme gene and are
resistant to P. syringae.
Example 4
A plasmid designated pMONT4Ly was constructed
corresponding to the partial restriction map seen in Fig.
4. pDR105 was obtained from A. Poteete and has the
lysozyme gene from phage T4 as an approximately 685 bp
fragment between the AvaII and HindIII restriction sites.
pDR105 was digested with AvaII and HindIII, and the
lysozyme fragment was filled in with Klenow en~yme and
purified from the gel. This fragment was then ligated
into pUC19 between the HincII and LiH sites and designated
pUCT4Ly. The orientation was checked by digestion with
EcoRI, and all 18 clones had obtained clockwise rotation.
pUCT4Ly was digested with BamHI, DraI and AvaII to avoid
fragments of similar length to the BamHI-DraI fragment.
The 646 bp Bc~mHI-DraI fragment was ligated into pMON530
between the ~glII and SmaI sites adjacent to the LiH
region, and the resulting plasmid was designated pMONT4Ly.
The proper or:ientation was confirmed with EcoRI which gave
a 353 bp band, and HpaI and EcoRV which gave bands of
.
,
,
132~5~
approximately 8000, 4000 and 604 bp. pMONT4Ly was cloned
in E. coli and conjugated with A. tumefaciens which was
then cultured with tobacco leaf discs as described in
Example 1, but again no transformats were obtained.
As in Example 1, the leaf disc infection procedure is
repeated using the lysozyme gene cut from pMONT4Ly and
placed in pBI121. Callus eventually develops, and
plants regenerated therefrom e~press the T4 phage lysozyme
and are resistant to P. syringae.
Example 5
The plasmid pLYS1023 (Fig. 5) was digested with PstI
and SacI. A 336 bp fragment was isolated and digested
with MnlI to obtain the MnlI-SacI 311 bp fragment. This
was ligated into pIBI76 between the SacI and SmaI sites.
The resulting plasmid was designated p7~-311 (Fig. 5).
pLYS1023 was then digested with SacI and PstI, and a 252
bp fragment was isolated. This fragment was then digested
with DraI and the SacI-DraI 211 bp fragment was isolated
from agarose gel. The 211 bp ~ragment was then ligated
into pIBI20 between the SacI and HincII sites, and the
resuting plasmid was designated p20-211. p20-211 was
digested with EcoRV and SacI and the ~ragment was purified
from gel. This fragment was then ligated with the
HincII-SacI 326 bp fragment from the p76-311 also purified
from a gel, to obtain a plasmid designated pCHLy. The
lysozyme gene (549 bp~ was rescued from pCHLy with BamHI
and XhoI and ligated into pMON530 between the BglII and
XhoI sites to obtain pMONCHLy. The lysozyme gene is then
cut from pMONCHLy and placed in pCAMV2X as described in
Example 1. This plasmid is then cloned in E. coli and
conjugated with A. tumefaciens which is then cultured with
tobacco leaf discs also 2S described in Example 1. Callus
eventually develops, and plants regenerated therefrom
express the lysozyme gene and are resis-tant to P.
syringae.
:. , . : , ;. ,., . "
- ~
- :- : .. ...
- - ~ ~ ..
-33- 132~
Example 6
The lysozyme gene and protein was obtained from
HYalophora-derived plasmid pBR322, provided by Kleanthis
Xanthopoulos. The lysozyme gene was removed from the
plasmid pBR322 by digestion with the enzyme PstI. The
resultant fragment was purified and treated with the Bal31
enzyme to remove the 3' poly dG tail. Then the adapter
shown as follows:
GTTTCATGAAACAGATCTGTCGAC.D,CJATCTGTTTCATGAAAC
CAAAGTACTTTGTCTAGACAGCTGTCTAGACAAAGTACTTTG
was ligated to the fragment after digestion with enzyme
XmnI. Then the fragment was digested with SalI and cloned
into the plasmid pBR322. The lysozyme gene was rescued by
digestion with enzyme Bgl II and inserted into the plant
vector pMON237. The plasmid pMON237 is similar to pMON530
except that it has the l9s promoter instead of the 35s,
and has only the BglII and EcoRl restriction sites near bp
O. The procedure of Example l is then followed to place
the lysozyme gene in pCAMV2X and to obtain plant
transformants.
Example 7
The antibacterial protein-producing cecropin gene,
obtained in plasmid pBR322 received from Kleanthis
Xanthopoulos was first cut with restriction enzymes PstI
and HinPII to provide a plasmid fragment pCPFLl. The
resulting 260 base pair fragment was purified and treated
with T4 DNA polymerase to fill in the HinPII site. The
resultant fragment was then treated with T4 DNA ligase and
the synthetic adapter C3, which is identified as follows:
CTAGCATAAAGATCTGACGTCAGATCTTTATCCTAG
GATCGTATTTCTAGACTGCAGTCTAGAAATAGGATC,
was joined to the fragment. The new gene fragment was
then ligated to pBR322 which had been cleaved with
restriction enzymes XmnI and AatII. Clones containing the
correct ampicillin sensitive genotype were selected, cut
with XmnI ancl ligated with a synthetic adaptor identified
as C5, which has the following nucleotide sequence:
. . .-:
., ~ .., .. , ,. :
:
-34~ 13211~7
CTTTCCATTTCATGGTAGATCTACCATGAAATGG~AAG
GAAAGGTAAAGTACCATCTAGATGGTACTTTACCTTTC
The resultant fragment was retransformed into E. Coli.
The cecropin gene is rescued from E. Coli by digestion
with BglII and inserted into the plant vector pMON237.
Thus, the cecropin gene is regenerated without its leader
peptide and with an appropriate start methionine at the
amino terminus end and the correct translational
termination (stop) signal at the carboxy terminus end.
Example 8
In vitro procedures utilizing a 41 base DNA
oligonucleotide having the sequence:
GATCTCCACG&TTGTGC`CCATATAATCATCGTGTTTTTCAA
effectively blocked 98% of the translation of a virus
genom~. This procedure was carried out by hybridizing the
DNA to the virus in an 8 microliter reaction mixture
containing 20 mM Hepes, pH 7.6, 0.lM NaCl and lmM ETDA.
RNA concentration of the virus was 0.5 ~g/ml and the DNA
was added in a five-fold molar excess. In general, the
reaction mixtures were heated at 70C for 10 minutes
followed by incubation at 45C for 3 hours. The process
of determining viral RNA translation is in a cell-free
protein synthesis regime, such as in RMA rabbit
reticulocyte lysate system described in Shih et al,
Proceedings of the National Academy of Science of the
U.S.A., vol. 75, pp. 5807-5811 (1978) and in Journal of
Virolooy, vol. 30, pp. 472-480 (1979). As a result of
the hybridization, viral translation of Tobacco mosaic
virus was effectively blocked.
Example 9
In the case of viroids, replication was prevented in
the potato spindle tuber viroid tPSTV) by hybridization of
synthetic DNA fragments to the PSTV in the ~entral
conserved region which appears to be present in all known
viroids and is presumed to be important for replication.
c.
,.. . ~.
.
_35_ ~32~
The synthetic DNA fragments have the oligonucleotide
sequence and identification as follows:
GATCTAGGGATCCCCGGGGAAACCT PSTV1
GATCTAGGTTTCCCC~GGGATCCCT PSTV2
This hybridization was carried out by annealing the
various oligonucleotide fractions to purified, infectious
PSTV RNA. The sample mixture was heated to 90C for 5
minutes and then allowed to cool slowly to room
temperature. These mixtures ~ere then inoculated onto
PSTV sensitive tomato plants and symptoms were allowed to
develop. The results are shown in the Table below.
Table of Molar Ratio of Compositions
Inoculated in Tomato Plants
Infected
15 Innocula Molar Ratio Tomato Plants
(#infected/#innoculated)
PSTV + PSTVl 1:1 0/4
PSTV + PSTVl 10:1 0/4
PSTV + PSTVl 1:10 0/4
PSTV + PSTV2 1:1 0/4
PSTV + PSTV2 10:1 2/4
PSTV + PSTV2 1:10 0/4
PSTV + PSTVf* 1:1 1/4
PSTV + PSTVf 10:1 0/4
PSTV + PSTVf 1:10 0/4
PSTV alone 3/5
*PSTV is a full length DNA of PSTV.
When hybridization occurs, the further replication of the
PSTV molecule was blocked.
The synthetic DNA transcription blocking for viroids
and synthetic DNA translation blocking for viruses are
further inserted into the plant vector pCAMV2X and A.
tumefaciens to produce tobacco plants which demonstrated
expression by Southern blotting.
Example 10
The pCAMV2X of Example 1 is used to transform rice
plants (Oryza sativa). An aqueous solution of 100 ~g/ml
-
pCAMV2X is injected with a tuberculin syringe above each
tiller node until several drops of the solution comes out
of the tiller (about 300 ~l). The plants are injected
when the young tillers have 5 leaves, the flag leaf is
, .
. .
.. . . . . ..
-36- ~ 32~7
about one-third to one-half its normal size and they
contain a young influorescence of about 2 cm suitable for
injection. This corresponds to about 14 days pre-meiosis.
The plants are self-pollinate~ or cross-pollinated with
other treated plants and the floral tillers grown to
maturity.
The seeds from these plants are then surface
sterilized and germinated in g:lass containers having about
400 ml of Knop nutritive solution supplemented with
kanamycin sulfate at 10 ~g/ml. About 10 seeds are
positioned in a plastic net over the solution so that they
are just in contact therewith. The seeds are maintained
in a culture room at about 26C and receive about 2000 lux
for 16 hours per day. Control seeds germinated in the
kamaycin nutritive solution are albino after about 10
days. From about 3000 seeds obtained from the injection
of 100 plants, about 3-15 remain green after 10 days.
Approximately one-third of these plants show expression of
the attacin gene by blotting techniques and have enhanced
resistance to blast.
The procedure is repeated with the plasmids described
in Examples 2-9 with similar results.
Example 11
Protoplasts of rice are treated with pCAMV2X of
Example 1 to obtain disease-resistant plants. Calli are
cultured on a gyrating shaker at 27C in the dark in a
suspension of medium B5 supplemented with 1 mg/l
2,4-dichlorophenoxy-acetic acid and 30 g/l sucrose, and
subcultured twice a week by a 1:3 dilution with fresh
medium. Protoplasts are isolated the second or third day
following subculturing by sedimenting and incubating in a
cell wall digestive solution containing 1% Onozuka
Cellulase RS, 0.5% Macerozyme R10, 0.05% Pectolyase Y23,
0.6M mannitol and 5 mM CaCl2 at pH 5.7. Following
incubation at 27C for three or four hours, protoplasts
are separated from the solution by filtering through a
series of 100, 50 and 25 ~m stainless steel sieves, and
" .
. ~ , * TRADE-MARK
,` .
_37_ 1 3 ~ ri
repeated centrifugations with sea water at 700 mOsmol/kg
H20 ~
The protoplasts are resuspended at about 2 x 106/ml
in medium F, and one ml of protoplast suspension is mixed
with 0.4 ml of medium F containing 40% (w/v) polyethylene
glycol 1500 and 10-50 ~g pC~V2X. This mixture is
incubated in a laminar flow hood at 22-24C for 30
minutes, diluted stepwise over a period of 20 minutes with
medium F and protoplasts are collected by sedimentation in
sea water and culture medium at 1:1 (v/v). The
protoplasts are resuspended at 2-4 x 105 protoplasts/~l in
medium C8/IV, and placed in 2 ml aliguots in 5 cm2 dishes
maintained at 27C in the dark. About 5-7 days later, the
same volume of 1.2% agarose is added to the dish. The
solidified medium is placed in a larger dish to which is
added 20 ml of B5-1 medium containing 100 mg/l kanamycin
sulfate. The liquid medium in the resulting bead-type
culture is replaced every 10 days with fresh medium
containing the antibiotic. Proliferating colonies are
transferred to agar-solidified B5-1 medium with 100 mg/l
kanamycin sulfate about two months later. The cells
express the attacin gene as shown by standard blotting
techniques, and plants regenerated therefrom have superior
resistance to blast relative to control plants.
This procedure is repeated with the plasmids of
Examples 2-9 with similar results.
Example 12
Protoplasts of rice are prepared as described in the
foregoing Example 11, and suspended at 1 x 107/ml in 0.5
ml PBS. The mixture is heat shocked for five minutes at
45C and cooled on ice to room temperature. First, the
plasmid pCAMV2X of Example 1, and then polyethylene
glycol, are added to the mixture at 10-50 ~g/ml and about
8% w/v, respectively. Following incubation for 5 minutes,
the mixture is transferred to the chamber of a BIO-RAD
electroporator equipped with an ISCO 494 power supply.
The power supply is connected to the shock chamber, and
-38- ~2~7
set at 2000V and 0.9mA limit. The wattage and current
dials are set to 5% and the power supply ~capacitor) is
discharged. The electroporated protoplasts are then
maintained at 20C for about 10 minutes and dilu-ted with
growth medium and agar to form a bead type culture. The
protoplasts are cultured at 24C in the dark for one day
and in 500 lux continuous l:ight for 6 days. The
protoplasts are then cultured in the same medium
containing 50 mg/l kanamycin sulfate as described in
Example 11. Similar results are observed, except the
transformation efficiency is higher.
Example 13
The following DNA sequence was synthesized on an
Applied Biosystems DNA synthesizer:
AAGCTTGATCCAAC.AATGGAAAAATGGATGAAAGAAATCTGGAAAGTGCTTAAAGATATGATCGATAAAATGAAAACTTTC
CTAGGTTGTTACCTTTTTACCTACTTTCTTTAGACCTTTCACGAATTTCTATACTAGCTATTTTACTTTTGAAAG:
G.~TACTCTTTTCGAAATGATCACTAAACTTTTCACTGAAGTCGAAAAATGGATGAAAGAAATCTGGAAAGTGCTTAAA
CTATGAGAaAAGCTTTACTAGTGATTTGAAAAGTGACTTCAGCTTTTTACCTACTTTCTTTAGTCCTTTCACGAATTT
GATATGATCGATAAAATGAAAACTTTCGTGGATACTCTTTTCGAAATGTGGACTAAAGTGCTTACTGAAGTGGAaAaA
CTATACTAGATATTTTACTTTTGAAAGCACCTATGAGAaAAGCTTTACACCTGATTTCACGAATGACTTCACCTTTTT
TGGATGAAAGAaATCTGGAAATTCCTTAAAGATATGATCGATAAAATGAaAACTTTCTGGGATACTCTTCTTGAAATG
ACCTACTTTCTTTAGACCTTTAAGGAATTTCTATACTAGCTATTTTACTTTTGAAAGACCCTATGAGAAGAACTTTAC
ATCACTAAATGGTTCACTGAAGTGGAAAAATGGATGAAAGAAATCTGGAAAGTGCTTAAAGATATGATCGATAaAATG
TAC-TGATTTACCM GTGACTTCACCTTTTTACCTACTTTCTTTAGACCTTTCACGAATTTCTATACTAGCTATTTTAC
TGAGGATC
ACRCCTAGTTCGAA
This DNA sequence is then inserted into the HindIII site
of pCAMV2X, and used to transform potatoes using an A.
tumefaciens vector. The resulting potatoes express the
protein
MFKWMKEIWKVLKDMIDKMKTFIDTLFEM
ITKLFTEVEKWMKEIWKVLKDMIDKMKTFVDTLFEM
LEMITKWE'TEVEKWMKEIWKVLKDMIDKM
and have a relatively high essential amino acid content.
Example 14
The for~going Examples 1-9 and 13 are repeated
sequentially with potatoes to obtain a potato with a wide
, - : - ' ,; ~ ,
_39_ 132~ ~ ~7
spectrum of microbial resistance to bacteria, fungi,
viruses and viroids and enhanced limiting essential amino
acid content. First, potato leaf discs are used instead
of tobacco according to the procedure of Example 1.
Transformants obtained thereby are then treated with a
pCAMV2X vector similar to that of Example 2 except that a
different antibiotic resistance marker is present in the
plasmid construct for selection of transformants.
Transformants expressing the antimicrobial genes of
Examples 1 and 2 are then sequentially transformed in the
same manner with the vectors of Examples 3-9 and 13, using
a different selectable marker in each transformation
stage. The resulting potato plants express attacin,
lysozyme, cecropin, and the protein of Example 13, and
have resistance to a wide spectrum of bacteria, fungi and
viruses, as well as an enhanced essential amino acid
content.
Example 15
The procedure of Example 14 is followed, except that
rice is used instead of potatoes, and the electroporation
of rice protoplasts described in Example 12 is used
instead of the Agrobacterium vector. The resulting rice
plants have a similar microbial resistance and enhanced
nutritional quality.
Example 16
The nucleotide sequence and polypeptide se~uence of
some synthetic polypeptide genes are designated Sp44 and
Sp47. These genes were constructed symmetrically so that
the correct reading frame is maintained in either
direction. Hence, there are two possible polypeptides for
each synthetic gene.
Single strand DNA sequences were constructed via an
automated DNA synthesizer which uses a solid support
phosphite synthesis method of the triester method of
synthesis. The following synthetic oligonucleotides were
constructed: 5'(AAGAAATGGATG)3l, 5'(CATCCA)3',
5'(TTTCTT)3', 5'(ACGATC)3', 5'(GATCGT)3', and
. - .~
. .. , , ........................... :
- ., :
,:
~40- ~32~1~7
5'(CCCGAATTCGGG)3'. The synthetic oligodeoxynucleotides
were phosphorylated in the terminal 5'-OH position by the
action of T4 polynucleotide kinase. The labeled
oligodeoxynucleotides were added to a final volume of 25 ~1
with the following molar concentrations: AAGAAATGGATG,
TTTCTT, and CATCCA were at 61 ~M each and ACGATC and
GATCGT were at 40 ~M each. The sequence containing the
EcoRl recognition sequence was at a concentration of
2.4 ~M. This mixture was boiled for 2 minutes and cooled
to room temperature for over a period of 3 hours.
Ligation buffer was added followed by the enzyme T4 DNA
ligase and the resultant mixture incubated at 13C
overnight. Following ligation, the synthetic genes were
precipitated with ethanol, redissolved in water and
digested wtih EcoRl. After digestion ~he sample was
passed over a Sephadex*G-75 column in order to separate
the small molecular weight linked fragments from the
synthetic genes. The high molecular weight fractions were
pooled, precipitated and ligated with EcoRl-digested
pBR325. Competent E. coli strain RRl cells were
transformed with the above ligation mixture.
Approximately 45 clones which were ampicillin-resistant
and chloramphenicol-sensitive were obtained from one
ligation. Two recombinant DNA strains designated Sp44 and
Sp47 were selected for DNA sequence analysis by the method
of Sanger.
As a result of the sequence analysis, the nucleotide
sequence and amino acid composition of the peptide
identified as Sp47 and the peptide identified as Sp44 were
determined and are set forth below.
* TRADE-MARK
, .j
-41- 1 3211 ~7
Sp~7
NUCLEOTIDE SEQUENCE AND AMINO ACID COMPOSITION
SP47(1)
AATTCGGGGATCGTAAGAMTGGATGGATCGTCATC('ATTTCTTCATCCATTTCTTACGATCCATCCATTTCTT
GCCCCTAGCATTCTTTACCTACCTAGCAGTAGGTAMGMGTAGGTAAAGMTGCTAGGTAGGTAAACAA
AAGAAATGGATGMGAAATGGATGACGATCCATCCATTTCTTCATCCATTTCTTCATCCATTTCTTACCATCAA
TTCTTTACCTACTTCTTTACCTACTGCTACGTAGGTAMGMGTAGGTAAAGAAGTAGGTAMGAATGCTAGTT
GAAATGGATGMGAAATGGATGAAGMATGGATGAAGAAATGGATGCATCCATTTCTTMGAAATGGATGMGA
CTTTACCTACTTCTTTACCTACTTCTTTACCTACTTCTTTACCTACGTAGGTAAAGAATTCTTTACCTACTTCT
lO AATGGATGAAGAMTGGATGACGATCGATCGTAAGAAATGGATGACGATCCATCCATTTCTTACGATCCCCG
TTACCTACTTCTTTACCTACTGCTAGCTAGCATTCTTTACCTACTGCTAGGTAGGTAAAGAATGCTAGGGGCTTAA
SP47
SP47 (1)
GDP~P~LTIPFL~KWM~I
TIHPFLHPFLHPFLTIKKWM~l~WMHPF
L}~lKKWk~TIDRKKWMTIHPFLTIP
SP47 ~2~
GlyAspArgLyslysTrpMetAspArgHisProPheLeuThrIleAspArglHisProPheLeuHis
ProPheLeulHisProPheLeuLysTrpMetHisProPheLeulHisProPheLeulHisProPheLeuHis
20 ProPheLeuAspArgLysLysTrpMetLysLysTrpMetLysLysTrpMetAspArgHisProPheLeuHis
ProPheLeuLysLysTrpMetAspArgLysLysTrpMetLysLysTrpMetThrIlelHisProPheLeuThr
IlePro
ARG ASP GLY HIS ILE LEU LYS MET PHE PRO T~
SP47(1) 3 3 1 8 6 8 22 11 8 9 6
,~ 3.13.11.08.36.38.323.011.58.39.4 6.3
SP47(2) 6 6 111 311 16 8 11 12 3
% 6.36.31.011.53.111.516.78.3 ~1.512.53.1
Mol. Wt.
SP47(1) 14,322Da
30 SP47(2) 14,232Da
SP 44
NUCLEOTIDE SEQUENCE AND AMINO ACID COMPOSITION
AATTCGGGACGATCACGATCCATCCATTTCTTAAGAAATGGATGACGATCCATCCATTTCTTAAGAAATGGATG
GCCCTGCTAGTGCTAGGTAGGTAAAGMTTCTTTACCTACTGCTAGGTAGGTAAAGAATTCTTTACCTAC
ACGATCCATCCATTTCTTCCCG
TGCTAGGTAGGTAAAGAAGGGCTTAA
,. . , . ~ .
-42 ~32~ ~7
SP44(1)
GTITIHPFLKKWMTIHPFLKKWMTIHPFLP
SP44(2)
GlyLysLysTrpMetAspArfHisProPheLeuLysI,ysTrpMetAspArgHisProPheLeuLysLys
TrpMetAspArgAspArgPro
ARG ASP GLY HIS IIE LEU LYS MET PHE PR0 THR TRP
SP44 (1) 0 0 l 3 4 3 4 2 3 4 4 2
~ 0 0 3.3 9.9 13.3 9.9 13.3 6.6 9.9 13.3 13.3 6.6
SP44 ~2) 4 4 l 2 ~ 2 6 3 3 3 0 3
% 13.3 13.3 3.3 6.~ 0 6.6 20.0 9.9 9.9 9.9 0 9.9
In order to determine the amount and type of
polypeptide produced by the recombinant cells, E. coli
cells retaining the plasmids were grown in M9 media
supplemented with . 5% casamino acids and 1.0 g. per ml
thiamine, at 37C. Ten mls of each culture was placed in
100 mls of the supplemented M9 medium and grown again at
37. After this time approximately 10 ~Ci each of 3H
lysine and 35S cysteine were added to the cells and 1 ml
was withdrawn hourly and the amount of label incorporated
into the polypeptide were determined. The recombinant
microbes used contained Sp47 and Sp44 genes on the plasmid
pBR325. The control was a bacteria which contained pBR325
alone. Hourly 1 ml samples were withdrawn and the counts
incorporated into polypeptide determined. The ratio (3H
lysine/3 5 S cysteine) of uptake was substantially higher in
cells that contained pSP44 and pSP47 plasmids than those
with pBR325. This indicates that polypeptides with higher
levels of lysine are being synthesized by bacteria
containing the recombinant plasmids pSP44 and pSP47.
The pSP47 ( 2 ) plasmids were inserted into lotononis
using an Agrobacterium vector. Plants selected at random
had increasecl essential amino acid levels as indicated in
Fig. 11.
Having described the invention above, many variations
from the illllstrated details will occur to those skilled
in the art. It is intended that all such variations
within the scope and spirit of the appended claims be
embraced thereby.
- : . . , : . , .: :, , ~ -
