Note: Descriptions are shown in the official language in which they were submitted.
CA 02280196 2002-09-09
TITLE
CHIMERIC GENES AND METHODS FOR INCREASING
THE LYSINE CONTENT OF THE SEEDS OF PLANTS
FIELD OF THE INVENTION
This invention relates to chimeric genes and methods for increasing the
lysine content of the seeds of plants and, in particular, to two chimeric
genes, a
first encoding plant lysine ketoglutarate reductase (LKR) and a second
encoding
lysine-insensitive dihydrodipicolinic acid synthase (DHDPS) which is operably
linked to a plant chloroplast transit sequence, all operably linked to plant
seed-
specific regulatory sequences.
BACKGROUND OF THE INVENTION
Many vertebrates, including man, lack the ability to manufacture a number
of amino acids and, therefore, require these amino acids preformed in the
diet.
These are called essential amino acids. Human food and animal feed derived
from
many grains are deficient in some of the ten essential amino acids. In com
(Zea
mays L.), lysine is the most limiting amino acid for the dietary requirements
of
many animals. Soybean (Glycine max L.) meal is used as an additive to com
based animal feeds primarily as a lysine supplement. Thus, an increase in the
lysine content of either corn or soybean would reduce or eliminate the need to
supplement mixed grain feeds with lysine produced via fermentation of
microbes.
Plant breeders have long been interested in using naturally occurring
variations to improve protein quality and quantity in crop plants. Maize lines
containing higher than normal levels of lysine (70%) have been identified
[Mertz
et al. (1964) Science 145:279, Mertz et al. (1965) Science 150:1469-70].
However, these lines which incorporate a mutant gene, opaque-2, exhibit poor
agronomic qualities (increased susceptibility to disease and pests, 8-14%
reduction in yield, low kernel weight, slower drying, lower dry milling yield
of
flaking grits, and increased storage problems) and thus are not commercially
useful [Deutscher (1978) Adv. Exp. Medicine and Biology 105:281-300]. Quality
Protein Maize (QPM) bred at CIMMYT using the opaque-2 and sugary-2 genes
and associated modifiers has a hard endosperm and enriched levels of lysine
and
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tryptophan in the kernels [Vasal, S. K., et al. Proceedings of the 3rd seed
protein
symposium, Gatersieben, August 31 - September 2, 1983]. However, the gene
pools represented in the QPM lines are tropical and subtropical. Quality
Protein
Maize is a genetically complex trait and the existing lines are not easily
adapted to
the dent germplasm in use in the United States, preventing the adoption of QPM
by corn breeders.
The amino acid content of seeds is determined primarily (90-99%) by the
amino acid composition of the proteins in the seed and to a lesser extent (1-
10%)
by the free amino acid pools. The quantity of total protein in seeds varies
from
about 10% of the dry weight in cereals to 20-40% of the dry weight of legumes.
Much of the protein-bound amino acids is contained in the seed storage
proteins
which are synthesized during seed development and which serve as a major
nutrient reserve following germination. In many seeds the storage proteins
account for 50% or more of the total protein.
To improve the amino acid composition of seeds genetic engineering
technology is being used to isolate, and express genes for storage proteins in
transgenic plants. For example, a gene from Brazil nut for a seed 2S albumin
composed of 26% sulfur-containing amino acids has been isolated [Altenbach et
al. (1987) Plant Mol. Biol. 8:239-250] and expressed in the seeds of
transformed
tobacco under the control of the regulatory sequences from a bean phaseolin
storage protein gene. The accumulation of the sulfur-rich protein in the
tobacco
seeds resulted in an up to 30% increase in the level of methionine in the
seeds
[Altenbach et al. (1989) Plant Mol. Biol. 13:513-522]. However, no plant seed
storage proteins similarly enriched in lysine relative to average lysine
content of
plant proteins have been identified to date, preventing this approach from
being
used to increase lysine.
An alternative approach is to increase the production and accumulation of
specific free amino acids such as lysine via genetic engineering technology.
However, little guidance is available on the control of the biosynthesis and
metabolism of lysine in the seeds of plants.
Lysine, along with threonine, methionine and isoleucine, are amino acids
derived from aspartate, and regulation of the biosynthesis of each member of
this
family is interconnected. Regulation of the metabolic flow in the pathway
appears
to be primarily via end products. The first step in the pathway is the
phosphorylation of aspartate by the enzyme aspartokinase (AK), and this enzyme
has been found to be an important target for regulation in many organisms.
However, detailed physiological studies on the flux of 4-carbon molecules
through the aspartate pathway have been carried out in the model plant system
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Lemna paucicostata [Giovanelli et al. (1989) Plant Physiol. 90:1584-1599]. It
was stated in this reference that "These data now provide definitive evidence
that
the step catalyzed by aspartokinase is not normally an important site for
regulation
of the entry of 4-carbon units into the aspartate family of amino acids [in
plants]."
The aspartate family pathway is also believed to be regulated at the branch-
point reactions. For lysine this is the condensation of aspartyl A-
semialdehyde
with pyruvate catalyzed by dihydrodipicolinic acid synthase (DHDPS), while for
threonine and methionine the reduction of aspartyl 0-semialdehyde by
homoserine
dehydrogenase (HDH) followed by the phosphorylation of homoserine by
homoserine kinase (IHK) are important points of control.
The E. coli dapA gene encodes a DHDPS enzyme that is about 20-fold less
sensitive to inhibition by lysine than a typical plant DHDPS enzyme, e.g.,
wheat
germ DHDPS. The E. coli dapA gene has been linked to the 35S promoter of
Cauliflower Mosaic Virus and a plant chloroplast transit sequence. The
chimeric
gene was introduced into tobacco cells via transformation and shown to cause a
substantial increase in free lysine levels in leaves [Glassman et al. (1989)
U.S. Patent No.
5,258,300, Canadian Patent No. 1,338,349 and WO 8911789 (published December
14, 1989),
Shaul et al. 1992 Plant Jour. 2:203-209, Galili et al. (1992) European Patent
No. 485970
published May 20, 1992 and U.S. Patent No. 5,367,110]. However the lysine
content of the
seeds was not increased in any of the transformed plants described in these
studies. The same
chimeric gene was also introduced into potato cells and lead to small
increases in free lysine
leaves, roots and tubers of regenerated plants [Galili et al. (1992) European
Patent No. 485970
published May 20, 1992 and U.S. Patent No. 5,367,11. 0, Perl et al. (1992)
Plant Mol. Biol.
19:815-823]. These workers have also reported on the introduction of an E.
coli 1ysC gene that
encodes a lysine-insensitive AK enzyme into tobacco cells via transformation
[Galili et al.
(1992) European Patent No. 485970 published May 20, 1992 and U.S. Patent No.
5,367,110;
Shaul et al. (1992) Plant Physiol. 100:1157-1163]. Expression of the E. coli
enzyme results in
increases in the levels of free threonine in the leaves and seeds of
transformed plants. Crosses
of plants expressing E. coli DHDPS and AK resulting in progeny
that accumulated more free lysine in leaves than the parental DHDPS plant, but
less free threonine in leaves than the parental AK plant. No evidence for
increased levels of free lysine in seeds was presented.
The limited understanding of the details of the regulation of the biosynthetic
pathway in plants makes the application of genetic engineering technology,
Particularly to seeds, uncertain. There is little information available on the
source
of the aspartate-derived amino acids in seeds. It is not known, for example,
whether they are synthesized in seeds, or transported to the seeds from
leaves, or
both, from most plants. In addition, free amino acids make up only a small
fraction of the total amino acid content of seeds. Therefore, over-
accumulation of
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free amino acids must be many-fold in order to significantly affect the total
amino
acid composition of the seeds. Furthermore, little is known about catabolism
of
free amino acids in seeds. Catabolism of free lysine has been observed in
developing endosperm of corn and barley. The first step in the catabolism of
lysine is believed to be catalyzed by lysine-ketoglutarate reductase (LKR)
[Brochetto-Braga et at. (1992) Plant Physiol. 98:1139-1147]. This protein is
actually a bifunctional enzyme that is also responsible for catalysis of the
presumed second reaction in the catabolism of lysine, saccharopine
dehydrogenase (SDH) [Goncalves-Butruille et al. (1996) Plant Physiol. 110:765-
771]. There are only a few reports of the isolation of genomic or cDNA clones
encoding various portions of LKR/SDH proteins from plants. GenBank accession
ATU9579 presents the sequence of a full-length cDNA clone for the bifunctional
enzyme from Arabidopsis thaliana. The protein encoded by this clone is a
homologue of both LKR and SDH proteins from fungal organisms. The DNA
sequence for the genomic clone from Arabidopsis is also available as GenBank
accession U95758 (Tang, et at. (1997) Plant Cell 9:1305-1316 and Epelbaum, et
al. (1997) Plant Mol. Biol. 35: 735-748). GenBank accession AF003551 discloses
a cDNA from corn which would direct the synthesis of a polypeptide from within
the SDH domain of LKR/SDH proteins. GenBank accession AF042184 discloses
the sequence of a cDNA from Brassica napus that is homologous to a relatively
short portion of the full length clone from Arabidodpsis. However, whether
such
catabolic pathways are widespread in plants and whether they affect the level
of
accumulation of free amino acids is unknown. Finally, the effects of over-
accumulation of a free amino acid such as lysine or threonine on seed
development and viability is not known.
Heretofore, no method to increase the level of lysine in seeds via genetic
engineering was known. Thus, there is a need for genes, chimeric genes, and
methods for expressing them in seeds so that an over-accumulation of lysine in
seeds will result in an improvement in nutritional quality.
SUMMARY OF THE INVENTION
This invention concerns an isolated nucleic acid fragment comprising a
nucleic acid sequence encoding all or part of lysine ketoglutarate reductase.
In another embodiment this invention concerns a chimeric gene
comprising the aforesaid nucleic acid fragment encoding all or part of lysine
ketoglutarate reductase, or a subfragment thereof, operably linked to suitable
seed-specific regulatory sequences wherein said chimeric gene reduces lysine
ketoglutarate reductase activity in seeds of transformed plants, as well as a
plant
cell or plant seed transformed with the aforesaid chimeric gene..
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In a third embodiment this invention concerns a plant cell wherein lysine
ketoglutarate reductase activity is reduced due to a mutation in a gene
encoding
lysine ketoglutarate reductase.
In a fourth embodiment this invention concerns a plant seed wherein
lysine ketoglutarate reductase activity is reduced due to a mutation in a gene
encoding lysine ketoglutarate reductase.
In a fifth embodiment this invention concerns a method for reducing
lysine ketoglutarate reductase activity in a plant seed which comprises:
(a) transforming plant cells with the chimeric gene comprising the
aforesaid nucleic acid fragment encoding all or part of lysine
ketoglutarate reductase or a subfragment thereof, operably linked to
suitable seed-specific regulatory sequences wherein said chimeric gene
reduces lysine ketoglutarate reductase activity in seeds of transformed
plants;
(b) regenerating fertile mature plants from the transformed plant
cells obtained from step (a) under conditions suitable to obtain seeds;
(c) screening progeny seed of step (b) for reduced lysine
ketoglutarate reductase activity; and
(d) selecting those lines whose seeds contain for reduced lysine
ketoglutarate reductase activity.
In a sixth embodiment. this invention concerns a nucleic acid fragment
comprising
(a) a first chimeric gene comprising the aforesaid nucleic acid
fragment encoding all or part of lysine ketoglutarate or a subfragment
thereof, operably linked to suitable seed-specific regulatory sequences
wherein said chimeric gene reduces lysine ketoglutarate reductase
activity in seeds of transformed plants and
(b) a second chimeric gene wherein a nucleic acid fragment encoding
dihydrodipicol.inic acid synthase which is insensitive to inhibition
by lysine is operably linked to a plant chloroplast transit
sequence and to a plant seed-specific regulatory sequence.
A seventh embodiment of this invention concerns a plant and a seed
comprising in its genome the aforesaid nucleic acid fragments or the first and
second aforesaid chimeric genes.
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BRIEF DESCRIPTION OF THE
DRAWINGS AND SEQUENCE DESCRIPTIONS
The invention can be more fully understood from the following detailed
description and the accompanying drawings and the sequence descriptions which
form a part of this application.
Figure 1 shows an alpha helix from the side and top views.
Figure 2 shows end (Figure 2a) and side (Figure 2b) views of an alpha
helical coiled-coil structure.
Figure 3 shows the chemical structure of leucine and methionine
emphasizing their similar shapes.
Figure 4a shows a schematic representation of a leaf gene expression
cassette; Figure 4b shows a schematic representation of a seed-specific gene
expression cassette.
Figure 5 shows a map of the binary plasmid vector pZS97K.
Figure 6 shows a map of the binary plasmid vector pZS97.
Figure 7A shows a map of the binary plasmid vector pZS 199; Figure 7B
shows a map of the binary plasmid vector pFS926; Figure 7C shows a map of the
binary plasmid vector pBT593; Figure 7D shows a map of the binary plasmid
vector pBT597.
Figure 8A shows a map of the plasmid vector pBT603; Figure 8B shows a
map of the plasmid vector pBT614.
Figure 9 shows the amino acid sequence similarity between the polypeptides
encoded by two plant cDNAs and fungal SDH (glutamate-forming).
Figure 10 depicts the strategy for creating a vector (pSK5) for use in
construction and expression of the SSP gene sequences.
Figure 11 shows the strategy for inserting oligonucleotide sequences into the
unique Ear I site of the base gene sequence.
Figure 12 shows the insertion of the base gene oligonucleotides into the
Nco I/EcoR I sites of pSK5 to create the plasmid pSK6. This base gene sequence
was used as in Figure 8 to insert the various SSP coding regions at the unique
Ear I site to create the cloned segments listed.
Figure 13 shows the insertion of the 63 bp "segment" oligonucleotides used
to create non-repetitive gene sequences for use in the duplication scheme in
Figure 12.
Figure 14 (A and B) shows the strategy for multiplying non-repetitive gene
"segments" utilizing in-frame fusions.
Figure 15 shows the vectors containing seed specific promoter and 3'
sequence cassettes. SSP sequences were inserted into these vectors using the
Nco I and Asp718 sites.
6
SUBSTITUTE SHEET (RULE 26)
CA 02280196 2002-09-09
Figure 16 shows a map of the plasmid vector pML63.
Figure 17 shows a map of the plasmid vector pBT680.
Figure 18 shows a map of the plasmid vector pBT68 1.
Figure 19 shows a map of the plasmid vector pLH 104.
Figure 20 shows a map of the plasmid vector pLH 105.
Figure 21 shows a map of the plasmid vector pBT739.
Figure 22 shows a map of the plasmid vector pBT756.
SEQ ID NO:1 shows the nucleotide and amino acid sequence of the coding
region of the wild type E. coil lysC gene, which encodes AKIII, described in
Example 1.
SEQ ID NOS:2 and 3 were used in Example 2 to create an Nco I site at the
translation start codon of the E. colllysC gene.
SEQ ID NOS:4 and 5 were used in Example 3 as PCR primers for the
isolation of the Corynebacterium daI Ai gene.
SEQ ID NO:6 shows the nucleotide and amino acid sequence of the coding
region of the wild type Corynebacterium AAA gene, which encodes lysine-
insensitive DHDPS, described in Example 3.
SEQ ID NO:7 was used in Example 4 to create an Nco I site at the
translation start codon of the E. coil daDA gene.
SEQ ID NOS:8, 9, 10 and 11 were used in Example 6 to create a chloroplast
transit sequence and link the sequence to the E. cols Ivs E. coli IvsC-M4, E.
coil
d pA and Corynebacteria danA genes.
SEQ ID NOS: 12 and 13 were used in Example 6 to create a Kpn I site
immediately following the translation stop codon of the E. coli danA gene.
SEQ ID NOS:14 and 15 were used in Example 6 as PCR primers to create a
chloroplast transit sequence and link the sequence to the Corynebacterium danA
gene.
SEQ ID NOS:16-92 represent nucleic acid fragments and the polypeptides
they encode that are used to create chimeric genes for lysine-rich synthetic
seed
storage proteins suitable for expression in the seeds of plants.
SEQ ID NO:93 was used in Example 6 as a constitutive expression cassette
for corn.
SEQ ID NOS:94-99 were used in Example 6 to create a corn chloroplast
transit sequence and link the sequence to the E. coli ly.Q-M4 gene.
SEQ ID NOS: 100 and 101 were used in Example 6 as PCR primers to create
a corn chloroplast transit sequence and link the sequence to the E. coli gene.
SEQ ID NOS: 102 and 103 are partial cDNAs for plant lysine ketoglutarate
reductase/saccharopine dehydrogenase from Arabidopsis thaliana.
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-8-
SEQ ID NOS:104 and 105 are polypeptides encoded by SEQ 1D NOS:102 and 103,
respectively that are homologous to fungal saccharopine dehydrogenase
(glutamate-forming).
SEQ ID NOS:106 and 107 were used in Example 25 as PCR primers to add Nco I and
Kpn I sites at the 5' and 3' ends of the corn DHDPS gene.
SEQ ID NOS:108 and 109 were used for PCR amplification of a 2.24 kb DNA
fragment from genomic Arabidopsis I)NA.
SEQ ID NO:110 shows the sequence of the Arabidopsis lysine ketoglutarate
reductase/saccharopine dehydrogenase genomic DNA fragment.
SEQ ID NO:111 shows the sequence of a full length cDNA for plant lysine
ketoglutarate reductase/saccharopine dehydrogenase from Arabidopsis thaliana.
SEQ ID NO:112 shows the deduced amino acid sequence of Arabidopsis lysine
ketoglutarate reductase/saccharopine dehydrogenase protein.
SEQ ID NOS:113 and 114 were used for PCR amplification of soybean and corn
lysine
ketoglutarate reductase/saccharopine dehydrogenase cDNA fragment.
SEQ ID NO: 115 shows the sequence of a soybean lysine ketoglutarate
reductase/saccharopine dehydrogenase cDNA fragment.
SEQ ID NO: 116 shows the sequence of a corn lysine ketoglutarate
reductase/saccharopine dehydrogenase cDNA fragment.
SEQ ID NO: 117 shows the partial amino acid sequence of soybean lysine
ketoglutarate
reductase/saccharopine dehydrogenase protein deduced from SEQ ID NO: 115.
SEQ ID NO: 118 shows the partial amino acid sequence of corn lysine
ketoglutarate
reductase/saccharopine dehydrogenase protein deduced from SEQ ID NO: 116.
SEQ ID NO: 119 shows the sequence of a 2582 nucleotide partial cDNA from
soybean
for a lysine ketoglutarate reductase/saccharopine dehydrogenase protein.
SEQ ID NO:120 shows the sequence of it 3265 nucleotide partial cDNA from corn
for a
lysine ketoglutarate reductase/saccharopine dehydrogenase protein.
SEQ ID NO:121 shows the deduced partial amino acid sequence of soybean lysine
ketoglutarate reductase/saccharopine dehydrogenase protein encoded by
nucleotides 3
through 2357 of SEQ ID NO:1 19.
SEQ ID NO:122 shows the deduced partial amino acid sequence of corn lysine
ketoglutarate reductase/saccharopine dehydrogenase protein encoded by
nucleotides 3
through 3071 of SEQ ID NO:120.
SEQ ID NO: 123 is a nucleotide sequence corresponding to nucleotides 1 through
1908
of SED ID NO:120.
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- 8a -
SEQ ID NO:124 is the deduced amino acid sequence from SEQ ID NO:123.
SEQ ID NO:125 shows the sequence of a 720 nucleotide lysine ketoglutarate
reductase/saccharopine dehydrogenase cDNA from rice,
SEQ ID NO:126 shows the deduced partial amino acid sequence of rice lysine
ketoglutarate reductase/saccharopine dehydrogenase protein encoded by
nucleotides 2
through 720 of SEQ ID NO:125.
SEQ ID NO:127 shows the sequence of a 308 nucleotide lysine ketoglutarate
reductase/saccharopine dehydrogenase cDNA from rice.
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SEQ ID NO: 128 shows the deduced partial amino acid sequence of rice
lysine ketoglutarate reductase/saccharopine dehydrogenase protein encoded by
nucleotides 1
through 129 of SEQ ID NO: 127.
SEQ ID NO: 129 shows the sequence of a 429 nucleotide cDNA from wheat.
SEQ ID NO: 130 shows the deduced partial amino acid sequence of wheat
lysine ketoglutarate reductase/saccharopine dehydrogenase protein encoded by
nucleotides 1 through 252 of SEQ ID NO:129.
SEQ ID NO: 131 shows the SDH coding region of the Arabidopsis cDNA
clone.
SEQ ID NO: 132 shows the amino acid sequence of the saccharopine dehydrogenase
domain of the Arabidopsis lysine ketoglutarate reductase/saccharopine
dehydrogenase
protein.
The Sequence Descriptions contain the one letter code for nucleotide sequence
characters and the three letter codes for amino acids as defined in conformity
with the
IUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030(1985) and
in the
Biochemical Journal 219 (No. 2):345-373(1984).
An aspect of the invention is to provide a chimeric gene which causes an
increased level of
lysine in seeds obtained from a transformed corn plant, the chimeric gene
comprising: (a) an
isolated nucleic acid fragment comprising a nucleic acid sequence for use in
antisense
inhibition or sense suppression of endogenous lysine ketoglutarate
reductase/saccharopine
dehydrogenase activity in a corn plant or corn plant cell wherein said
isolated nucleic acid
fragment comprises all or a part of the nucleic acid sequence of SEQ ID NO:
120, said part
being sufficient in length for use in antisense inhibition or sense
suppression; and (b) at least
one regulatory sequence operably linked to said fragment.
Another aspect of the invention is to provide a plant cell transformed with
the chimeric gene
described above.
Another aspect of the invention is to provide a method for increasing lysine
content in a plant
seed which comprises: (a) transforming plant cells with the chimeric gene
described above; (b)
regenerating fertile mature plants from the transformed corn plant cells
obtained from step (a)
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CA 02280196 2012-01-16
under conditions suitable to obtain seeds; (c) screening progeny seed of step
(b) for increased
lysine content; and (d) selecting those lines whose seeds have increased
lysine content.
Another aspect of the invention is to provide a method for obtaining reduced
lysine
ketoglutarate reductase and saccharopine dehydrogenase activity in a plant
seed, which
comprises: (a) transforming plant cells with the chimeric gene described
above; (b)
regenerating fertile mature plants from the transformed plant cells obtained
from step (a) under
conditions suitable to obtain seeds; (c) screening progeny seed of step (b)
for reduced lysine
ketoglutarate reductase and saccharopine dehydrogenase activity; and (d)
selecting those lines
whose seeds contain reduced lysine ketoglutarate reductase and saccharopine
dehydrogenase
activity.
Another aspect of the invention is to provide a nucleic acid fragment
comprising (a) a first
chimeric gene described above, and (b) a second chimeric gene encoding
dihydrodipicolinic
acid synthase which is insensitive to inhibition by lysine wherein said second
chimeric gene is
operably linked to a plant chloroplast transit sequence and to a plant seed-
specific regulatory
sequence.
Another aspect of the invention is to provide a plant cell having reduced
lysine ketoglutarate
reductase and saccharopine dehydrogenase activity comprising in its genome a
mutation in an
endogenous lysine ketoglutarate reductase/saccharopine dehydrogenase gene
wherein said
gene comprises the nucleic acid sequence SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:110,
SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 119, SEQ ID NO:120,
SEQ
ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129 or SEQ ID NO:131.
The plant cells described above can be Arabidopsis, corn, soybean, rapeseed,
wheat or rice.
DETAILED DESCRIPTION OF THE INVENTION
Nucleic acid fragments and procedures are described which are useful for
increasing the accumulation of lysine in the seeds of transformed plants, as
compared
to levels of lysine in untransformed plants. In order to increase the
accumulation of free
lysine in the seeds of plants via genetic engineering, a determination was
made of which
enzymes in this pathway controlled the pathway in the seeds of plants. In
order to
accomplish this, genes encoding enzymes in the pathway were isolated from
bacteria. In
some cases, mutations in the genes were obtained so that the enzyme encoded
was made
9a
CA 02280196 2012-01-16
insensitive to end-product inhibition. Intracellular localization sequences
and suitable
regulatory sequences for expression in the seeds of plants were linked to
create chimeric
genes. The chimeric genes were then introduced into plants via transformation
and
assessed for their ability to elicit accumulation of the lysine in seeds.
A unique first nucleic acid fragment is provided which comprises two nucleic
acid subfragments (subsequences), one encoding LKR and the other encoding
DHDPS
which is substantially insensitive to feedback inhibition by lysine. For the
purposes of
the present application, the term substantially insensitive will mean at least
20-fold
less sensitive to feedback inhibition by lysine than a typical plant enzyme
catalyzing
the same reaction. It has been found that a combination of subfragments
successfully
increases the lysine accumulated in seeds of transformed plants as compared to
untransformed host plants.
It also has been discovered that the full potential for accumulation of excess
free
lysine in seeds is reduced by lysine catabolism. Furthermore, it has been
discovered that
lysine catabolism results in the accumulation of lysine breakdown
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products such as saccharopine and a-amino adipic acid. Provided herein are two
alternative routes to reduce the loss of excess lysine due to catabolism and
to
reduce the accumulation of lysine breakdown products. In the first approach,
lysine catabolism is prevented through reduction in the activity of the enzyme
lysine ketoglutarate reductase (LKR), which catalyzes the first step in lysine
breakdown. This can be accomplished by introducing a mutation that reduces or
eliminates enzyme function in the plant gene that encodes LKR. Such mutations
can be identified in lysine over-producer lines by screening mutants for a
failure
to accumulate the lysine breakdown products, saccharopine and a-amino adipic
acid. Alternatively, several procedures to isolate plant LKR genes are
provided;
nucleic acid fragments containing plant LKR cDNAs are also provided. Chimeric
genes for expression of antisense LKR RNA or for cosuppression of LKR in the
seeds of plants can then be created. The chimeric LKR gene is linked to the
chimeric genes encoding lysine insensitive DHDPS and both are introduced into
plants via transformation simultaneously, or the chimeric genes are brought
together by crossing plants transformed independently with each of the
chimeric
genes.
In the second approach, excess free lysine is incorporated into a form that is
insensitive to breakdown, e.g., by incorporating it into a di-, tri- or
oligopeptide,
or preferably a lysine-rich storage protein. The lysine-rich storage protein
chosen
should contain higher levels of lysine than average proteins. Ideally, these
storage
proteins should contain at least 15% lysine by weight. The design of a
preferred
class of polypeptides which can be expressed in vivo to serve as lysine-rich
seed
storage proteins is provided. Genes encoding the lysine-rich synthetic storage
proteins (SSP) are synthesized and chimeric genes wherein the SSP genes are
linked to suitable regulatory sequences for expression in the seeds of plants
are
created. The SSP chimeric gene is then linked to the chimeric DHDPS gene and
both are introduced into plants via transformation simultaneously, or the
genes are
brought together by crossing plants transformed independently with each of the
chimeric genes.
A method for transforming plants is taught herein wherein the resulting
seeds of the plants have at least ten percent, preferably ten percent to four-
fold
greater, lysine than do the seeds of untransformed plants. Provided as
examples
herein are transformed rapeseed plants with seed lysine levels increased by
100%
over untransformed plants and soybean plants with seed lysine levels increased
by
four-fold over lysine levels of untransformed plants, and corn plants with
seed
lysine levels increased by 130%.
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In the context of this disclosure, a number of terms shall be utilized. As
used herein, the term "nucleic acid" refers to a large molecule which can be
single-stranded or double-stranded, composed of monomers (nucleotides)
containing a sugar, phosphate and either a purine or pyrimidine. A "nucleic
acid
fragment" is a fraction of a given nucleic acid molecule. In higher plants,
deoxyribonucleic acid (DNA) its the genetic material while ribonucleic acid
(RNA) is involved in the transfer of the information in DNA into proteins. A
"genome" is the entire body of genetic material contained in each cell of an
organism. The term "nucleotide sequence" refers to a polymer of DNA or RNA
which can be single- or double-stranded, optionally containing synthetic, non-
natural or altered nucleotide bases capable of incorporation into DNA or RNA
polymers.
As used herein, the term "homologous to" refers to the complementarity
between the nucleotide sequence of two nucleic acid molecules or between the
amino acid sequences of two protein molecules. Quantitative estimates of
homology are provided by either DNA-DNA or DNA-RNA hybridization under
conditions of stringency as is well understood by those skilled in the art [as
described in Hames and Higgins (eds.) Nucleic Acid Hybridisation, IRL Press,
Oxford, U.K.]; or by the comparison of sequence similarity between two nucleic
acids or proteins.
As used herein, "essentially similar" refers to DNA sequences that may
involve base changes that do not cause a change in the encoded amino acid, or
which involve base changes which may alter one or more amino acids, but do not
affect the functional properties of the protein encoded by the DNA sequence.
It is
therefore understood that the invention encompasses more than the specific
exemplary sequences. Modifications to the sequence, such as deletions,
insertions, or substitutions in the sequence which produce silent changes that
do
not substantially affect the functional properties of the resulting protein
molecule
are also contemplated. For example, alteration in the gene sequence which
reflect
the degeneracy of the genetic code, or which result in the production of a
chemically equivalent amino acid at a given site, are contemplated; thus, a
codon
for the amino acid alanine, a hydrophobic amino acid, may be substituted by a
codon encoding another less hydrophobic residue, such as glycine, or a more
hydrophobic residue, such as valine, leucine, or isoleucine. Similarly,
changes
which result in substitution of one negatively charged residue for another,
such as
aspartic acid for glutamic acid, or one positively charged residue for
another, such
as lysine for arginine, can also be expected to produce a biologically
equivalent
product. Nucleotide changes which result in alteration of the N-terminal and
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C-terminal portions of the protein molecule would also not be expected to
alter the
activity of the protein. In some cases, it may in fact be desirable to make
mutants
of the sequence in order to study the effect of alteration on the biological
activity
of the protein. Each of the proposed modifications is well within the routine
skill
in the art, as is determination of retention of biological activity of the
encoded
products. Moreover, the skilled artisan recognizes that "essentially similar"
sequences encompassed by this invention are also defined by their ability to
hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65 C), with the
sequences exemplified herein.
"Gene" refers to a nucleic acid fragment that expresses a specific protein,
including regulatory sequences preceding (5' non-coding) and following (3' non-
coding) the coding region. "Native" gene refers to the gene as found in nature
with its own regulatory sequences. "Chimeric" gene refers to a gene comprising
heterogeneous regulatory and coding sequences. "Endogenous" gene refers to the
native gene normally found in its natural location in the genome. A "foreign"
gene refers to a gene not normally found in the host organism but that is
introduced by gene transfer.
"Coding sequence" refers to a DNA sequence that codes for a specific
protein and excludes the non-coding sequences.
"Initiation codon" and "termination codon" refer to a unit of three adjacent
nucleotides in a coding sequence that specifies initiation and chain
termination,
respectively, of protein synthesis (mRNA translation). "Open reading frame"
refers to the amino acid sequence encoded between translation initiation and
termination codons of a coding sequence.
"RNA transcript" refers to the product resulting from RNA polymerase-
catalyzed transcription of a DNA sequence. When the RNA transcript is a
perfect
complementary copy of the DNA sequence, it is referred to as the primary
transcript or it may be a RNA sequence derived from posttranscriptional
processing of the primary transcript. "Messenger RNA (mRNA) refers to RNA
that can be translated into protein by the cell. "cDNA" refers to a double-
stranded
DNA that is complementary to and derived from mRNA. "Sense" RNA refers to
RNA transcript that includes the mRNA. "Antisense RNA" refers to a RNA
transcript that is complementary to all or part of a target primary transcript
or
mRNA and that blocks the expression of a target gene by interfering with the
processing, transport and/or translation of its primary transcript or mRNA.
The
complementarity of an antisense RNA may be with any part of the specific gene
transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence,
introns, or
the coding sequence. In addition, as used herein, antisense RNA may contain
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regions of ribozyme sequences that increase the efficacy of antisense RNA to
block gene expression. "Ribozyme" refers to a catalytic RNA and includes
sequence-specific endoribonucleases.
As used herein, suitable "regulatory sequences" refer to nucleotide
sequences located upstream (:5'), within, and/or downstream (3') to a coding
sequence, which control the transcription and/or expression of the coding
sequences, potentially in conjunction with the protein biosynthetic apparatus
of
the cell. These regulatory sequences include promoters, translation leader
sequences, transcription termination sequences, and polyadenylation sequences.
"Promoter" refers to a DNA sequence in a gene, usually upstream (5') to its
coding sequence, which controls the expression of the coding sequence by
providing the recognition for RNA polymerase and other factors required for
proper transcription. A promoter may also contain DNA sequences that are
involved in the binding of protein factors which control the effectiveness of
transcription initiation in response to physiological or developmental
conditions.
It may also contain enhancer elements.
An "enhancer" is a DNA sequence which can stimulate promoter activity. It
may be an innate element of the promoter or a heterologous element inserted to
enhance the level and/or tissue-specificity of a promoter. "Constitutive
promoters" refers to those that direct gene expression in all tissues and at
all times.
"Organ-specific" or "development-specific" promoters as referred to herein are
those that direct gene! expression almost exclusively in specific organs, such
as
leaves or seeds, or at specific development stages in an organ, such as in
early or
late embryogenesis, respectively.
The term "operably linked" refers to nucleic acid sequences on a single
nucleic acid molecule which are associated so that the function of one is
affected
by the other. For example, a promoter is operably linked with a structure gene
(i.e., a gene encoding aspartokinase that is lysine-insensitive as given
herein)
when it is capable of affecting the expression of that structural gene (i.e.,
that the
structural gene is undler the transcriptional control of the promoter).
The term "expression", as used herein, is intended to mean the production of
the protein product encoded by a gene. More particularly, "expression" refers
to
the transcription and stable accumulation of the sense (mRNA) or antisense RNA
derived from the nucleic acid fragment(s) of the invention that, in
conjunction
with the protein apparatus of the cell, results in altered levels of protein
product.
"Antisense inhibition." refers to the production of antisense RNA transcripts
capable of preventing the expression of the target protein. "Overexpression"
refers to the production of a gene product in transgenic organisms that
exceeds
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levels of production in normal or non-transformed organisms. "Cosuppression"
refers to the expression of a foreign gene which has substantial homology to
an
endogenous gene resulting in the suppression of expression of both the foreign
and the endogenous gene. "Altered levels" refers to the production of gene
product(s) in transgenic organisms in amounts or proportions that differ from
that
of normal or non-transformed organisms.
The "3' non-coding sequences" refers to the DNA sequence portion of a
gene that contains a polyadenylation signal and any other regulatory signal
capable of affecting mRNA processing or gene expression. The polyadenylation
signal is usually characterized by affecting the addition of polyadenylic acid
tracts
to the 3' end of the mRNA precursor.
The "translation leader sequence" refers to that DNA sequence portion of a
gene between the promoter and coding sequence that is transcribed into RNA and
is present in the fully processed mRNA upstream (5') of the translation start
codon. The translation leader sequence may affect processing of the primary
transcript to mRNA, mRNA stability or translation efficiency.
"Mature" protein refers to a post-translationally processed polypeptide
without its targeting signal. "Precursor" protein refers to the primary
product of
translation of mRNA. A "chloroplast targeting signal" is an amino acid
sequence
which is translated in conjunction with a protein and directs it to the
chloroplast.
"Chloroplast transit sequence" refers to a nucleotide sequence that encodes a
chloroplast targeting signal.
"Transformation" herein refers to the transfer of a foreign gene into the
genome of a host organism and its genetically stable inheritance. Examples of
methods of plant transformation include Agrobacterium-mediated transformation
and particle-accelerated or "gene gun" transformation technology.
"Amino acids" herein refer to the naturally occurring L amino acids
(Alanine, Arginine, Aspartic acid, Asparagine, Cystine, Glutamic acid,
Glutamine,
Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Proline,
Phenylalanine, Serine, Threonine, Tryptophan, Tyrosine, and Valine).
"Essential
amino acids" are those amino acids which cannot be synthesized by animals. A
"polypeptide" or "protein" as used herein refers to a molecule composed of
monomers (amino acids) linearly linked by amide bonds (also known as peptide
bonds).
"Synthetic protein" herein refers to a protein consisting of amino acid
sequences that are not known to occur in nature. The amino acid sequence may
be
derived from a consensus of naturally occurring proteins or may be entirely
novel.
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"Primary sequence" refers to the connectivity order of amino acids in a
polypeptide chain without regard to the conformation of the molecule. Primary
sequences are written from the amino terminus to the carboxy terminus of the
polypeptide chain by convention.
"Secondary structure" herein refers to physico-chemically favored regular
backbone arrangements of a polypeptide chain without regard to variations in
side
chain identities or conformations. "Alpha helices" as used herein refer to
right-
handed helices with approximately 3.6 residues per turn of the helix. An
"amphipathic helix" refers herein to a polypeptide in a helical conformation
where
one side of the helix is predominantly hydrophobic and the other side is
predominantly hydrophilic.
"Coiled-coil" herein refers to an aggregate of two parallel right-handed
alpha helices which are wound around each other to form a left-handed
superhelix.
"Salt bridges" as discussed here refer to acid-base pairs of charged amino
acid side chains so arranged in space that an attractive electrostatic
interaction is
maintained between two parts of a polypeptide chain or between one chain and
another.
"Host cell" means the cell that is transformed with the introduced genetic
material.
Isolation of AK Genes
The E. coli IyG gene has been cloned, restriction endonuclease mapped and
sequenced previously [Cassan et al. (1986) J. Biol. Chem. 261:1052-1057]. For
the present invention 'the 1ysC gene was obtained on a bacteriophage lambda
clone
from an ordered library of 3400 overlapping segments of cloned E. coli DNA
constructed by Kohara, Akiyama and Isono [Kohara et al. (1987) Cell
50:595-508]. The E. coli lysC gene encodes the enzyme AKIII, which is
sensitive
to lysine inhibition. Mutations were obtained in the 1ysC gene that cause the
AKIII enzyme to be resistant to lysine.
To determine the molecular basis for lysine-resistance, the sequence of the
wild type lysC gene and three mutant genes were determined. The sequence of
the cloned wild type Inc gene, indicated in SEQ ID NO: 1:, differed from the
published lysC sequence in the coding region at 5 positions.
The sequences of the three mutant 1YsC genes that encoded lysine-
insensitive aspartokinase each differed from the wild type sequence by a
single
nucleotide, resulting in a single amino acid substitution in the protein. One
mutant (M2) had an A substituted for a G at nucleotide 954 of SEQ ID NO:1:
resulting in an isoleucine for methionine substitution in the amino acid
sequence
of AKIII and two mutants (M3 and M4) had identical T for C substitutions at
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nucleotide 1055 of SEQ ID NO:1 resulting in an isoleucine for threonine
substitution.
Other mutations could be generated, either in vivo as described in Example I
or in vitro by site-directed mutagenesis by methods known to those skilled in
the
art, that result in amino acid substitutions for the methionine or threonine
residue
present in the wild type AKIII at these positions. Such mutations would be
expected to result in a lysine-insensitive enzyme. Furthermore, the method
described in Example 1 could be used to easily isolate and characterize as
many
additional mutant lysC genes encoding lysine insensitive AKIII as desired.
A number of other AK genes have been isolated and sequenced. These
include the thrA gene of E. coli (Katinka et al. (1980) Proc. Natl. Acad. Sci.
USA
77:5730-5733], the metL gene of E. coli (Zakin et al. (1983) J. Biol. Chem.
258:3028-3031 ], the HOM3 gene of S. cerevisiae [Rafalski et al. (1988) J.
Biol.
Chem. 263:2146-2151 ]. The thrA gene of E. coli encodes a bifunctional
protein,
AKI-HDHI. The AK activity of this enzyme is insensitive to lysine, but
sensitive
to threonine. The metL gene of E. coli also encodes a bifunctional protein,
AKII-HDHII, and the AK activity of this enzyme is also insensitive to lysine.
The
HOM3 gene of yeast encodes an AK which is insensitive to lysine, but sensitive
to
threonine.
In addition to these genes, several plant genes encoding lysine-insensitive
AK are known. In barley lysine plus threonine-resistant mutants bearing
mutations in two unlinked genes that result in two different lysine-
insensitive AK
isoenzymes have been described [Bright et al. (1982) Nature 299:278-279,
Rognes et al. (1983) Planta 157:32-38, Arruda et al. (1984) Plant Physiol.
76:442-446]. In corn, a lysine plus threonine-resistant cell line had AK
activity
that was less sensitive to lysine inhibition than its parent line [Hibberd et
al.
(1980) Planta 148:183-1871. A subsequently isolated lysine plus threonine-
resistant corn mutant is altered at a different genetic locus and also
produces
lysine-insensitive AK [Diedrick et al. (1990) Theor. Appl. Genet. 79.209-215,
Dotson et al. (1990) Planta 182:546-552]. In tobacco there are two AK enzymes
in leaves, one lysine-sensitive and one threonine-sensitive. A lysine plus
threonine-resistant tobacco mutant that expressed completely lysine-
insensitive
AK has been described [Frankard et al. (1991) Theor. Appl. Genet. 82:273-282].
These plant mutants could serve as sources of genes encoding lysine-
insensitive
AK and used, based on the teachings herein, to increase the accumulation of
lysine and threonine in the seeds of transformed plants.
A partial amino acid sequence of AK from carrot has been reported [Wilson
et al. (1991) Plant Physiol.' 97:1323:1328]. Using this information a set of
16
CA 02280196 2002-09-09
degenerate DNA oligonucleotides could be designed, synthesized and used as
hybridization probes to permit the isolation of the carrot AK gene. Recently
the
carrot AK gene has been isolated and its nucleotide sequence has been
determined
[Matthews et al. (1991) U.S. Patent No. 5,451,516]. This gene can be used as a
heterologous hybridization probe to isolate the genes encoding lysine-
insensitive
AK described above.
High level expression of wild,ty eland
mutant lysC genes in E. coli
To achieve high level expression of the i genes in E. coli, a bacterial
expression vector which employs the bacteriophage 17 RNA polymerase/T7
promoter system [Rosenberg et al. (1987) Gene .56:125-135] was used. The
expression vector and lvsC gene were modified as described in Example 2 to
construct a LC expression vector. For expression of the mutant j genes (M2,
M3 and M4), the wild type 1vsC gene was replaced with the mutant genes as
described in Example 2.
For high level expression, each of the expression vectors was transformed
into E. coli strain B121(DE3) [Studier et al. (1986).. Mol. Biol. 189:113-
130].
Cultures were grown, expression was induced, cells were collected, and
extracts
were prepared as described in Example 2. Supernatant and pellet fractions of
extracts from uninduced and induced cultures were analyzed by SDS
polyacrylamide gel electrophoresis and by AK enzyme assays as described in
Example 2. The major protein visible by Coomassie blue staining in the
supernatant and pellet fractions of induced cultures was AKIII. About 80% of
the
AKIII protein was in the supernatant and AKIII represented 10-20% of the total
E. coli protein in the extract.
Approximately 80% of the AKIII enzyme activity was in the supernatant
fraction. The specific activity of wild type and mutant crude extracts was
5-7 moles product per minute per milligram total protein. Wild type AKIII was
sensitive to the presence of L-lysine in the assay. Fifty percent inhibition
was
found at a concentration of about 0.4 mM and 90 percent inhibition at about
0.1 mM. In contrast, mutants AKIII-M2, M3 and M4 were not inhibited at all by
l5 mM L-lysine.
Wild type AKIII protein was purified from the supernatant of an induced
culture as described in Example 2. Rabbit antibodies were raised against the
purified AKIII protein.
Many other microbial expression vectors have been described in the
literature. One skilled in the art could make use of any of these to construct
lysC
expression vectors. These 1vsC expression vectors could then be introduced
into
17
CA 02280196 2002-09-09
appropriate microorganisms via transformation to provide a system for high
level
expression of AKUI.
Isolation of DHDPS genes
The E. coli danA gene (ecodanA) has been cloned, restriction endonuclease
mapped and sequenced previously [Richaud et al. (1986) J. Bacteriol.
166:297-300]. For the present invention the d> gene was obtained on a
bacteriophage lambda clone from an ordered library of 3400 overlapping
segments of cloned E. coll. DNA constructed by Kohara, Akiyama and Isono
[Kohara et al. (1987) Cell 50:595-508]. The ecodanA gene encodes a DHDPS
enzyme that is sensitive to lysine inhibition. However, it is about 20-fold
less
sensitive to inhibition by lysine than a typical plant DHDPS, e.g., wheat germ
DHDPS.
The Corynebacterium daDA gene (cordapA) was isolated from genomic
DNA from ATCC strain 13032 using polymerase chain reaction (PCR). The
nucleotide sequence of the Corynebacterium danA gene has been published
[Bonnassie et al. (1990) Nucleic Acids Res, 18:6421). From the sequence it was
possible to design oligonucleotide primers for polymerase chain reaction (PCR)
that would allow amplification of a DNA fragment containing the gene, and at
the
same time add unique restriction endonuclease sites at the start codon and
just past
the stop codon of the gene to facilitate further constructions involving the
gene.
The details of the isolation of the cordanA gene are presented in Example 3.
The
corddapA gene encodes a DHDPS enzyme that is insensitive to lysine inhibition.
In addition to introducing a restriction endonuclease site at the translation
start codon, the PCR primers also changed the second codon of the cordgpA gene
from AGC coding for serine to GCT coding for alanine. Several cloned DNA
fragments that expressed active, lysine-insensitive DHDPS were isolated,
indicating that the second codon amino acid substitution did not affect enzyme
activity.
The PCR-generated Corynebacterium davA gene was subcloned into the
phagemid vector pGEM -9Zf(-) from Promega, and single-stranded DNA was
generated and sequenced (SEQ ID NO:6). Aside from the differences in the
second codon already mentioned, the sequence-matched the published sequence
except at two positions, nucleotides 798 and 799. In the published sequence
these
are TC, while in the gene shown in SEQ ID NO:6 they are CT. This change
results in an amino acid substitution of leucine for serine. The reason for
this
difference is not known. The difference has no apparent effect on DHDPS
enzyme activity.
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The isolation of other genes encoding DHDPS has been described in the
literature. A cDNA encoding DHDPS from wheat [Kaneko et al. (1990) J. Biol.
Chem. 265:17451-17455], and a cDNA encoding DHDPS from corn [Frisch et al.
(1991) Mol. Gen. Genet. 228:287-293] are two examples. These genes encode
wild type lysine-sensitive DHDPS enzymes. However, Negrutui et al. [(1984)
Theor. App!. Genet. 68:11-20], obtained two AEC-resistant tobacco mutants in
which DHDPS activity was less sensitive to lysine inhibition than the wild
type
enzyme. These genes could be isolated using the methods already described for
isolating the wheat or corn genes or, alternatively, by using the wheat or
corn
genes as heterologous hybridization probes.
Still other genes encoding DHDPS could be isolated by one skilled in the art
by using either the ecodapA gene, the cordapA gene, or either of the plant
DHDPS
genes as DNA hybridization probes. Alternatively, other genes encoding DHDPS
could be isolated by functional complementation of an E. coli dapA mutant, as
was done to isolate the cordapA gene [Yeh et al. (1988) Mol. Gen. Genet.
212:105-111] and the corn DHDPS gene.
High level expression of ecodapA and
cordapA genes in E. coli
To achieve high level expression of the ecodapA and cordapA genes in
E. coli, a bacterial expression vector which employs the bacteriophage T7 RNA
polymerase/T7 promoter system [Rosenberg et al. (1987) Gene 56:127-135] was
used. The vector and dapA genes were modified as described below to construct
ecodapA and cordapA, expression vectors.
For high level expression each of the expression vectors was transformed
into E. coli strain BL2.1(DE3) [Studier et al. (1986) J. Mol. Biol. 189:113-
130].
Cultures were grown, expression was induced, cells were collected, and
extracts
were prepared as described in Example 4. Supernatant and pellet fractions of
extracts from uninduced and induced cultures were analyzed by SDS
polyacrylamide gel electrophoresis and by DHDPS enzyme assays as described in
Example 4. The major protein visible by Coomassie blue staining in the
supernatant and pellet fractions of both induced cultures had a molecular
weight
of 32-34 kd, the expected size for DHDPS. Even in the uninduced cultures this
protein was the most prominent protein produced.
In the induced culture with the ecodapA gene about 80% of the DHDPS
protein was in the supernatant and DHDPS represented 10-20% of the total
protein in the extract. In the induced culture with the cordapA gene more than
50% of the DHDPS protein was in the pellet fraction. The pellet fractions in
both
cases were 90-95% pure DHDPS, with no other single protein present in
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significant amounts. Thus, these fractions were pure enough for use in the
generation of rabbit antibodies.
The specific activity of E. coli DHDPS in the supernatant fraction of
induced extracts was about 50 OD540 units per milligram protein. E. coli DHDPS
was sensitive to the presence of L-lysine in the assay. Fifty percent
inhibition was
found at a concentration of about 0.5 mM. For Corynebacterium DHDPS,
enzyme activity was measured in the supernatant fraction of uninduced
extracts,
rather than induced extracts. Enzyme activity was about 4 OD530 units per
minute
per milligram protein. In contrast to E. coli DHDPS, Corynebacterium DHDPS
was not inhibited at all by L-lysine, even at a concentration of 70 mM.
Many other microbial expression vectors have been described in the
literature. One skilled in the art could make use of any of these to construct
ecodapA or cordapA expression vectors. These expression vectors could then be
introduced into appropriate microorganisms via transformation to provide a
system for high level expression of DHDPS.
Excretion of amino acids by E. coli expressing
high levels of DHDPS and/or AKIII
The E. coli expression cassettes were inserted into expression vectors and
then transformed into E. coli strain BL21 (DE3) [Studier et al. (1986) J. Mol.
Biol.
189:113-130] to induce E. coli to produce and excrete amino acids. Details of
the
procedures used and results are presented in Example 5.
Other microbial expression vectors known to those skilled in the art could
be used to make and combine expression cassettes for the 1vsC and dapA genes.
These expression vectors could then be introduced into appropriate
microorganisms via transformation to provide alternative systems for
production
and excretion of lysine, threonine and methionine.
Construction of Chimeric Genes for Expression in Plants
A preferred class of heterologous hosts for the expression of the chimeric
genes of this invention are eukaryotic hosts, particularly the cells of higher
plants.
Preferred among the higher plants and the seeds derived from them are soybean,
rapeseed (Brassica napus, B. campestris), sunflower (Helianthus annus), cotton
(Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago
sativa), wheat (Triticum sp), barley (Hordeum vulgare), oats (Avena sativa,
L),
sorghum (Sorghum bicolor , rice (Oryza sativa), and forage grasses. Expression
in plants will use regulatory sequences functional in such plants. The
expression
of foreign genes in plants is well-established [De Blaere et al. (1987) Meth.
Enzymol. 143:277-291 ]. Proper level of expression of the different chimeric
genes of this invention in plant cells may be achieved through the use of many
CA 02280196 1999-08-05
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different promoters. Such chimeric genes can be transferred into host plants
either
together in a single expression vector or sequentially using more than one
vector.
The origin of promoter chosen to drive the expression of the coding
sequence is not critical as long as it has sufficient transcriptional activity
to
accomplish the invention by expressing translatable mRNA or antisense RNA in
the desired host tissue. Preferred promoters for expression in all plant
organs, and
especially for expression in leaves include those directing the 19S and 35S
transcripts in Cauliflower mosaic virus [Odell et al.(1985) Nature 313:810-
812;
Hull et al. (1987) Virology 86:482-493], small subunit of ribulose
1,5-bisphosphate carboxylase [Morelli et al.(1985) Nature 315:200; Broglie et
al.
(1984) Science 224:838; Hererra-Estrella et al.(1984) Nature 310:115; Coruzzi
et al.(1984) EMBO J. 3:1671; Faciotti et al.(1985) Bio/Technology 3:241],
maize
zein protein [Matzke et al.(1984) EMBO J. 3:1525], and chlorophyll a/b binding
protein [Lampa et al.(l986) Nature 316:750-752].
Depending upon the application, it may be desirable to select promoters that
are specific for expression in one or more organs of the plant. Examples
include
the light-inducible promoters of the small subunit of ribulose 1,5-
bisphosphate
carboxylase, if the expression is desired in photosynthetic organs, or
promoters
active specifically in seeds.
Preferred promoters are those that allow expression specifically in seeds.
This may be especially useful,, since seeds are the primary source of
vegetable
amino acids and also since seed-specific expression will avoid any potential
deleterious effect in non-seed organs. Examples of seed-specific promoters
include, but are not limited to, the promoters of seed storage proteins. The
seed
storage proteins are strictly regulated, being expressed almost exclusively in
seeds
in a highly organ-specific and stage-specific manner [Higgins et al.(1984)
Ann.
Rev. Plant Physiol. 35:191-221; Goldberg et al.(1989) Cell 56:149-160;
Thompson et al. (1989) BioEssays 10:108-113]. Moreover, different seed storage
proteins may be expressed at different stages of seed development.
There are currently numerous examples for seed-specific expression of seed
storage protein genes in transgenic dicotyledonous plants. These include genes
from dicotyledonous plants for bean P-phaseolin [Sengupta-Goplalan et al.
(1985)
Proc. Natl. Acad. Sci. USA 82:3320-3324; Hoffman et al. (1988) Plant Mol.
Biol.
11:717-729], bean lectin [Voelker et al. (1987) EMBOJ. 6: 3571-3577], soybean
lectin [Okamuro et al. (1986) .Prot. Natl. Acad. Sci. USA 83:8240-8244],
soybean
kunitz trypsin inhibitor [Perez-Grau et al. (1989) Plant Cell 1:095-11091,
soybean
(3-conglycinin [Beachy et al. (1985) EMBO J 4:3047-3 053; Barker et al. (1988)
Proc. Natl. Acad. Sci. USA 85:458-462; Chen et al. (1988) EMBO 1 7:297-302;
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Chen et al. (1989) Dev. Genet. 10:112-122; Naito et al. (1988) Plant Mol.
Biol.
11:109-123], pea vicilin [Higgins et al. (1988) Plant Mol. Biol. 11:683-695],
pea
convicilin [Newbigin et al. (1990) Planta 180:461], pea legumin [Shirsat et
al.
(1989) Mol. Gen. Genetics 215:326]; rapeseed napin [Radke et al. (1988) Theor.
Appl. Genet. 75:685-694] as well as genes from monocotyledonous plants such as
for maize 15 kD zein [Hoffman et al. (1987) EMBO J. 6:3213-3221; Schernthaner
et al. (1988) EMBO J. 7:1249-1253; Williamson et al. (1988) Plant Physiol.
88:1002-1007], barley 0-hordein [Marris et al. (1988) Plant Mol. Biol.
10:359-366] and wheat glutenin [Colot et al. (1987) EMBOJ 6:3559-3564].
Moreover, promoters of seed-specific genes, operably linked to heterologous
coding sequences in chimeric gene constructs, also maintain their temporal and
spatial expression pattern in transgenic plants. Such examples include
Arabidopsis thaliana 2S seed storage protein gene promoter to express
enkephalin
peptides in Arabidopsis and B. napus seeds [Vandekerckhove et al. (1989)
Bio/Technology 7:929-932], bean lectin and bean (3-phaseolin promoters to
express luciferase [Riggs et al. (1989) Plant Sci. 63:47-57], and wheat
glutenin
promoters to express chloramphenicol acetyl transferase [Colot et al. (1987)
EMBO J 6:3559-3564].
Of particular use in the expression of the nucleic acid fragment of the
invention will be the heterologous promoters from several extensively-
characterized soybean seed storage protein genes such as those for the Kunitz
trypsin inhibitor [Jofuku et al. (1989) Plant Cell 1: 1079-1093; Perez-Grau et
al.
(1989) Plant Cell 1:1095-1109], glycinin [Nielson et al. (1989) Plant Cell
1:313-328], 0-conglycinin [Harada et al. (1989) Plant Cell 1:415-4251.
Promoters
of genes for a'- and [3-subunits of soybean 0-conglycinin storage protein will
be
particularly useful in expressing mRNAs or antisense RNAs in the cotyledons at
mid- to late-stages of soybean seed development [Beachy et al. (1985) EMBO J.
4:3047-3053; Barker et al. (1988) Proc. Natl. Acad. Sci. USA 85:458-462; Chen
et al. (1988) EMBO J. 7:297-302; Chen et al. (1989) Dev. Genet. 10:112-122;
Naito et al. (1988) Plant Mol. Biol. 11:109-123] in transgenic plants, since:
a) there is very little position effect on their expression in transgenic
seeds, and
b) the two promoters show different temporal regulation: the promoter for the
a'-subunit gene is expressed a few days before that for the (3-subunit gene.
Also of particular use in the expression of the nucleic acid fragments of the
invention will be the heterologous promoters from several extensively
characterized corn seed storage protein genes such as endosperm-specific
promoters from the 10 kD zein [Kirihara et al. (1988) Gene 71:359-370], the
27 kD zein [Prat et al. (1987) Gene 52:51-49; Gallardo et al. (1988) Plant
Sci.
22
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54:211-281], and the 19 kD zein [Marks et al. (1985) J. Biol. Chem.
260:16451-16459]. The relative transcriptional activities of these promoters
in
corn have been reported [Kodrzyck et al. (1989) Plant Cell 1: 105-114]
providing
a basis for choosing a promoter for use in chimeric gene constructs for corn.
For
expression in corn embryos, the strong embryo-specific promoter from the GLB 1
gene [Kriz (1989) Biochemical Genetics 27:239-251, Wallace et al. (1991) Plant
Physiol. 95:973-975] can be used.
It is envisioned that the introduction of enhancers or enhancer-like elements
into other promoter constructs will also provide increased levels of primary
transcription to accomplish the invention. These would include viral enhancers
such as that found in the 35S promoter [Odell et al. (1988) Plant Mol. Biol.
10:263-272], enhancers from the opine genes [Fromm et al. (1989) Plant Cell
1:977-984], or enhancers from any other source that result in increased
transcription when placed into a promoter operably linked to the nucleic acid
fragment of the invention.
Of particular importance is the DNA sequence element isolated from the
gene for the a'-subunit of (3-conglycinin that can confer 40-fold seed-
specific
enhancement to a constitutive promoter [Chen et al. (1988) EMBO J. 7:297-302;
Chen et al. (1989) Dev. Genet. 10:112-122]. One skilled in the art can readily
isolate this element and insert it within the promoter region of any gene in
order to
obtain seed-specific enhanced expression with the promoter in transgenic
plants.
Insertion of such an element in any seed-specific gene that is expressed at
different times than the [3-conglycinin gene will result in expression in
transgenic
plants for a longer period during seed development.
Any 3' non-coding region capable of providing a polyadenylation signal and
other regulatory sequences that may be required for the proper expression can
be
used to accomplish the invention. This would include the 3' end from any
storage
protein such as the 3' end of the bean phaseolin gene, the 3' end of the
soybean
P-conglycinin gene, the 3' end from viral genes such as the 3' end of the 35S
or the
19S cauliflower mosaic virus transcripts, the 3' end from the opine synthesis
genes, the 3' ends of iibulose '1,5-bisphosphate carboxylase or chlorophyll
a/b
binding protein, or 3' end sequences from any source such that the sequence
employed provides the necessary regulatory information within its nucleic acid
sequence to result in the proper expression of the promoter/coding region
combination to which it is operably linked. There are numerous examples in the
art that teach the usefulness of different 3' non-coding regions [for example,
see
Ingelbrecht et al. (1989) Plant Cell1:671-680].
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DNA sequences coding for intracellular localization sequences may be
added to the 1ysC and dapA coding sequence if required for the proper
expression
of the proteins to accomplish the invention. Plant amino acid biosynthetic
enzymes are known to be localized in the chloroplasts and therefore are
synthesized with a chloroplast targeting signal. Bacterial proteins such as
DHDPS
and AKIII have no such signal. A chloroplast transit sequence could,
therefore, be
fused to the dapA and ysC coding sequences. Preferred chloroplast transit
sequences are those of the small subunit of ribulose 1,5-bisphosphate
carboxylase,
e.g. from soybean [Berry-Lowe et al. (1982) J. Mol. Appl. Genet. 1:483-498]
for
use in dicotyledonous plants and from corn [Lebrun et al. (1987) Nucleic Acids
Res. 15:4360] for use in monocotyledonous plants.
Introduction of Chimeric Genes into Plants
Various methods of introducing a DNA sequence (i.e., of transforming) into
eukaryotic cells of higher plants are available to those skilled in the art
(see EPO
publications 0 295 959 A2 and 0 138 341 Al). Such methods include those based
on transformation vectors based on the Ti and Ri plasmids of Agrobacterium
spp.
It is particularly preferred to use the binary type of these vectors. Ti-
derived
vectors transform a wide variety of higher plants, including monocotyledonous
and dicotyledonous plants, such as soybean, cotton and rape [Pacciotti et al.
(1985) Bio/Technology 3:241; Byrne et at. (1987) Plant Cell, Tissue and Organ
Culture 8:3; Sukhapinda et al. (1987) Plant Mol. Biol. 8:209-216; Lorz et al.
(1985) Mol. Gen. Genet. 199:178; Potrykus (1985) Mol. Gen. Genet. 199:183].
For introduction into plants the chimeric genes of the invention can be
inserted into binary vectors as described in Examples 7-12 and 14-16. The
vectors
are part of a binary Ti plasmid vector system [Bevan, (1984) Nucl. Acids. Res.
12:8711-8720] of Agrobacterium tumefaciens.
Other transformation methods are available to those skilled in the art, such
as direct uptake of foreign DNA constructs [see EPO publication 0 295 959 A2],
techniques of electroporation [see Fromm et al. (1986) Nature (London)
319:7911
or high-velocity ballistic bombardment with metal particles coated with the
nucleic acid constructs [see Kline et al. (1987) Nature (London) 32 7:70, and
see
U.S. Pat. No. 4,945,050]. Once transformed, the cells can be regenerated by
those
skilled in the art.
Of particular relevance are the recently described methods to transform
foreign genes into commercially important crops, such as rapeseed [see De
Block
et al. (1989) Plant Physiol. 91:694-701], sunflower [Everett et al. (1987)
Bio/Technology 5:1201], soybean [McCabe et al. (1988) Bio/Technology 6:923;
Hinchee et al. (1988) Bio/Technology 6:915; Chee et al. (1989) Plant Physiol.
24
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91:1212-1218; Christou et al. (1989) Proc. Natl. Acad. Sci USA 86:7500-7504;
EPO Publication 0 301 749 A2], and corn [Gordon-Kamm et al. (1990) Plant Cell
2:603-618; Fromm et al. (1990) Biotechnology 8:833-8391.
For introduction into plants by high-velocity ballistic bombardment, the
chimeric genes of the invention can be inserted into suitable vectors as
described
in Example 6. Transformed plants can be obtained as described in
Examples 17-19.
Expression of lysC and dapA Chimeric Genes
in Tobacco Plants
To assay for expression of the chimeric genes in leaves or seeds of the
transformed plants, the AKIII or DHDPS proteins can be detected and
quantitated
enzymatically and/or immunologically by methods known to those skilled in the
art. In this way lines producing high levels of expressed protein can be
easily
identified.
In order to measure the free amino acid composition of the leaves, free
amino acids can be extracted by various methods including those as described
in
Example 7. To measure the free or total amino acid composition of seeds,
extracts
can be prepared by various methods including those as described in Example 8.
There was no significant effect of expression of AKIII or AKIII-M4 (with a
chloroplast targeting signal) on the free lysine or threonine (or any other
amino
acid) levels in the leaves (see Table 2 in Example 7). Since AKIII-M4 is
insensitive to feedback inhibition by any of the end-products of the pathway,
this
indicates that control must be exerted at other steps in the biosynthetic
pathway in
leaves.
In contrast, expression of the AKIII or AKIII-M4 (with a chloroplast
targeting signal) in the seeds resulted in 2 to 4-fold or 4 to 23-fold
increases,
respectively, in the level of free threonine in the seeds compared to
untransformed
plants and 2 to 3-fold increases in the level of free lysine in some cases
(Table 3,
Example 8). There was a good correlation between transformants expressing
higher levels of AKIII or AKIII-M4 protein and those having higher levels of
free
threonine, but this was not the case for lysine. The relatively small
increases of
free threonine or lysine achieved with the AKIII protein were not sufficient
to
yield detectable increases compared to untransformed plants, in the levels of
total
threonine or lysine in the seeds. The larger increases of free threonine
achieved
via expression of the AKIII-M4 protein were sufficient to yield detectable
increases, compared to seeds from untransformed plants, in the levels of total
threonine in the seeds. Sixteen to twenty-five percent increases in total
threonine
content of the seeds were observed. The lines that showed increased total
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threonine were the same ones that showed the highest levels of increase in
free
threonine and high expression of the AKIII-M4 protein.
The above teachings show that amino acid biosynthesis takes place in seeds
and can be modulated by the expression of foreign genes encoding amino acid
biosynthetic enzymes. Furthermore, they show that control of an amino acid
biosynthetic pathway can differ markedly from one plant organ to another, e.g.
seeds and leaves. The importance of this observation is emphasized upon
considering the different effects of expressing a foreign DHDPS in leaves and
seeds described below. It can be concluded that threonine biosynthesis in
seeds is
controlled primarily via end-product inhibition of AK. Therefore, threonine
accumulation in the seeds of plants can be increased by expression of a gene,
introduced via transformation, that encodes AK which is insensitive to lysine
inhibition and which is localized in the chloroplast.
The above teachings also demonstrate that transformed plants which express
higher levels of the introduced enzyme in seeds accumulate higher levels of
free
threonine in seeds. Furthermore, the teachings demonstrate that transformed
plants which express a lysine-insensitive AK in seeds accumulate higher levels
of
free threonine in seeds than do transformed plants which express similar
levels of
a lysine-sensitive AK. To achieve commercially valuable increases in free
threonine, a lysine-insensitive AK is preferred.
These teachings indicate that the level of free lysine in seeds controls the
accumulation of another aspartate-derived amino acid, threonine, through end-
product inhibition of AK. In order to accumulate high levels of free lysine
itself,
it will be necessary to bypass lysine inhibition of AK via expression of a
lysine-
insensitive AK.
Expression of active E. coli DHDPS enzyme was achieved in both young
and mature leaves of the transformed tobacco plants (Table 4, Example 9). High
levels of free lysine, 50 to 100-fold higher than normal tobacco plants,
accumulated in the young leaves of the plants expressing the enzyme with a
chloroplast targeting signal, but not without such a targeting signal.
However, a
much smaller accumulation of free lysine (2 to 8-fold) was seen in the larger
leaves. Experiments that measure lysine in the phloem suggest that lysine is
exported from the large leaves. This exported lysine may contribute to the
accumulation of lysine in the small growing leaves, which are known to take
up,
rather than export nutrients. No effect on the free lysine levels in the seeds
of
these plants was observed even though E. coli DHDPS enzyme was expressed in
the seeds as well as the leaves.
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High level seed-specific expression of E. coli DHDPS enzyme, either with
or without a chloroplast targeting signal, had no effect on the total, or
free, lysine
or threonine (or any other amino acid) composition of the seeds in any
transformed line (Table 5, Example 10). These results demonstrate that
expression in seeds of a DHD13S enzyme that is substantially insensitive to
lysine
inhibition is not sufficient to lead to increased production or accumulation
of free
lysine.
These teachings, from transformants expressing the E. coli DHDPS enzyme
indicate that lysine biosynthesis in leaves is controlled primarily via end-
product
inhibition of DHDPS,, while in seeds there must be at least one additional
point of
control in the pathway. The teachings from transformants expressing the E.
coli
AKIII and AKIII-M4 enzymes indicate that the level of free lysine in seeds
controls the accumulation of all aspartate-derived amino acids through end-
product inhibition of AK. AK is therefore an additional control point.
To achieve simultaneous, high level expression of both E. coli DHDPS and
AKIII-M4 in leaves and seeds, plants that express each of the genes could be
crossed and hybrids that express both could be selected. Another method would
be to construct vectors that contain both genes on the same DNA fragment and
introduce the linked genes into plants via transformation. This is preferred
because the genes would remain linked throughout subsequent plant breeding
efforts. Representative vectors carrying both genes on the same DNA fragment
are described in Examples 11, 12, 15, 16, 18, 19, and 25.
Tobacco plants transformed with a vector carrying both E. coli DHDPS and
AKIII-M4 genes linked to the 35S promoter are described in Example 11. In
transformants that express little or no AKIII-M4, the level of expression of
E. coli
DHDPS determines the level of lysine accumulation in leaves (Example 11,
Table 6). However, in transformants that express both AKIII-M4 and E. coli
DHDPS, the level of expression of each protein plays a role in controlling the
level of lysine accumulation. Transformed lines that express DHDPS at
comparable levels accumulate more lysine when AKIII-M4 is also expressed
(Table 6, compare lines 564-18A, 564-56A, 564-36E, 564-55B, and 564-47A).
Thus, expression of a lysine-insensitive AK increases lysine accumulation in
leaves when expressed in concert with a DHDPS enzyme that is 20-fold less
sensitive to lysine than the endogenous plant enzyme.
These leaf results, taken together with the seed results derived from
expressing E. coli AEJII-M4 and E. coli DHDPS separately in seeds, suggest
that
simultaneous expression of both E. coli AKIII-M4 and E. coli DHDPS in seeds
would lead to increased accumulation of free lysine and would also lead to an
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increased accumulation of free threonine. Tobacco plants transformed with a
vector carrying both E. coli DHDPS and AKIII-M4 genes linked to the phaseolin
promoter are described in Example 12. There is an increased accumulation of
free
lysine and free threonine in these plants. The increased level of free
threonine was
4-fold over normal seeds, rather than the 20-fold increase seen in seeds
expressing
AKIII-M4 alone. The reduction in accumulation of free threonine indicates that
pathway intermediates are being diverted down the lysine branch of the
biosynthetic pathway. The increased level of free lysine was 2-fold over
normal
seeds (or seeds expressing E. coli DHDPS alone). However, the lysine increase
in
seeds is not equivalent to the 100-fold increase seen in leaves.
The E. coli DHDPS enzyme is less sensitive to lysine inhibition than plant
DHDPS, but is still inhibited by lysine. The above teachings on the AK
proteins
indicate that expression of a completely lysine-insensitive enzyme can lead to
a
much greater accumulation of the aspartate pathway end-product threonine than
expression of an enzyme which, while less sensitive than the plant enzyme, is
still
inhibited by lysine. Therefore vectors carrying both Corynebacterium DHDPS
and AKIII-M4 genes linked to the seed-specific promoters were constructed as
described in Examples 15 and 19. Tobacco plants transformed with vectors
carrying both Corynebacterium DHDPS and AKIII-M4 genes linked to seed-
specific promoters are described in Example 15. As shown in Table 9, these
plants did not show a greater accumulation of free lysine in seeds than
previously
described plants expressing the E. coli DHDPS enzyme in concert with the
lysine-
insensitive AK. In hindsight this result can be explained by the fact that
lysine
accumulation in seeds never reached a level high enough to inhibit the E. coli
DHDPS, so replacement of this enzyme with lysine-insensitive Corynebacterium
DHDPS had no effect.
In transformed lines expressing high levels of E. coli AKIII-M4 and E. coli
DHDPS or Corynebacterium DHDPS, it was possible to detect substantial
amounts of a-aminoadipic acid in seeds. This compound is thought to be an
intermediate in the catabolism of lysine in cereal seeds, but is normally
detected
only via radioactive tracer experiments due to its low level of accumulation.
The
discovery of high levels of this intermediate, comparable to levels of free
amino
acids, indicates that a large amount of lysine is being produced in the seeds
of
these transformed lines and is entering the catabolic pathway. The build-up of
a-aminoadipic acid was not observed in transformants expressing only E. coli
DHDPS or only AKIII-M4 in seeds. These results show that it is necessary to
express both enzymes simultaneously to produce high levels of free lysine in
seeds. To accumulate high levels of free lysine it may also be necessary to
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prevent lysine catabolism. Alternatively, it may be desirable to convert the
high
levels of lysine produced into a form that is insensitive to breakdown, e.g.
by
incorporating it into a di-, tri- or oligopeptide, or a lysine-rich storage
protein.
Ex cession of lysC and dapA Chimeric Genes
in Rapeseed and Soybean Plants
To analyze for expression of the chimeric lysC and dapA genes in seeds of
transformed rapeseed and soybean and to determine the consequences of
expression on the amino acid content in the seeds, a seed meal can be prepared
as
described in Examples 16 or 119 or by any other suitable method. The seed meal
can be partially or completely defatted, via hexane extraction for example, if
desired. Protein extracts can be prepared from the meal and analyzed for AK
and/or DHDPS enzyme activity. Alternatively the presence of the AK and/or
DHDPS protein can be tested for immunologically by methods well-known to
those skilled in the art. To measure free amino acid composition of the seeds,
free
amino acids can be extracted from the meal and analyzed by methods known to
those skilled in the ail (see Examples 8, 16 and 19 for suitable procedures).
All of the rapeseed transformants obtained from a vector carrying the
cordayA gene expressed the C'orynebacterium DHDPS protein, and six of eight
transformants obtained from a vector carrying the lysC-M4 gene expressed the
AKIII-M4 protein (Example 1.6, Table 12). Thus it is straightforward to
express
these proteins in oilseed rape seeds. Trsformants expressing DHDPS protein
showed a greater than 100-fold increase in free lysine level in their seeds.
There
was a good correlation between transformants expressing higher levels of DHDPS
protein and those having higher levels of free lysine. One transformant that
expressed AKIII-M4 in the absence of Corynebacteria DHDPS showed a 5-fold
increase in the level of free threonine in the seeds. Concomitant expression
of
both enzymes resulted in accumulation of high levels of free lysine, but not
threonine.
A high level of a-aminoadipic acid, indicative of lysine catabolism, was
observed in many of the transformed lines, especially lines expressing the
highest
levels of DHDPS and AKIII protein. Thus, prevention of lysine catabolism by
inactivation of lysine ketoglularate reductase should further increase the
accumulation of free lysine in the seeds. Alternatively, incorporation of
lysine
into a peptide or lysine-rich protein would prevent catabolism and lead to an
increase in the accumulation of lysine in the seeds.
To measure the total amino acid composition of mature rapeseed seeds,
defatted meal was analyzed as described in Example 16. Relative amino acid
levels in the seeds were compared as percentages of lysine to total amino
acids.
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Seeds with a 5-100% increase in the lysine level, compared to the
untransformed
control, were observed. The transformant with the highest lysine content
expressed high levels of both E. coli AKIII-M4 and Corynebacterium DHDPS. In
this transformant lysine makes up about 13% of the total seed amino acids,
considerably higher than any previously known rapeseed seed.
Six of seven soybean transformants expressed the DHDPS protein. In the
six transformants that expressed DHDPS, there was excellent correlation
between
expression of GUS and DHDPS in individual seeds. Therefore, the GUS and
DHDPS genes are integrated at the same site in the soybean genome. Four of
seven transformants expressed the AKIII protein, and again there was excellent
correlation between expression of AKIII, GUS and DHDPS in individual seeds.
Thus, in these four transformants the GUS, AKIII and DHDPS genes are
integrated at the same site in the soybean genome.
Soybean transformants expressing Corynebacteria DHDPS alone and in
concert with E. coli AKIII-M4 accumulated high levels of free lysine in their
seeds. A high level of saccharopine, the first metabolic product of lysine
catabolism, was also observed in seeds that contained high levels of lysine.
Lesser amounts of a-amino adipic acid were also observed. Thus, prevention of
lysine catabolism by inactivation of lysine ketoglutarate reductase should
further
increase the accumulation of free lysine in the soybean seeds. Alternatively,
incorporation of lysine into a peptide or lysine-rich protein would prevent
catabolism and lead to an increase in the accumulation of lysine in the
soybean
seeds.
Analyses of free lysine levels in individual seeds from transformants in
which the transgenes segregated as a single locus revealed that the increase
in free
lysine level was significantly higher in about one-fourth of the seeds. Since
one-
fourth of the seeds are expected to be homozygous for the transgene, it is
likely
that the higher lysine seeds are the homozygotes. Furthermore, this indicates
that
the level of increase in free lysine is dependent upon the transgene copy
number.
Therefore, lysine levels could be further increased by making hybrids of two
different transformants, and obtaining progeny that are homozygous at both
transgene loci.
The soybean seeds expressing Corynebacteria DHDPS showed substantial
increases in accumulation of total seed lysine. Seeds with a 5-35% increase in
total lysine content, compared to the untransformed control, were observed. In
these seeds lysine makes up 7.5-7.7% of the total seed amino acids.
Soybean seeds expressing Corynebacteria DHDPS in concert with E. coli
AKIII-M4 showed much greater accumulation of total seed lysine than those
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expressing Corynebacteria DHDPS alone. Seeds with a more than four-fold
increase in total lysine content were observed. In these seeds lysine makes up
20-25% of the total seed amino acids, considerably higher than any previously
known soybean seed.
Expression of lysC and dapA Chimeric Genes
in Corn Plants
Corn plants regenerated from transformed callus can be analyzed for the
presence of the intact 1ysC and dapA transgenes via Southern blot or PCR.
Plants
carrying the genes are either selfed or outcrossed to an elite line to
generate F 1
seeds. Six to eight seeds are pooled and assayed for expression of the
Corynebacterium DHDPS protein and the E. coli AKIII-M4 protein by western
blot analysis. The free amino acid composition and total amino acid
composition
of the seeds are determined as described above.
Expression of the Corynebacterium DHDPS protein, and/or the E. coli
AKIII-M4 protein can be obtained in the embryo of the seed using regulatory
sequences active in the embryo, preferably derived from the globulin 1 gene,
or in
the endosperm using regulatory sequences active in the endosperm, preferably
derived from the glutelin 2 gene or the 10 kD zein gene (see Example 26 for
details). Free lysine levels in the seeds is increased from about 1.4% of free
amino acids in control seeds to 15-27% in seeds of transformants expressing
Corynebacterium DHDPS alone from the globulin 1 promoter. The increased free
lysine was localized to the embryo in seeds expressing Corynebacterium DHDPS
from the globulin 1 promoter.
The large increases in free lysine result in significant increases in the
total
seed lysine content. Total lysine levels can be increased at least 130% in
seeds
expressing Corynebacterium DHDPS from the globulin 1 promoter. Greater
increases in free lysine levels can be achieved by expressing E. coli AKIII-M4
protein from the globulin I promoter in concert with Corynebacterium DHDPS.
Lysine catabolism is expected to be much greater in the corn endosperm
than the embryo. Thus, to achieve significant lysine increases in the
endosperm it
is preferable to express both Corynebacterium DHDPS and the E. coli AKIII-M4
in the endosperm and to reduce lysine catabolism by reducing the level of
lysine
ketoglutarate reductase as described below.
Isolation of a Plant
Lysine Ketoglutarate Reductase Gene
It may be desirable to prevent lysine catabolism in order to accumulate
higher levels of free lysine and to prevent accumulation of lysine breakdown
products such as saccharopine and a-amino adipic acid. Evidence indicates that
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lysine is catabolized in plants via the saccharopine pathway. The first
enzymatic
evidence for the existence of this pathway was the detection of lysine
ketoglutarate reductase (LKR) activity in immature endosperm of developing
maize seeds [Arruda et al. (1982) Plant Physiol. 69:988-989]. LKR catalyzes
the
first step in lysine catabolism, the condensation of L-lysine with a-
ketoglutarate
into saccharopine using NADPH as a cofactor. LKR activity increases sharply
from the onset of endosperm development in corn, reaches a peak level at about
20 days after pollination, and then declines [Arruda et al. (1983)
Phytochemistry
22:2687-2689]. In order to prevent the catabolism of lysine it would be
desirable
to reduce or eliminate LKR expression or activity. This could be accomplished
by
cloning the LKR gene, preparing a chimeric gene for cosuppression of LKR or
preparing a chimeric gene to express antisense RNA for LKR, and introducing
the
chimeric gene into plants via transformation. Alternatively, plant mutants
could
be obtained wherein LKR enzyme activity is absent.
Several methods to clone a plant LKR gene are available to one skilled in
the art. The protein can be purified from corn endosperm, as described in
Brochetto-Braga et al. [(1992) Plant Physiol. 98:1139-1147] and used to raise
antibodies. The antibodies can then be used to screen an cDNA expression
library
for LKR clones. Alternatively the purified protein can be used to determine
amino acid sequence at the amino-terminal of the protein or from protease
derived
internal peptide fragments. Degenerate oligonucleotide probes can be prepared
based upon the amino acid sequence and used to screen a plant cDNA or genomic
DNA library via hybridization.
Another method makes use of an E. coli strain that is unable to grow in a
synthetic medium containing 20 g/ml, of L-lysine. Expression of LKR full-
length cDNA in this strain will reverse the growth inhibition by reducing the
lysine concentration. Construction of a suitable E. coli strain and its use to
select
clones from a plant cDNA library that lead to lysine-resistant growth is
described
in Example 20.
Yet another method relies upon homology between plant LKR and
saccharopine dehydrogenase. Fungal saccharopine dehydrogenase (glutamate-
forming) and saccharopine dehydrogenase (lysine-forming) catalyze the final
two
steps in the fungal lysine biosynthetic pathway. Plant LKR and fungal
saccharopine dehydrogenase (lysine-forming) catalyze both forward and reverse
reactions, use identical substrates and use similar co-factors. Similarly,
plant
saccharopine dehydrogenase (glutamate-forming), which catalyzes the second
step
in the lysine catabolic pathway, works in both forward and reverse reactions,
uses
identical substrates and uses similar co-factors as fungal saccharopine
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dehydrogenase (glutamate-forming). Several genes for fungal saccharopine
dehydrogenases have been isolated and sequenced and are readily available to
those skilled in the art [Xuan et al. (1990) Mol. Cell. Biol. 10:4795-4806,
Feller
et al. (1994) Mol. Cell. Biol. 14:6411-6418]. These genes could be used as
heterologous hybridization probes to identify plant LKR and plant saccharopine
dehydrogenase (glutamate-forming) nucleic acid fragments, or alternatively to
identify homologous :protein coding regions in plant cDNAs.
Biochemical and genetic evidence derived from human and bovine studies
has demonstrated that mammalian LKR and saccharopine dehydrogenase
(glutamate-forming) enzyme activities are present on a single protein with a
monomer molecular weight of about 117,000. This contrasts with the fungal
enzymes which are carried on separate proteins, saccharopine dehydrogenase
(lysine-forming) with a molecular weight of about 44,000, and saccharopine
dehydrogenase (glutamate-forming) with a molecular weight of about 51,000.
Plant LKR has been reported to have a molecular weight of about 140,000
indicating that it is like the animal catabolic protein wherein both LKR and
saccharopine dehydrogenase (glutamate-forming) enzyme activities are present
on
a single protein.
Two plant saccharopine dehydrogenase (glutamate-forming) nucleic acid
fragments (SEQ ID NOS:102 and 103) containing cDNA derived from
Arabidopsis thaliana are provided. These were identified as cDNAs that encode
proteins homologous to fungal. saccharopine dehydrogenase (glutamate-forming).
These nucleic acid fragments were used to design and synthesize
oligonucleotide
primers (SEQ ID NO: 108 and SEQ ID NO: 109). The primers were synthesized
and used for PCR amplification of a 2.24 kb DNA fragment from genomic
Arabidopsis DNA. This DNA fragment was used to isolate a larger genomic
DNA fragment, which included the entire coding region, as well as 5' and 3'
flanking regions, via hybridization to a genomic DNA library. The sequence of
this genomic DNA fragment is provided (SEQ ID NO:I 10); oligonucleotides were
synthesized based on this sequence and used to isolate a full length cDNA via
RT-PCR. The sequence of the full length cDNA (SEQ ID NO: 111) is provided.
These nucleic acid fragments can be used as hybridization probes to identify
and
isolate genomic DNA fragments or cDNA fragments encoding both LKR and
saccharopine dehydrogenase (glutamate-forming) enzyme activities from any
plant desired.
The deduced amino acid sequence of Arabidopsis LKR/SDH protein is
shown in SEQ ID NO:112. The amino acid sequence shows that in plants LKR
and SDH enzyme activities are carried on a single bi-functional protein, and
that
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the protein lacks an N-terminal targeting sequence indicating that the lysine
degradative pathway is located in the plant cell cytosol. The amino acid
sequence
of Arabidopsis LKR/SDH protein was compared to that of other LKR and SDH
proteins thus revealing regions of conserved amino acid sequence. Degenerate
oligonucleotides can be designed based upon this information and used to
amplify
genomic or cDNA fragments via PCR from other organisms, preferably plants.
As an example of this, SEQ ID NO: 113 and SEQ ID NO: 114 were designed and
used to amplify soybean and corn LKR/SDH cDNA fragments. The sequence of a
partial soybean LKRISDH cDNA is shown in SEQ ID NO: 115, and the sequence
of a partial corn cDNA is shown in SEQ ID NO: 116. These DNA fragments can
be used to isolate larger genomic DNA fragments, which include the entire
coding
region, as well as 5' and 3' flanking regions, via hybridization to corn or
soybean
genomic DNA or cDNA libraries, as was done for Arabidopsis. More complete
sequence information from the coding regions for soybean and corn LKR/SDH
was obtained using the sequences in SEQ ID NOS:115 and 116 as starting
materials in protocols such as 5' RACE and hybridization to cDNA libraries. A
near full-length cDNA for soybean LKR/SDH is shown in SEQ ID NO: 119, and a
near full-length cDNA for corn LKRJSDH is shown in SEQ ID NO:120. A
truncated version of the LKR/SDH cDNA from corn is set forth in SEQ ID
NO:123.
The deduced partial amino acid sequences of soybean LRR/SDH protein is
shown in SEQ ID NOS:117 and 121 and the deduced partial amino acid sequences
of corn LK.R/SDH protein is shown in SEQ ID NO: 118, 122 and 124. These
amino acid sequences can be compared to other LKR/SDH protein sequences,
e.g., the Arabidopsis LKR/SDH protein sequence, thus revealing regions of
conserved amino acid sequence. With this information oligonucleotide primers
can be designed and synthesized to permit isolation of LKR/SDH genomic or
cDNA fragments from any plant source.
The availibility of sequence information for plant LKR/SDH proteins from
Arabidopsis, soybean, and corn allowed comparisons of those sequences to EST
sequences obtained from other plants, including ESTs from rice and wheat. SEQ
ID NOS:125 and 127 set forth sequences for partial cDNA clones encoding
LKR/SDH from rice, and SEQ ID NO:129 set forth the sequence of a partial
cDNA encoding a ffragment of LKR/SDH from wheat. The prdicted protein
fragments encoded by the sequences presented in SEQ ID NOS:125, 127 and 129
are set forth in SEQ ID NOS:126, 128 and 130, respectively,
The availability of plant LKR/SDH genes makes it possible to block
expression of the LKR/SDH gene in transformed plants. To accomplish this a
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chimeric gene designed for cosuppression of LKR can be constructed by linking
the LKR gene or gene fragment to any of the plant promoter sequences described
above. (See U.S. Patent No. 5,231,020 for methodology to block plant gene
expression via cosuppression.) Alternatively, a chimeric gene designed to
express
antisense RNA for all or part of the LKR gene can be constructed by linking
the
LKR gene or gene fragment in reverse orientation to any of the plant promoter
sequences described above. (See U.S. Patent 5,107,065 for methodology to block
plant gene expression via antisense RNA.) Either the cosuppression or
antisense
chimeric gene can be introduced into plants via transformation. Transformants
wherein expression of the endogenous LKR gene is reduced or eliminated are
then
selected.
Preferred promoters for the chimeric genes would be seed-specific
promoters. For soybean, rapeseed and other dicotyledonous plants, strong seed-
specific promoters from a bean phaseolin gene, a soybean P-conglycinin gene,
glycinin gene, Kunitz; trypsin inhibitor gene, or rapeseed napin gene would be
preferred. For corn and other monocotyledonous plants, a strong endosperm-
specific promoter, e.g., the 10 kD or 27 kD zein promoter, or a strong embryo-
specific promoter, e.g., the FLB I promoter, would be preferred.
Transformed plants containing any of the chimeric LKR genes can be
obtained by the methods described above. In order to obtain transformed plants
that express a chimeric gene for cosuppression of LKR or antisense LKR, as
well
as a chimeric gene encoding substantially lysine-insensitive DHDPS, the
cosuppression or antisense LKR gene could be linked to the chimeric gene
encoding substantially lysine-insensitve DHDPS and the two genes could be
introduced into plants via transformation. Alternatively, the chimeric gene
for
cosuppression of LKR or antisense LKR could be introduced into previously
transformed plants that express substantially lysine-insensitive DHDPS, or the
cosuppression or antisense LKR gene could be introduced into normal plants and
the transformants obtained could be crossed with plants that express
substantially
lysine-insensitive DHDPS.
The availability of plant LKR/SDH genes makes it possible to express the
proteins in heterologous systems. To demonstrate this, a DNA fragment which
includes the Arabidopsis SDHi coding region (SEQ ID NO: 119) was generated
using PCR primers and ligated into a prokaryotic expression vector. High level
expression of Arabidopsis SDH was achieved in E. coli and the SDH protein has
been purified from the bacterial extracts, and used to raise rabbit antibodies
to the
protein. These antibodies can be used to screen for plant mutants in order to
find
variants which do not produce LKR/SDH protein, or produce reduced amounts of
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the protein compared to the parent plant. The plant mutants that express
reduced
LKR/SDH protein, or no protein at all, could be crossed with plants that
express
substantially lysine-insensitive DHDPS.
Design of Lysine-Rich Polypeptides
It may be desirable to convert the high levels of lysine produced into a form
that is insensitive to breakdown, e.g., by incorporating it into a di-, tri-
or
oligopeptide, or a lysine-rich storage protein. No natural lysine-rich
proteins are
known.
One aspect of this invention is the design of polypeptides which can be
expressed in vivo to serve as lysine-rich seed storage proteins. Polypeptides
are
linear polymers of amino acids where the a-carboxyl group of one amino acid is
covalently bound to the a-amino group of the next amino acid in the chain. Non-
covalent interactions among the residues in the chain and with the surrounding
solvent determine the final conformation of the molecule. Those skilled in the
art
must consider electrostatic forces, hydrogen bonds, Van der Waals forces,
hydrophobic interactions, and conformational preferences of individual amino
acid residues in the design of a stable folded polypeptide chain [see for
example:
Creighton, (1984) Proteins, Structures and Molecular Properties, W. H. Freeman
and Company, New York, pp 133-197, or Schulz et al., (1979) Principles of
Protein Structure, Springer Verlag, New York, pp 27-45]. The number of
interactions and their complexity suggest that the design process may be aided
by
the use of natural protein models where possible.
The synthetic storage proteins (SSPs) embodied in this invention are chosen
to be polypeptides with the potential to be enriched in lysine relative to
average
levels of proteins in plant seeds. Lysine is a charged amino acid at
physiological
pH and is therefore found most often on the surface of protein molecules
[Chothia,
(1976) Journal of Molecular Biology 105:1-14]. To maximize lysine content,
Applicants chose a molecular shape with a high surface-to-volume ratio for the
synthetic storage proteins embodied in this invention. The alternatives were
either
to stretch the common globular shape of most proteins to form a rod-like
extended
structure or to flatten the globular shape to a disk-like structure.
Applicants chose
the former configuration as there are several natural models for long rod-like
proteins in the class of fibrous proteins [Creighton, (1984) Proteins,
Structures
and Molecular Properties, W.H. Freeman and Company, New York, p 191].
Coiled-coils constitute a well-studied subset of the class of fibrous proteins
[see Cohen et al., (1986) Trends Biochem. Sci. 11:245-248]. Natural examples
are
found in a-keratins, paramyosin, light meromyosin and tropomyosin. These
protein molecules consist of two parallel alpha helices twisted about each
other in
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a left-handed supercoil. The repeat distance of this supercoil is 140 A
(compared
to a repeat distance of 5.4 A for one turn of the individual helices). The
supercoil
causes a slight skew (10 ) between the axes of the two individual alpha
helices.
In a coiled coil there are 3.5 residues per turn of the individual helices
resulting in an exact '7 residue periodicity with respect to the superhelix
axis (see
Figure 1). Every seventh amino acid in the polypeptide chain therefore
occupies
an equivalent position with respect to the helix axis. Applicants refer to the
seven
positions in this heptad unit of the invention as (d e f g a b c) as shown in
Figures 1 and 2a. This conforms to the conventions used in the coiled-coil
literature.
The a and d amino acids of the heptad follow a 4,3 repeat pattern in the
primary sequence and fall on one side of an individual alpha helix (See Figure
1).
If the amino acids on one side of an alpha helix are all non-polar, that face
of the
helix is hydrophobic and will associate with other hydrophobic surfaces as,
for
example, the non-polar face of another similar helix. A coiled-coil structure
results when two helices dimerize such that their hydrophobic faces are
aligned
with each other (See :Figure 2a).
The amino acids on the external faces of the component alpha helices (b, c,
e, f, g) are usually polar in natural coiled-coils in accordance with the
expected
pattern of exposed and buried residue types in globular proteins [Schulz, et
al.,
(1979) Principles of Protein Structure. Springer Verlag, New York, p 12;
Talbot,
et al, (1982) Acc. Chem. Res. 15:224-230; Hodges et al., (1981) Journal of
Biological Chemistry 256:1214-1224]. Charged amino acids are sometimes found
forming salt bridges between positions e and g' or positions g and e' on the
opposing chain (see Figure 2a).
Thus, two amphipathic helices like the one shown in Figure 1 are held
together by a combination of hydrophobic interactions between the a, a', d,
and d'
residues and by salt bridges between e and g' and/or g and e' residues. The
packing of the hydrophobic residues in the supercoil maintains the chains "in
register". For short polypeptides comprising only a few turns of the component
alpha helical chains, the 10 skew between the helix axes can be ignored and
the
two chains treated as parallel (as shown in Figure 2a).
A number of synthetic coiled-coils have been reported in the literature (Lau
et al., (1984) Journal of Biological Chemistry 259:13253-13261; Hodges et al.,
(1988) Peptide Research 1:19-30; DeGrado et at., (1989) Science 243:622-628;
O'Neil et al., (1990) Science 2'50:646-651 ]. Although these polypeptides vary
in
size, Lau et al. found that 29 amino acids were sufficient for dimerization to
form
the coiled-coil structure [Lau et al., (1984) Journal of Biological Chemistry
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259:13253-13261]. Applicants constructed the polypeptides in this invention as
28-residue and larger chains for reasons of conformational stability.
The polypeptides of this invention are designed to dimerize with a coiled-
coil motif in aqueous environments. Applicants have used a combination of
hydrophobic interactions and electrostatic interactions to stabilize the
coiled-coil
conformation. Most nonpolar residues are restricted to the a and d positions
which creates a hydrophobic stripe parallel to the axis of the helix. This is
the
dimerization face. Applicants avoided large, bulky amino acids along this face
to
minimize steric interference with dimerization and to facilitate formation of
the
stable coiled-coil structure.
Despite recent reports in the literature suggesting that methionine at
positions a and d is destabilizing to coiled-coils in the leucine zipper
subgroup
[Landschulz et al., (1989) Science 243:1681-1688 and Hu et al., (1990) Science
250:1400-1403], Applicants chose to substitute methionine residues for leucine
on
the hydrophobic face of the SSP polypeptides. Methionine and leucine are
similar
in molecular shape (Figure 3). Applicants demonstrated that any
destabilization
of the coiled-coil that may be caused by methionine in the hydrophobic core
appears to be compensated in sequences where the formation of salt bridges (e-
g'
and g-e') occurs at all possible positions in the helix (i.e., twice per
heptad).
To the extent that it is compatible with the goal of creating a polypeptide
enriched in lysine, Applicants minimized the unbalanced charges in the
polypeptide. This may help to prevent undesirable interactions between the
synthetic storage proteins and other plant proteins when the polypeptides are
expressed in vivo.
The polypeptides of this invention are designed to spontaneously fold into a
defined, conformationally stable structure, the alpha helical coiled-coil,
with
minimal restrictions on the primary sequence. This allows synthetic storage
proteins to be custom-tailored for specific end-user requirements. Any amino
acid
can be incorporated at a frequency of up to one in every seven residues using
the
b, c, and f positions in the heptad repeat unit. Applicants note that up to
43% of
an essential amino acid from the group isoleucine, leucine, lysine,
methionine,
threonine, and valine can be incorporated and that up to 14% of the essential
amino acids from the group phenylalanine, tryptophan, and tyrosine can be
incorporated into the synthetic storage proteins of this invention.
In the SSPs only Met, Leu, Ile, Val or Thr are located in the hydrophobic
core. Furthermore, the e, g, e', and g' positions in the SSPs are restricted
such that
an attractive electrostatic interaction always occurs at these positions
between the
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two polypeptide chains in an SSP dimer. This makes the SSP polypeptides more
stable as dimers.
Thus, the novel synthetic storage proteins described in this invention
represent a particular subset of possible coiled-coil polypeptides. Not all
polypeptides which adopt an amphipathic alpha helical conformation in aqueous
solution are suitable for the applications described here.
The following rules derived from Applicants' work define the SSP
polypeptides that Applicants use in their invention:
The synthetic polypeptide comprises n heptad units (d e f g a b c), each
heptad being either the same or different, wherein:
n is at least 4;
a and (I are independently selected from the group consisting of
Met, Leu, Val, Ile and Thr;
e and g are independently selected from the group consisting of the
acid/base pairs Glu/Lys, Lys/Glu, Arg/Glu, Arg/Asp,
Lys/Asp, Glu/Arg, Asp/Arg and Asp/Lys; and
b, c and f are independently any amino acids except Gly or Pro and
at least two amino acids of b, c and fin each heptad are
selected from the group consisting of Glu, Lys, Asp, Arg,
His, Thr, Ser, Asn, Gln, Cys and Ala.
Chimeric Genes Encoding Lysine-Rich Polynentides
DNA sequences which encode the polypeptides described above can be
designed based upon the genetic code. Where multiple codons exist for
particular
amino acids, codons should be chosen from those preferable for translation in
plants. Oligonucleotides corresponding to these DNA sequences can be
synthesized using an.ABI DNA synthesizer, annealed with oligonucleotides
corresponding to the complementary strand and inserted into a plasmid vector
by
methods known to those skilled in the art. The encoded polypeptide sequences
can be lengthened by inserting; additional annealed oligonucleotides at
restriction
endonuclease sites engineered into the synthetic gene. Some representative
strategies for constructing genes encoding lysine-rich polypeptides of the
invention, as well as DNA and amino acid sequences of preferred embodiments
are provided in Example 21.
A chimeric gene designed to express RNA for a synthetic storage protein
gene encoding a lysine-rich polypeptide can be constructed by linking the gene
to
any of the plant promoter sequences described above. Preferred promoters would
be seed-specific promoters. For soybean, rapeseed and other dicotyledonous
plants strong seed-specific promoters from a bean phaseolin gene, a soybean
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¾-conglycinin gene, glycinin gene, Kunitz trypsin inhibitor gene, or rapeseed
napin gene would be preferred. For corn or other monocotyledonous plants, a
strong endosperm-specific promoter, e.g., the 10 kD or 27 kD zein promoter, or
a
strong embyro-specific promoter, e.g., the corn globulin I promoter, would be
preferred.
In order to obtain plants that express a chimeric gene for a synthetic storage
protein gene encoding a lysine-rich polypeptide, plants can be transformed by
any
of the methods described above. In order to obtain plants that express both a
chimeric SSP gene and chimeric genes encoding substantially lysine-insensitive
DHDPS and AK, the SSP gene could be linked to the chimeric genes encoding
substantially lysine-insensitive DHDPS and AK and the three genes could be
introduced into plants via transformation. Alternatively, the chimeric SSP
gene
could be introduced into previously transformed plants that express
substantially
lysine-insensitive DHDPS and AK, or the SSP gene could be introduced into
normal plants and the transformants obtained could be crossed with plants that
express substantially lysine-insensitive DHDPS and AK.
Results from genetic crosses of transformed plants containing lysine
biosynthesis genes with transformed plants containing lysine-rich protein
genes
(see Example 23) demonstrate that the total lysine levels in seeds can be
increased
by the coordinate expression of these genes. This result was especially
striking
because the gene copy number of all of the transgenes was reduced in the
hybrid.
It is expected that the lysine level would be further increased if the
biosynthesis
genes and the lysine-rich protein genes were all homozygous.
Use of the cts/lysC-M4 Chimeric Gene as a
Selectable Marker for Plant Transformation
Growth of cell cultures and seedlings of many plants is inhibited by high
concentrations of lysine plus threonine. Growth is restored by addition of
methionine (or homoserine which is converted to methionine in vivo). Lysine
plus
threonine inhibition is thought to result from feedback inhibition of
endogenous
AK, which reduces flux through the pathway leading to starvation for
methionine.
In tobacco there are two AK enzymes in leaves, one lysine-sensitive and one
threonine sensitive.[Negrutui et al. (1984) Theor. Appl. Genet. 68:11-20].
High
concentrations of lysine plus threonine inhibit growth of shoots from tobacco
leaf
disks and inhibition is reversed by addition of low concentrations of
methionine.
Thus, growth inhibition is presumably due to inhibition of the two AK
isozymes.
Expression of active lysine and threonine insensitive AKIII-M4 also
reverses lysine plus threonine growth inhibition (Table 2, Example 7). There
is a
good correlation between the level of AKIII-M4 protein expressed and the
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resistance to lysine plus threonine. Expression of lysine-sensitive wild type
AKIII
does not have a similar effect. Since expression of the AKIII-M4 protein
permits
growth under normally inhibitory conditions, a chimeric gene that causes
expression of AKIII-M4 in plants can be used as a selectable genetic marker
for
transformation as illustrated in Examples 13 and 17.
EXAMPLES
The present invention is further defined in the following Examples, in which
all parts and percentages are by weight and degrees are Celsius, unless
otherwise
stated. It should be understood that these Examples, while indicating
preferred
embodiments of the invention., are given by way of illustration only. From the
above discussion and these Examples, one skilled in the art can ascertain the
essential characteristics of this invention, and without departing from the
spirit
and scope thereof, can make various changes and modifications of the invention
to
adapt it to various usages and conditions.
EXAMPLE 1
Isolation of the E. coli lysC Gene and mutations
in lysC resulting in lysine-insensitive AKIII
The E. coli lysC. gene has been cloned, restriction endonuclease mapped and
sequenced previously [Cassan et al. (1986) J. Biol. Chem. 261:1052-1057]. For
the present invention the lysC gene was obtained on a bacteriophage lambda
clone
from an ordered library of 3400 overlapping segments of cloned E. coli DNA
constructed by Kohara, Akiyama and Isono [Kohara et al. (1987) Cell
50:595-508]. This library provides a physical map of the whole E. coli
chromosome and ties the physical map to the genetic map. From the knowledge
of the map position of lvsC at 90 min on the E. coli genetic map [Theze et al.
(1974) J. Bacteriol. 1.17:133-143], the restriction endonuclease map of the
cloned
gene [Cassan et al. (1986) J. Biol. Chem. 261:1052-1057], and the restriction
endonuclease map of the cloned DNA fragments in the E. coli library [Kohara
et al. (1987) Cell 50:595-508], it was possible to choose lambda phages 4E5
and
7A4 [Kohara et al. (1987) Cell 50:595-508] as likely candidates for carrying
the
lysC gene. The phages were grown in liquid culture from single plaques as
described [see Current Protocols in Molecular Biology (1987) Ausubel et al.
Eds.
John Wiley & Sons New York] using LE392 as host [see Sambrook et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press].
Phage DNA was prepared by phenol extraction as described [see Current
Protocols in Molecular Biology (1987) Ausubel et al. eds. John Wiley & Sons
New York].
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From the sequence of the gene several restriction endonuclease fragments
diagnostic for the 1ysC gene were predicted, including an 1860 bp EcoR I-Nhe I
fragment, a 2140 bp EcoR I-Xmn I fragment and a 1600 bp EcoR I-BamH I
fragment. Each of these fragments was detected in both of the phage DNAs
confirming that these carried the lysC gene. The EcoR I-Nhe I fragment was
isolated and subcloned in plasmid pBR322 digested with the same enzymes,
yielding an ampicillin-resistant, tetracycline-sensitive E. coli transformant.
The
plasmid was designated pBT436.
To establish that the cloned 1vsC gene was functional, pBT436 was
transformed into E. coli strain Gifl 06M1 (E. coli Genetic Stock Center strain
CGSC-5074) which has mutations in each of the three E. coli AK genes [Theze
et al. (1974) J. Bacteriol. 117:133-143]. This strain lacks all AK activity
and
therefore requires diaminopimelate (a precursor to lysine which is also
essential
for cell wall biosynthesis), threonine and methionine. In the transformed
strain all
these nutritional requirements were relieved demonstrating that the cloned
tysC
gene encoded functional AKIII.
Addition of lysine (or diaminopimelate which is readily converted to lysine
in vivo) at a concentration of approximately 0.2 mM to the growth medium
inhibits the growth of Gifl 06M 1 transformed with pBT436. M9 media [see
Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory Press] supplemented with the arginine and isoleucine,
required
for Gifl 06M1 growth, and ampicillin, to maintain selection for the pBT436
plasmid, was used. This inhibition is reversed by addition of threonine plus
methionine to the growth media. These results indicated that AKIII could be
inhibited by exogenously added lysine leading to starvation for the other
amino
acids derived from aspartate. This property of pBT436-transformed Gifl 06M 1
was used to select for mutations in lysC that encoded lysine-insensitive
AKIII.
Single colonies of Gifl06M1 transformed with pBT436 were picked and
resuspended in 200 L of a mixture of 100 L I% lysine plus 100 L of M9
media. The entire cell suspension containing 107-108 cells was spread on a
petri
dish containing M9 media supplemented with the arginine, isoleucine, and
ampicillin. Sixteen petri dishes were thus prepared. From 1 to 20 colonies
appeared on 11 of the 16 petri dishes. One or two (if available) colonies were
picked and retested for lysine resistance and from this nine lysine-resistant
clones
were obtained. Plasmid DNA was prepared from eight of these and re-
transformed into Gifl 06M 1 to determine whether the lysine resistance
determinant was plasmid-borne. Six of the eight plasmid DNAs yielded lysine-
resistant colonies. Three of these six carried 1ysQ genes encoding AKIII that
was
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uninhibited by 15 mM lysine, whereas wild type AKIII is 50% inhibited by
0.3-0.4 mM lysine and >90% inhibited by 1 mM lysine (see Example 2 for
details).
To determine the molecular basis for lysine-resistance the sequences of the
wild type lysC gene and three mutant genes were determined. A method for
"Using mini-prep plasmid DNA for sequencing double stranded templates with
SequenaseTM" [Kraft et al. (1988) BioTechniques 6:544-545] was used.
Oligonucleotide primers, based on the published 1ysC sequence and spaced
approximately every 200 bp, were synthesized to facilitate the sequencing. The
sequence of the wild type lysC' gene cloned in pBT436 (SEQ ID NO:1) differed
from the publishedsC sequence in the coding region at 5 positions. Four of
these nucleotide differences were at the third position in a codon and would
not
result in a change in the amino acid sequence of the AKIII protein. One of the
differences would result in a cysteine to glycine substitution at amino acid
58 of
AKIII. These differences are probably due to the different strains from which
the
lvsC genes were cloned.
The sequences of the three mutant lysC genes that encoded lysine-
insensitive AK each differed from the wild type sequence by a single
nucleotide,
resulting in a single amino acid substitution in the protein. Mutant M2 had an
A
substituted for a G at nucleotide 954 of SEQ ID NO: I resulting in an
isoleucine
for methionine substitution at amino acid 318 and mutants M3 and M4 had
identical T for C substitutions at nucleotide 1055 of SEQ ID NO:1 resulting in
an
isoleucine for threonine substitution at amino acid 352. Thus, either of these
single amino acid substitutions is sufficient to render the AKIII enzyme
insensitive to lysine inhibition.
EXAMPLE 2
High level expression of wild type and mutant lysC genes in E. coli
An Nco I (CCATGG) site was inserted at the translation initiation codon of
the lvsC gene using the following oligonucleotides:
SEQ ID NO:2:
GATCCATGGC TGAAATTGTT GTCTCCAAAT TTGGCG
SEQ ID NO:3:
GTACCGCCAA ATTTGGAGAC AACAATTTCA GCCATG
When annealed these oligonucleotides have BamH I and Asp718 "sticky" ends.
The plasmid pBT436 was digested with BamH I, which cuts upstream of the lysC
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coding sequence and Asp718 which cuts 31 nucleotides downstream of the
initiation codon. The annealled oligonucleotides were ligated to the plasmid
vector and E. soli transformants were obtained. Plasmid DNA was prepared and
screened for insertion of the oligonucleotides based on the presence of an Nco
I
site. A plasmid containing the site was sequenced to assure that the insertion
was
correct, and was designated pBT457. In addition to creating an Nco I site at
the
initiation codon of IysC, this oligonucleotide insertion changed the second
codon
from TCT, coding for serine, to GCT, coding for alanine. This amino acid
substitution has no apparent effect on the AKIII enzyme activity.
To achieve high level expression of the IysC genes in E. coli, the bacterial
expression vector pBT430 was used. This vector is a derivative of pET-3a
[Rosenberg et al. (1987) Gene 56:125-135] which employs the bacteriophage T7
RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first
destroying the EcoR I and Hind III sites in pET-3a at their original
positions. An
oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at
the
BamH I site of pET-3a. This created pET-3aM with additional unique cloning
sites for insertion of genes into the expression vector. Then, the Nde I site
at the
position of translation initiation was converted to an Nco I site using
oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this
region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430.
The IysC gene was cut out of plasmid pBT457 as a 1560 bp Nco I-EcoR I
fragment and inserted into the expression vector pBT430 digested with the same
enzymes, yielding plasmid pBT461. For expression of the mutant IysC genes
(M2, M3 and M4) pBT461 was digested with Kpn I-EcoR I, which removes the
wild type IysC gene from about 30 nucleotides downstream from the translation
start codon, and inserting the homologous Kpn I-EcoR I fragments from the
mutant genes yielding plasmids pBT490, pBT491 and pBT492, respectively.
For high level expression each of the plasmids was transformed into E. coli
strain BL21(DE3) [Studier et al. (1986) J. Mol. Biol. 189:113-130]. Cultures
were
grown in LB medium containing ampicillin (100 mg/L) at 25 C. At an optical
density at 600 nm of approximately 1, IPTG (isopropylthio-[i-galactoside, the
inducer) was added to a final concentration of 0.4 mM and incubation was
continued for 3 h at 25 . The cells were collected by centrifugation and
resuspended in 1/20th (or 1/100th) the original culture volume in 50 mM NaCl;
50 mM Tris-Cl, pH 7.5; 1 mM EDTA, and frozen at -20 . Frozen aliquots of
1 mL were thawed at 37 and sonicated, in an ice-water bath, to lyse the
cells.
The lysate was centrifuged at 4 for 5 min at 15,000 rpm. The supernatant was
removed and the pellet was resuspended in 1 mL of the above buffer.
44
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The supernatant and pellet fractions of uninduced and IPTG-induced
cultures of BL2I (DE3)/pBT461 were analyzed by SDS polyacrylamide gel
electrophoresis. The major protein visible by Coomassie blue staining in the
supernatant of the induced culture had a molecular weight of about 48 kd, the
expected size for AKIII. About 80% of the AKIII protein was in the supernatant
and AKIII represented 10-20% of the total E. coil protein in the extract.
AK activity was assayed as shown below:
Assay mix (for 12 assay tubes):
4.5 mL H2O
1.0 mL 8M KOH
1.0 mL 8M NH2OH-HC1
1.0 mL I M Tris-HCI pH 8.0
0.5 mL 0.2M ATP (121 mg/mL in 0.2M NaOH)
50 p.L I M MgSO4
Each 1.5 mL eppendorf assay tube contained:
0.64 mL assay mix
0.04 mL 0.2 M L-aspartic acid or 0.04 mL H2O
0.0005-0.12 mL extract
H2O to total volume 0.8 mL
Assay tubes were incubated at 30 for desired time (10-60 min). Then
0.4 mL FeCl3 reagent (10% w/v FeC13, 3.3% trichloroacetic acid, 0.7 M HCI) was
added and the material centrifuged for 2 min in an eppendorf centrifuge. The
supernatant was decanted. The OD was read at 540 rim and compared to the
aspartyl-hydroxamate standard.
Approximately 80% of the AKIII activity was in the supernatant fraction.
The specific activity of wild type and mutant crude extracts was 5-7 M
product
per min per milligram total protein. Wild type AKIII was sensitive to the
presence of L-lysine in the assay. Fifty percent inhibition was found at a
concentration of about 0.4 mM and 90% inhibition at about 1.0 mM. In contrast,
mutants AKIII-M2, M3 and M4 (see Example 1) were not inhibited at all by
15 mM L-lysine.
Wild type AKIII protein was purified from the supernatant of the IPTG-
induced culture as follows. To 1 mL of extract, 0.25 mL of 10% streptomycin
sulfate was added and kept at 4 overnight. The mixture was centrifuged at 4
for
15 min at 15,000 rpm. The supernatant was collected and desalted using a
SephadexTM G-25 M column (Column PD-10, Pharmacia). It was then run on a
CA 02280196 2002-09-09
Mono-QTM HPLC column and eluted with a 0-1 M NaC 1 gradient. The two 1 mL
fractions containing most of the AKIII activity were pooled, concentrated,
desalted and run on an HPLC sizing column (TSK G3000SW). Fractions were
eluted in 20 mM KPO4 buffer, pH7.2, 2 mM MgSO4, 10 mM [i-mercaptoethanol,
0.15 M KCI, 0.5 mM L-lysine and were found to be >95% pure by SDS
polyacrylamide gel electrophoresis. Purified AKIII protein was sent to
Hazelton
Research Facility (310 Swampridge Road, Denver, PA 17517) to have rabbit
antibodies raised against the protein.
EXAMPLE 3
Isolation of the E. coli and Corynebacteriumgiutamicum dapA genes
The E. coli dapA gene (ecoda A has been cloned, restriction endonuclease
mapped and sequenced previously [Richaud et al. (1986) J Bacteriol.
166:297-300]. For the present invention the dapA gene was obtained on a
bacteriophage lambda clone from an ordered library of 3400 overlapping
segments of cloned E. coli DNA constructed by Kohara, Akiyama and Isono
[Kohara et al. (1987) Cell 50:595-508, see Example I]. From the knowledge of
the map position of dapA at 53 min on the E. coli genetic map [Bachman (1983)
Microbiol. Rev. 47:180-230], the restriction endonuclease map of the cloned
gene
[Richaud et al. (1986) J. Bacteriol. 166:297-300], and the restriction
endonuclease
map of the cloned DNA fragments in the E. coli library [Kohara et al. (1987)
Cell
50:595-508], it was possible to choose lambda phages 4C1I and 5A8 [Kohara
et at. (1987) Cell 50:595-508) as likely candidates for carrying the dapA
gene.
The phages were grown in liquid culture from single plaques as described [see
Current Protocols in Molecular Biology (1987) Ausubel et al. eds., John Wiley
&
Sons New York] using LE392 as host [see Sambrook et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press]. Phage
DNA was prepared by phenol extraction as described [see Current Protocols in
Molecular Biology (1987) Ausubel et al. eds., John Wiley & Sons New York].
Both phages contained an approximately 2.8 kb Pst I DNA fragment expected for
the daDA gene [Richaud et al. (1986) J. Bacteriol. 166:297-300]. The fragment
was isolated from the digest of phage 5A8 and inserted into Pst I digested
vector
pBR322 yielding plasmid pBT427.
ggA) was isolated from genomic
The Corynebacterium dapA gene (cord
DNA from ATCC strain 13032 using polymerase chain reaction (PCR). The
nucleotide sequence of the Corynebacterium daDA gene has been published
[Bonnassie et al. (1990) Nucleic Acids Res. 18:64211. From the sequence it was
possible to design oligonucleotide primers for PCR that would allow
amplification
of a DNA fragment containing the gene, and at the same time add unique
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restriction endonuclease sites at the start codon (Nco I) and just past the
stop
codon (EcoR I) of the gene. The oligonucleotide primers used were:
SEQ ID NO:4:
CCCGGGCCAT GGCTACAGGT TTAACAGCTA AGACCGGAGT AGAGCACT
SEQ ID NO:5:
GATATCGAAT TCTCATTATA GAACTCCAGC TTTTTTC
PCR was performed using a Perkin-Elmer Cetus kit according to the
instructions of the vendor on a. thermocycler manufactured by the same
company.
The reaction product, when run on an agarose gel and stained with ethidium
bromide, showed a strong DNA band of the size expected for the
Corynebacterium dapA gene, about 900 bp. The PCR-generated fragment was
digested with restriction endonucleases Nco I and EcoR I and inserted into
expression vector pBT430 (see Example 2) digested with the same enzymes. In
addition to introducing an Nco I site at the translation start codon, the PCR
primers also resulted in a change of the second codon from AGC coding for
serine
to GCT coding for alanine. Several clones that expressed active, lysine-
insensitive DHDPS (see Example 4) were isolated, indicating that the second
codon amino acid substitution did not affect activity; one clone was
designated
FS766.
The Nco I to EcoR I fragment carrying the PCR-generated Corynebacterium
dapA gene was subcloned into the phagemid vector pGEM-9Zf(-) from Promega,
single-stranded DNA was prepared and sequenced. This sequence is shown in
SEQ ID NO:6.
Aside from the differences in the second codon already mentioned, the
sequence matched the published sequence except at two positions, nucleotides
798
and 799. In the published sequence these are TC, while in the gene shown in
SEQ
ID NO:6 they are CT. This change results in an amino acid substitution of
leucine
for serine. The reason for this difference is not known. It may be due to an
error
in the published sequence, the difference in strains used to isolate the gene,
or a
PCR-generated error. The latter seems unlikely since the same change was
observed in at least 3 independently isolated PCR-generated dapA genes. The
difference has no apparent effect on DHDPS enzyme activity (see Example 4).
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EXAMPLE 4
High level expression of the E. coli and
Corynebacterium glutamicum dapA genes in E. coli
An Nco I (CCATGG) site was inserted at the translation initiation codon of =
the E. coli daps gene using oligonucleotide-directed mutagenesis. The 2.8 kb
Pst I DNA fragment carrying the dapA gene in plasmid pBT427 (see Example 3)
was inserted into the Pst I site of phagemid vector pTZI 8R (Pharmacia)
yielding
pBT43 1. The orientation of the dapA gene was such that the coding strand
would
be present on the single-stranded phagemid DNA. Oligonucleotide-directed
mutagenesis was carried out using a Muta-Gene kit from Bio-Rad according to
the
manufacturer's protocol with the mutagenic primer shown below:
SEQ ID NO:7:
CTTCCCGTGA CCATGGGCCA TC
Putative mutants were screened for the presence of an Nco I site and a
plasmid,
designated pBT437, was shown to have the proper sequence in the vicinity of
the
mutation by DNA sequencing. The addition of an Nco I site at the translation
start codon also resulted in a change of the second codon from TTC coding for
phenylalanine to GTC coding for valine.
To achieve high level expression of the genes in E. coli the bacterial
expression vector pBT430 (see Example 2) was used. The E. coil dapA gene was
cut out of plasmid pBT437 as an 1150 bp Nco I-Hind III fragment and inserted
into the expression vector pBT430 digested with the same enzymes, yielding
plasmid pBT442. For expression of the Corynebacterium dapA gene, the 910 bp
Nco I to EcoR I fragment of SEQ ID NO:6 inserted in pBT430 (pFS766, see
Example 3) was used.
For high level expression each of the plasmids was transformed into E. coil
strain BL21(DE3) [Studier et al. (1986) J. Mol. Biol. 189:113-130]. Cultures
were
grown in LB medium containing ampicillin (100 mg/L) at 25 . At an optical
density at 600 nm of approximately 1, IPTG (isopropylthio-a-galactoside, the
inducer) was added to a final concentration of 0.4 mM and incubation was
continued for 3 h at 25 . The cells were collected by centrifugation and
resuspended in 1/20th (or 1/100th) the original culture volume in 50 mM NaCl;
50 mM Tris-Cl, pH 7.5; 1 mM EDTA, and frozen at -20 . Frozen aliquots of
1 mL were thawed at 37 and sonicated, in an ice-water bath, to lyse the
cells.
The lysate was centrifuged at 4 for 5 min at 15,000 rpm. The supernatant was
removed and the pellet was resuspended in I mL of the above buffer.
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The supernatant and pellet fractions of uninduced and IPTG-induced
cultures of BL21(DIE3)/pBT442 or BL21(DE3)/pFS766 were analyzed by SDS
polyacrylamide gel electrophoresis. The major protein visible by Coomassie
blue
staining in the supernatant and pellet fractions of both induced cultures had
a
molecular weight of 32-34 kd, the expected size for DHDPS. Even in the
uninduced cultures this protein was the most prominent protein produced.
In the BL21(DE3)/pBT442 IPTG-induced culture about 80% of the DHDPS
protein was in the supernatant and DHDPS represented 10-20% of the total
protein in the extract. In the BL21(DE3)/pFS766 IPTG-induced culture more than
50% of the DHDPS protein was in the pellet fraction. The pellet fractions in
both
cases were 90-95% pure DHDPS, with no other single protein present in
significant amounts. Thus, these fractions were pure enough for use in the
generation of antibodies. The pellet fractions containing 2-4 mg of either E.
coli
DHDPS or Coryneb=terium DHDPS were solubilized in 50 mM NaCl; 50 mM
Tris-Cl, pH 7.5; 1 mM EDTA, 0.2 mM dithiothreitol, 0.2% SDS and sent to
Hazelton Research Facility (310 Swampridge Road, Denver, PA 17517) to have
rabbit antibodies raised against the proteins.
DHDPS enzyme activity was assayed as follows:
Assay mix (for 10 X 1.0 mL assay tubes or 40 X 0.25 mL for microtiter dish);
made fresh, just before use:
2.5 mL H ,!O
0.5 mL 1.0 M Tris-11C1 pH8.0
0.5 mL 0.1 M Na Pyruvate
0.5 mL o-Aminobenzaldehyde (10mg/mL in ethanol)
25 L 1.OM DL-Aspartic-o-semialdehyde (ASA) in 1.ON
HCI
Assay (1.0 mL): MicroAssay (0.25mL):
DHDPS assay mix 0.40 mL 0.10 mL
enzyme extract + H20; 0.10 mL .025 mL
mM L-lysine 5 L or 20 L I pL or 5 gL
Incubate at 30 for desired time. Stop by addition of.
1.0 N HC1 0.50 mL 0.125 mL
Color allowed to develop for 30-60 min. Precipitate spun down in eppendorf
centrifuge. OD540 vs 0 min read as blank. For MicroAssay, aliquot 0.2 mL into
microtiter well and read at OD530=
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The specific activity of E. coli DHDPS in the supernatant fraction of
induced extracts was about 50 OD54o units per minute per milligram protein in
a
1.0 mL assay. E. coli DHDPS was sensitive to the presence of L-lysine in the
assay. Fifty percent inhibition was found at a concentration of about 0.5 mM.
For
Corynebacterium DHDPS, the activity was measured in the supernatant fraction
of uninduced extracts, rather than induced extracts. Enzyme activity was about
4
OD530 units per min per milligram protein in a 0.25 mL assay. In contrast to
E. coli DHDPS, Corynebacterium DHDPS was not inhibited at all by L-lysine,
even at a concentration of 70 mM.
EXAMPLE 5
Excretion of amino acids by E. coli expressing huh levels of DHDPS and/or
AKIII
The E. coli expression cassette with the E. coli dapA gene linked to the T7
RNA polymerase promoter was isolated by digesting pBT442 (see Example 4)
with Bgl II and BamH I separating the digestion products via agarose gel
electrophoresis and eluting the approximately 1250 bp fragment from the gel.
This fragment was inserted into the BamH I site of plasmids pBT461 (containing
the T7 promoter/1vsC gene) and pBT492 (containing the T7 promoter/ivsC-M4
gene). Inserts where transcription of both genes would be in the same
direction
were identified by restriction endonuclease analysis yielding plasmids pBT517
(T7/dapA + T7/1vsC-M4) and pBT519 (T7/dapA + T7/1 sC .
In order to induce E. coli to produce and excrete amino acids, these
plasmids, as well as plasmids pBT442, pBT461 and pBT492 (and pBR322 as a
control) were transformed into E. coli strain BL21(DE3) [Studier et al. (1986)
J.
Mol. Biol. 189:113-130]. All of these plasmids, but especially pBT517 and
pBT519, are somewhat unstable in this host strain, necessitating careful
maintenance of selection for ampicillin resistance during growth.
All strains were grown in minimal salts M9 media [see Sambrook et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory
Press] supplemented with ampicillin to maintain selection for the plasmids
overnight at 37 . Cultures were collected when they reached an OD600 of 1.
Cells were removed by centrifugation and the supernatants (3 mL) were passed
through 0.2 micron filters to remove remaining cells and large molecules. Five
microliter aliquots of the supernatant fractions were analyzed for amino acid
composition with a Beckman Model 6300 amino acid analyzer using post-column
ninhydrin detection. Results are shown in Table 1.
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TABLE I
Amino Acid Concentration in Culture Supernatants [mM]
Plasmid LYS Thr Met Ala Val Asp Glu
pBR322 0 0 0 0.05 0.1 0 0
pBT442 0.48 0 0 0.04 0.06 0 0
pBT461 0.14 0.05 0 0.02 0.03 0 0
pBT492 0.16 0.07 0 0.02 0.03 0 0
pBT517 0.18 0 0.01 0 0 0.02 0.02
pBT519 0.14 0 0.01 0 0 0.01 0
All of the plasmids, except the pBR322 control, lead to the excretion of
lysine into the culture medium. Expression of the lysC or the lysC-M4 gene
lead
to both lysine and threonine excretion. Expression of lysC-M4 + dapA lead to
excretion of lysine, methionine, aspartic acid and glutamic acid, but not
threonine.
In addition, alanine and valine were not detected in the culture supernatant.
Similar results were obtained with lysC + dapA, except that no glutamic acid
was
excreted.
EXAMPLE 6
Construction of Chimeric dapA, lysC and lysC-M4 Genes for Expression in Plants
Several gene expression cassettes were used for construction of chimeric
genes for expression of ecoda~LA, cordapA= l and ~sC-M4 in plants. A leaf
expression cassette (Figure 4a) is composed of the 35S promoter of cauliflower
mosaic virus [Odell et al.(1985) Nature 313:810-812; Hull et al. (1987)
Virology
86:482-493], the translation leader from the chlorophyll alb binding protein
(Cab)
gene, [Dunsmuir (1985) Nucleic Acids Res. 13:2503-2518] and 3' transcription
termination region from the nopaline synthase (Nos) gene [Depicker et al.
(1982)
J Mol. Appl. Genet. 1:561-570]. Between the 5' and 3' regions are the
restriction
endonuclease sites Nco I (which includes the ATG translation initiation
codon),
EcoR I, Sma I and Kpn I. The entire cassette is flanked by Sal I sites; there
is also
a BamH I site upstream of the cassette.
A seed-specific expression cassette (Figure 4b) is composed of the promoter
and transcription terminator from the gene encoding the 0 subunit of the seed
storage protein phaseolin from. the bean Phaseolus vulgaris [Doyle et al.
(1986) J.
Biol. Chem. 261:9228-9238]. The phaseolin cassette includes about 500
nucleotides upstream (5') from. the translation initiation codon and about
1650
nucleotides downstream (3') from the translation stop codon of phaseolin.
Between the 5' and 3' regions are the unique restriction endonuclease sites
Nco I
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(which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I.
The entire cassette is flanked by Hind III sites.
A second seed expression cassette was used for the cordapA gene. This was
composed of the promoter and transcription terminator from the soybean Kunitz
tyrosine inhibitor 3 (KTI3) gene [Jofuku et al. (1989) Plant Cell 1:427-435].
The
KTI3 cassette includes about 2000 nucleotides upstream (5') from the
translation
initiation codon and about 240 nucleotides downstream (3') from the
translation
stop codon of phaseolin. Between the 5' and 3' regions are the unique
restriction
endonuclease sites Nco I (which includes the ATG translation initiation
codon),
Xba I, Kpn I and Sma I. The entire cassette is flanked by BamH I sites.
A constitutive expression cassette for corn was used for expression of the
lysC-M4 gene and the ecodapA gene. It was composed of a chimeric promoter
derived from pieces of two corn promoters and modified by in vitro site-
specific
mutagenesis to yield a high level constitutive promoter and a 3' region from a
corn
gene of unknown function. Between the 5' and 3' regions are the unique
restriction endonuclease sites Nco I (which includes the ATG translation
initiation
codon), Sma I and Bgl II. The nucleotide sequence of the constitutive corn
expression cassette is shown in SEQ ID NO:93.
Plant amino acid biosynthetic enzymes are known to be localized in the
chloroplasts and therefore are synthesized with a chloroplast targeting
signal.
Bacterial proteins such as DHDPS and AKIII have no such signal. A chloroplast
transit sequence (cts) was therefore fused to the ecodapA, cordapA, IysC, and
1ysC-M4 coding sequence in some chimeric genes. The cts used was based on the
cts of the small subunit of ribulose 1,5-bisphosphate carboxylase from soybean
[Berry-Lowe et al. (1982) J. Mol. Appl. Genet. 1:483-498]. The
oligonucleotides
SEQ ID NOS: 8-11 were synthesized and used as described below. For corn the
cts used was based on the cts of the small subunit of ribulose 1,5-
bisphosphate
carboxylase from corn [Lebrun et al. (1987) Nucleic Acids Res. 15:43601 and is
designated mcts to distinguish it from the soybean cts. The oligonucleotides
SEQ
ID NOS: 17-22 were synthesized and used as described below.
Fourteen chimeric genes were created:
No. 1) 35S promoter/Cab leader/lysC/Nos 3'
No. 2) 35S promoter/Cab leader/cts/1_ysC/Nos 3'
No. 3) 35S promoter/Cab leader/cts/lysC-M4/Nos 3'
No. 4) phaseolin 5' region/cts/lysC/phaseolin 3' region
No. 5) phaseolin 5' region/cts/lysC-M4/phaseolin 3' region
No. 6) 35S promoter/Cab leader/ecodapAA/Nos 3'
No. 7) 35S promoter/Cab leader/cts/ecodapAA/Nos 3
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No. 8) phaseolin 5' region/ecodarA/phaseolin 3' region
No. 9) phaseolin 5' region/cts/ecodapA/phaseolin 3' region
No. 10) 35S promoter/Cab leader/cts/cordapA/Nos 3
No. 11) phaseolin 5' region/cts/cordapA/phaseolin 3' region
No. 12) KTI3 5' region/cts/cord aapA/KTI3 3' region
No. 13) HH534 5' region/mcts/lysC-M4/HH2-1 3' region
No. 14) HH534 5' region/mcts/ecodayA/HH2-1 3' region
A 1440 bp Nco I-Hpa I fragment containing the entire 1ysC coding region
plus about 90 bp of 3' non-coding sequence was isolated from an agarose gel
following electrophoresis and inserted into the leaf expression cassette
digested
with Nco I and Sma I (chimeric gene No. 1), yielding plasmid pBT483.
Oligonucleotides SEQ ]:D NO:8 and SEQ ID NO:9, which encode the
carboxy terminal part of the chloroplast targeting signal, were annealed,
resulting
in Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and
inserted into Nco I digested pBT461. The insertion of the correct sequence in
the
correct orientation was verified by DNA sequencing yielding pBT496.
Oligonucleotides SEQ ID NO: 10 and SEQ ID NO: 11, which encode the amino
terminal part of the chloroplast targeting signal, were annealed, resulting in
Nco I
compatible ends, purified via polyacrylamide gel electrophoresis, and inserted
into
Nco I digested pBT496. The insertion of the correct sequence in the correct
orientation was verified by DNA sequencing yielding pBT521. Thus the cts was
fused to the lysC gene.
To fuse the cts to the l sC-M4 gene, pBT521 was digested with Sal I, and
an approximately 900 bp DNA fragment that included the cts and the amino
terminal coding region of lysC was isolated. This fragment was inserted into
Sal I
digested pBT492, effectively replacing the amino terminal coding region of
1vsC-M4 with the fused cts and the amino terminal coding region of 1vsC. Since
the mutation that resulted in lysine-insensitivity was not in the replaced
fragment,
the new plasmid, pBT523, carried the cts fused to 1ysC-M4.
The 1600 bp Nco I-Hpa I fragment containing the cts fused to lysC plus
about 90 bp of 3' non-coding sequence was isolated and inserted into the leaf
expression cassette digested with Nco I and Sma I (chimeric gene No. 2),
yielding
plasmid pBT541 and the seed-specific expression cassette digested with Nco I
and
Sma I (chimeric gene No. 4), yielding plasmid pBT543.
Similarly, the 1600 bp Nco I-Hpa I fragment containing the cts fused to
1 rsC-M4 plus about 90 bp of 3' non-coding sequence was isolated and inserted
into the leaf expression cassette digested with Nco I and Sma I (chimeric gene
No.
3), yielding plasmid pBT540 and the seed-specific expression cassette digested
with Nco I and Sma l (chimeric gene No. 5), yielding plasmid pBT544.
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Before insertion into the expression cassettes, the ecodapA gene was
modified to insert a restriction endonuclease site, Kpn I, just after the
translation
stop codon. The oligonucleotides SEQ ID NOS: 12-13 were synthesized for this
purpose:
SEQ ID NO:12:
CCGGTTTGCT GTAATAGGTA CCA
SEQ ID NO:13:
AGCTTGGTAC CTATTACAGC AAACCGGCAT G
Oligonucleotides SEQ ID NO:12 and SEQ ID NO:13 were annealed,
resulting in an Sph I compatible end on one end and a Hind III compatible end
on
the other and inserted into Sph I plus Hind III digested pBT437. The insertion
of
the correct sequence was verified by DNA sequencing yielding pBT443.
An 880 bp Nco I-Kpn I fragment from pBT443 containing the entire
ecodapA coding region was isolated from an agarose gel following
electrophoresis
and inserted into the leaf expression cassette digested with Nco I and Kpn I
(chimeric gene No. 6), yielding plasmid pBT450 and into the seed-specific
expression cassette digested with Nco I and Kpn I (chimeric gene No. 8),
yielding
plasmid pBT494.
Oligonucleotides SEQ ID NO:8 and SEQ ID NO:9, which encode the
carboxy terminal part of the chloroplast targeting signal, were annealed
resulting
in Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and
inserted into Nco I digested pBT450. The insertion of the correct sequence in
the
correct orientation was verified by DNA sequencing yielding pBT45 1. A 950 bp
Nco I-Kpn I fragment from pBT451 encoding the carboxy terminal part of the
chloroplast targeting signal fused to the entire ecodapA coding region was
isolated
from an agarose gel following electrophoresis and inserted into the seed-
specific
expression cassette digested with Nco I and Kpn I, yielding plasmid pBT495.
Oligonucleotides SEQ ID NO:10: and SEQ ID NO: 11:, which encode the amino
terminal part of the chloroplast targeting signal, were annealed resulting in
Nco I
compatible ends, purified via polyacrylamide gel electrophoresis, and inserted
into
Nco I digested pBT451 and pBT495. Insertion of the correct sequence in the
correct orientation was verified by DNA sequencing yielding pBT455 and
pBT520, respectively. Thus the cts was fused to the ecodapA gene in the leaf
expression cassette (chimeric gene No. 7) and the seed-specific expression
cassette (chimeric gene No. 9).
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An 870 bp Nco I-EcoR I fragment from pFS766 containing the entire
cordapA coding region was isolated from an agarose gel following
electrophoresis
and inserted into the leaf expression cassette digested with Nco I and EcoR I,
yielding plasmid pFS789. To attach the cts to the cordapA gene, a DNA fragment
containing the entire cts was prepared using PCR. The template DNA was
pBT540 and the oligonucleotide primers used were:
SEQ ID NO:14:
GCTTCCTCAA TGATCTCCTC CCCAGCT
SEQ ID NO:15:
CATTGTACTC TTCCACCGTT GCTAGCAA
PCR was performed using a Perkin-Elmer Cetus kit according to the
instructions of the vendor on a thermocycler manufactured by the same company.
The PCR-generated 160 bp fragment was treated with T4 DNA polymerase in the
presence of the 4 deoxyribonucleotide triphosphates to obtain a blunt-ended
fragment. The cts fragment was inserted into pFS789 which had been digested
with Nco I and treated with the Klenow fragment of DNA polymerase to fill in
the
5' overhangs. The inserted fragment and the vector/insert junctions were
determined to be correct by DNA sequencing, yielding pFS846 containing
chimeric gene No. 10.,
A 1030 bp Nco I-Kpn I fragment from pFS846 containing the cts attached to
the cordapA coding region was isolated from an agarose gel following electro-
phoresis and inserted into the phaseolin seed expression cassette digested
with
Nco I and Kpn I, yielding plasmid pFS889 containing chimeric gene No. 11.
Similarly, the 1030 bp Nco I-Kpn I fragment from pFS846 was inserted into the
KTI3 seed expression cassette digested with Nco I and Kpn I, yielding plasmid
pFS862 containing chimeric gene No. 12.
Oligonucleotides SEQ II) NO:94 and SEQ ID NO:95, which encode the
carboxy terminal part of the corn chloroplast targeting signal, were annealed,
resulting in Xba I and Nco I compatible ends, purified via polyacrylamide gel
electrophoresis, and inserted into Xba I plus Nco I digested pBT492 (see
Example
2). The insertion of the correct sequence was verified by DNA sequencing
yielding pBT556. Oligonucleotides SEQ ID NO:96 and SEQ ID NO:97, which
encode the middle part of the chloroplast targeting signal, were annealed,
resulting
in Bgl II and Xba I compatible ends, purified via polyacrylamide gel
electrophoresis, and inserted into Bgl II and Xba I digested pBT556. The
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insertion of the correct sequence was verified by DNA sequencing yielding
pBT557. Oligonucleotides SEQ ID NO:98 and SEQ ID NO:99, which encode the
amino terminal part of the chloroplast targeting signal, were annealed,
resulting in
Nco I and Afl II compatible ends, purified via polyacrylamide gel
electrophoresis,
and inserted into Nco I and Afl II digested pBT557. The insertion of the
correct
sequence was verified by DNA sequencing yielding pBT558. Thus the mcts was
fused to the lysC-M4 gene.
A 1.6 kb Nco I-Hpa I fragment from pBT558 containing the mots attached
to the lysC-M4 gene was isolated from an agarose gel following electrophoresis
and inserted into the constitutive corn expression cassette digested with Nco
I and
Sma I, yielding plasmid pBT573 containing chimeric gene No. 13.
To attach the mcts to the ecodapA gene a DNA fragment containing the
entire mots was prepared using PCR as described above. The template DNA was
pBT558 and the oligonucleotide primers used were:
SEQ ID NO:100:
GCGCCCACCG TGATGA
SEQ ID NO:101:
CACCGGATTC TTCCGC
The mots fragment was inserted into pBT450 (above) which had been
digested with Nco I and treated with the Klenow fragment of DNA polymerase to
fill in the 5' overhangs. The inserted fragment and the vector/insert
junctions were
determined to be correct by DNA sequencing, yielding pBT576. Plasmid pBT576
was digested with Asp718, treated with the Klenow fragment of DNA polymerase
to yield a blunt-ended fragment, and then digested with Nco I. The resulting
1030
bp Nco I-blunt-ended fragment containing the ecodagA gene attached to the mots
was isolated from an agarose gel following electrophoresis. This fragment was
inserted into the constitutive corn expression cassette digested with Bgl II,
treated
with the Klenow fragment of DNA polymerase to yield a blunt-ended fragment,
and then digested with Nco I, yielding plasmid pBT583 containing chimeric gene
No. 14.
EXAMPLE 7
Transformation of Tobacco with the 35S Promoter/lysC Chimeric Genes
Transformation of tobacco with the 35S promoter/1vsC chimeric genes was
effected according to the following:
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The 35S promoter/Cab leader/1vsC/Nos 3', 35S promoter/Cab
leader/cts/lysC/Nos 3', and 35S promoter/Cab leader/cts/lysC-M4/Nos 3'
chimeric
genes were isolated as 3.5-3.6 kb BamH I-EcoR I fragments and inserted into
BamH I-EcoR I digested vector pZS97K (Figure 5), yielding plasmids pBT497,
pBT545 and pBT542, respectively. The vector is part of a binary Ti plasmid
vector system [Bevan., (1984) IVucl. Acids. Res. 12:8711-8720] of
Agrobacterium
tumefaciens. The vector contains: (1) the chimeric gene nopaline synthase
promoter/neomycin phosphotransferase coding region (nos:NPT II) as a
selectable
marker for transformed plant cells [Bevan et al. (1983) Nature 304:184-186];
(2) the left and right borders of the T-DNA of the Ti plasmid [Bevan (1984)
Nucl.
Acids. Res. 12:8711-8720]; (3) the E. coli lacZ a-complementing segment [Viera
and Messing (1982) Gene 19:259-267] with unique restriction endonuclease sites
for EcoR I, Kpn I, BamH I and Sal I; (4) the bacterial replication origin from
the
Pseudomonas plasmid pVS1 [Itoh et al. (1984) Plasmid 11:206-220]; and (5) the
bacterial neomycin phosphotransferase gene from Tn5 [Berg et al. (1975) Proc.
Natl. Acad Sci. U.S.A. 72:3628-3632] as a selectable marker for transformed A.
tumefaciens.
The 35S promoter/Cab leader/cts/1vsC/Nos 3', and 35S promoter/Cab
leader/cts/lysC-M4/Nos 3' chimeric genes were also inserted into the binary
vector
pBT456, yielding pBT547 and pBT546, respectively. This vector is pZS97K, into
which the chimeric gene 35S promoter/Cab leader/cts/dapA/Nos 3' had previously
been inserted as a BamH I-Sal I fragment (see Example 9). In the cloning
process
large deletions of the dapA chimeric gene occurred. As a consequence these
plasmids are equivalent to pBT545 and pBT542, in that the only transgene
expressed in plants (other than the selectable marker gene, NPT II) was 35S
promoter/Cab leader/cts/1vsC/Nos 3' or 35S promoter/Cab leader/cts/lysC-M4/Nos
3'.
The binary vectors containing the chimeric lysC genes were transferred by
tri-parental matings [F:uvkin et: al. (1981) Nature 289:85-88] to
Agrobacterium
strain LBA4404/pAL4404 [Hockema et al (1983), Nature 303:179-180]. The
Agrobacterium transformants were used to inoculate tobacco leaf disks [Horsch
et
al. (1985) Science 227:1229-1231]. Transgenic plants were regenerated in
selective medium containing kanamycin.
To assay for expression of the chimeric genes in leaves of the transformed
plants, protein was extracted as follows. Approximately 2.5 g of young plant
leaves, with the midrib removed, were placed in a dounce homogenizer with 0.2
g
of polyvinyl polypyrrolidone and 11 mL of 50mM Tris-HCl pH8.0, 50mM NaCl,
1 mM EDTA (TNE) and ground thoroughly. The suspension was further
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homogenized by a 20 sec treatment with a Brinkman PolytronTM Homogenizer
operated at setting 7. The resultant suspensions were centrifuged at 16,000
rpm
for 20 min at 41 in a Dupont-Sorvall superspeed centrifuge using an SS34 rotor
to
remove particulates. The supernatant was decanted, the volume was adjusted to
be 10 mL by addition of THE if necessary, and 8 ml, of cold, saturated
ammonium sulfate was added. The mixture was set on ice for 30 min and
centrifuged as described above. The supernatant was decanted and the pellet,
which contained the AKIII protein, was resuspended in 1 mL of THE and desalted
by passage over a SephadexTM G-25 M column (Column PD- 10, Pharmacia).
For immunological characterization, three volumes of extract were mixed
with 1 volume of 4 X SDS-gel sample buffer (0.17M Tris-HC1 pH6.8, 6.7% SDS,
16.7% (v/v) (3-mercaptoethanol, 33% (v/v) glycerol) and 3 L from each extract
were run per lane on an SDS polyacrylamide gel, with bacterially produced
AKIII
serving as a size standard and protein extracted from untransformed tobacco
leaves serving as a negative control. The proteins were then
electrophoretically
blotted onto a nitrocellulose membrane (Western Blot). The membranes were
exposed to the AKIII antibodies prepared as described in Example 2 at a 1:5000
dilution of the rabbit serum using standard protocol provided by BioRad with
their
Immun-Blot Kit. Following rinsing to remove unbound primary antibody, the
membranes were exposed to the secondary antibody, donkey anti-rabbit Ig
conjugated to horseradish peroxidase (Amersham) at a 1:3000 dilution.
Following
rinsing to remove unbound secondary antibody, the membranes were exposed to
Amersham chemiluminescence reagent and X-ray film.
Seven of thirteen transformants containing the chimeric gene, 35S
promoter/Cab leader/ets/lysC-M4/Nos 3', and thirteen of seventeen
transformants
containing the chimeric gene, 35S promoter/Cab leader/cts/ sC/Nos 3', produced
AKIII protein (Table 2). In all cases protein which reacted with the AKIII
antibody was of several sizes. Approximately equal quantities of proteins
equal in
size to AKIII produced in E. col', and a protein about 6 kd larger were
evident in
all samples, suggesting that the chloroplast targeting signal had been removed
from about half of the protein synthesized. This further suggests that about
half of
the protein entered the chloroplast. In addition, a considerable amount of
protein
of higher molecular weight was observed. The origin of this protein is
unclear;
the total amount present was equal or slightly greater than the amounts of the
mature and putative AKIII precursor proteins combined.
The leaf extracts were assayed for AK activity as described in Example 2.
AKIII could be distinguished from endogenous AK activity, if it were present,
by
its increased resistance to lysine plus threonine. Unfortunately, however,
this
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assay was not sensitive enough to reliably detect AKIII activity in these
extracts.
Zero of four transformants containing the chimeric gene, 35S promoter/Cab
leader/lysC/Nos 3', showed AKIII activity. Only one extract, from a
transformant
containing the 35S promoter/Cab leader/cts/lysC-M4/Nos 3' gene, produced a
convincing level of enzyme activity. This came from transformant 546-49A, and
was also the extract that showed the highest level of AKIII-M4 protein via
Western blot.
An alternative method to detect the expression of active AKIII enzyme was
to evaluate the sensitivity or resistance of leaf tissue to high
concentrations of
lysine plus threonine. Growth of cell cultures and seedlings of many plants is
inhibited by high concentrations of lysine plus threonine; this is reversed by
addition of methionine (or homoserine which is converted to methionine in
vivo).
Lysine plus threonine inhibition is thought to result from feedback inhibition
of
endogenous AK, which reduces flux through the pathway leading to starvation
for
methionine. In tobacco there are two AK enzymes in leaves, one lysine-
sensitive
and one threonine sensitive [Negrutui et al. (1984) Theor. Appl. Genet. 68:11-
20].
High concentrations of lysine plus threonine inhibit growth of shoots from
tobacco leaf disks and. inhibition is reversed by addition of low
concentrations of
methionine. Thus, growth inhibition is presumably due to inhibition of the two
AK isozymes.
Expression of active lysine and threonine insensitive AKIII-M4 would be
predicted to reverse the growth inhibition. As can be seen in Table 2, this
was
observed. There is, in fact, a good correlation between the level of AKIII-M4
protein expressed and the resistance to lysine plus threonine inhibition.
Expression of lysine-sensitive wild type AKIII does not have a similar effect.
Only the highest expressing transformant showed any resistance to lysine plus
threonine inhibition, and this was much less dramatic than that observed with
AKIII-M4.
To measure free amino acid composition of the leaves, free amino acids
were extracted as follows. Approximately 30-40 mg of young leaf tissue was
chopped with a razor and dropped into 0.6 mL of methanol/ chloroform/water
mixed in ratio of 12v/5v/3v (MCW) on dry ice. After 10-30 min the suspensions
were brought to room temperature and homogenized with an Omni 1000
Handheld Rechargeable Homogenizer and then centrifuged in an eppendorf
microcentrifuge for 3 min. Approximately 0.6 mL of supernatant was decanted
and an additional 0.2 :mL of MCW was added to the pellet which was then
vortexed and centrifuged as above. The second supernatant, about 0.2 mL, was
added to the first. To this, 0.2mL of chloroform was added followed by 0.3 mL
of
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water. The mixture was vortexed and the centrifuged in an eppendorf
microcentrifuge for about 3 min, the upper aqueous phase, approximately 1.0
mL,
was removed, and was dried down in a Savant Speed Vac Concentrator.
One-tenth of the sample was run on a Beckman Model 6300 amino acid analyzer
using post-column ninhydrin detection. Relative free amino acid levels in the
leaves were compared as ratios of lysine or threonine to leucine, thus using
leucine as an internal standard. There was no consistent effect of expression
of
AKIII or AKIII-M4 on the lysine or threonine (or any other amino acid) levels
in
the leaves (Table 2).
TABLE 2
BT542 transformants: 35S promoter/Cab leader/cts/lysC-M4/Nos 3'
BT545 transformants: 35S promoter/Cab leader/cts/1vsC/Nos 3'
BT546 transformants: 35S promoter/Cab leader/cts/jsC-M4/Nos 3'
BT547 transformants: 35S promoter/Cab leader/cts/lysC/Nos 3'
FREE AMINO AKIII RESISTANCE
ACIDS/LEAF ACTIVITY WESTERN TO Lys 3mM
LINE K/L T/L U/MG/HR BLOT + Thr 3mM
542-5B 0.5 3.5 0 - -
542-26A 0.5 3.3 0 - -
542-27B 0.5 3.4 0 ++ +++
542-35A 0.5 4.3 0.01 - -
542-54A 0.5 2.8 0 - -
542-57B 0.5 3.4 0 - +
545-5A n.d. n.d. 0.02 ++
545-7B 0.5 3.4 0 +
545-17B 0.6 2.5 0.01 +
545-27A 0.6 3.5 0 ++
545-50E 0.6 3.6 0.03 ++
545-52A 0.5 3.6 0.02 -
546-4A 0.4 4.5 0 + +
546-24B 0.6 4.9 0.04 ++ ++
546-44A 0.5 6.0 0.03 + ++
546-49A 0.7 7.0 0.10 +++ +++
546-54A 0.5 6.4 0 + +
546-56B 0.5 4.4 0.01 - -
546-58B 0.6 8.0 0 + ++
547-3D 0.4 5.4 0 ++ -
547-8B 0.6 5.0 0.02 -
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547-9A O.:5 4.3 0.03 +++
547-12A 0.7 3.9 0 +++ +
547-15B 0.6 4.5 0 + -
547-16A 0.5 3.6 0 ++
547-18A O.5 4.0 +++ -
547-22A 0.8 4.4 -
547-25C O.5 4.3 + -
547-28C 0.6 5.6 -
547-29C 0.5 3.8 +++ +
EXAMPLE 8
Transformation of Tobacco with the Phaseolin Promoter/lysC Chimeric Genes
The phaseolin promoter/lysC chimeric gene cassettes, phaseolin 5'
region/cts/lysC/phaseolin 3' region, and phaseolin 5' region/cts/lysC-
M4/phaseolin
3' region (Example 6) were isolated as approximately 3.3 kb Hind III
fragments.
These fragments were inserted into the unique Hind III site of the binary
vector
pZS97 (Figure 6) yielding pBT548 and pBT549, respectively. This vector is
similar to pZS97K described in Example 7 except for the presence of two
additional unique cloning sites, Sma I and Hind III, and the bacterial (3-
lactamase
gene (causing ampicillin resistance) as a selectable marker for transformed
A. tumefaciens instead of the bacterial neomycin phosphotransferase gene.
The binary vectors containing the chimeric lysC genes were transferred by
tri-parental matings to Agrobacterium strain LBA4404/pAL4404, the
Agrobacterium transfbrmants were used to inoculate tobacco leaf disks and
transgenic plants regenerated by the methods set out in Example 7.
To assay for expression of the chimeric genes in the seeds of the
transformed plants, the plants were allowed to flower, self-pollinate and go
to
seed. Total proteins were extracted from mature seeds as follows.
Approximately
30-40 mg of seeds were put into a 1.5mL disposable plastic microfuge tube and
ground in 0.25 mL of 50 mM 'Tris-HC1 pH6.8, 2 mM EDTA, 1% SDS, 1% (v/v)
R-mercaptoethanol. The grinding was done using a motorized grinder with
disposable plastic shafts designed to fit into the microfuge tube. The
resultant
suspensions were centrifuged for 5 min at room temperature in a microfuge to
remove particulates. Three volumes of extract was mixed with 1 volume of 4 X
SDS-gel sample buffer (0.17 M Tris-HC1 pH 6.8, 6.7% SDS, 16.7% (v/v)
(i-mercaptoethanol, 33% (v/v) glycerol) and 5 L from each extract were run
per
lane on an SDS polyacrylamide gel, with bacterially produced AKIII serving as
a
size standard and protein extracted from untransformed tobacco seeds serving
as a
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negative control. The proteins were then electrophoretically blotted onto a
nitrocellulose membrane. The membranes were exposed to the AKIII antibodies
(prepared as described in Example 2) at a 1:5000 dilution of the rabbit serum
using standard protocol provided by BioRad with their Immun-Blot Kit.
Following rinsing to remove unbound primary antibody the membranes were
exposed to the secondary antibody, donkey anti-rabbit Ig conjugated to
horseradish peroxidase (Amersham) at a 1:3000 dilution. Following rinsing to
remove unbound secondary antibody, the membranes were exposed to Amersham
chemiluminescence reagent and X-ray film.
Ten of eleven transformants containing the chimeric gene, phaseolin 5'
region/cts/lysC/phaseolin 3' region, and ten of eleven transformants
containing the
chimeric gene, phaseolin 5' region/cts/lysC-M4/phaseolin 3' region, produced
AKIII protein (Table 3). In all cases protein which reacted with the AKIII
antibody was of several sizes. Approximately equal quantities of proteins
equal in
size to AKIII produced in E. coli, and about 6 kd larger were evident in all
samples, suggesting that the chloroplast targeting signal had been removed
from
about half of the protein synthesized. This further suggests that about half
of the
protein entered the chloroplast. In addition, some proteins of lower molecular
weight were observed, probably representing breakdown products of the AKIII
polypeptide.
To measure free amino acid composition of the seeds, free amino acids were
extracted from mature seeds as follows. Approximately 30-40 mg of seeds and an
approximately equal amount of sterilized sand were put into a 1.5 mL
disposable
plastic microfuge tube along with 0.2 mL of methanol/chloroform/water mixed in
ratio of 12v/5v/3v (MCW) at room temperature. The seeds were ground using a
motorized grinder with disposable plastic shafts designed to fit into the
microfuge
tube. After grinding an additional 0.5 mL of MCW was added, the mixture was
vortexed and then centrifuged in an eppendorf microcentrifuge for about 3 min.
Approximately 0.6 ml, of supernatant was decanted and an additional 0.2 mL of
MCW was added to the pellet which was then vortexed and centrifuged as above.
The second supernatant, about 0.2 mL, was added to the first. To this, 0.2 mL
of
chloroform was added followed by 0.3 mL of water. The mixture was vortexed
and then centrifuged in an eppendorf microcentrifuge for about 3 min, the
upper
aqueous phase, approximately 1.0 mL, was removed, and was dried down in a
Savant Speed Vac Concentrator. The samples were hydrolyzed in 6N
hydrochloric acid, 0.4% (v/v) f-mercaptoethanol under nitrogen for 24 h at
110-120 ; 1/4 of the sample was run on a Beckman Model 6300 amino acid
analyzer using post-column ninhydrin detection. Relative free amino acid
levels
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in the seeds were compared as ratios of lysine, methionine, threonine or
isoleucine
to leucine, thus using leucine as an internal standard.
To measure the total amino acid composition of the seeds, 6 seeds were
hydrolyzed in 6 N hydrochloric acid, 0.4% (v/v) P-mercaptoethanol under
nitrogen for 24 h at 110-120 ; 1 /10 of the sample was run on a Beckman Model
6300 amino acid analyzer using post-column ninhydrin detection. Relative amino
acid levels in the seeds were compared as ratios of lysine, methionine,
threonine
or isoleucine to leucine, thus using leucine as an internal standard. Because
the
transgene was segregating in these self-pollinated progeny of the primary
transformant and only six seeds were analyzed, there was expected to be some
sampling error. Therefore, the measurement was repeated multiple times for
some
of the lines (Table 3).
Expression of the cts/1vsC gene in the seeds resulted in a 2 to 4-fold
increase
in the level of free threonine in the seeds and a 2 to 3-fold increase in the
level of
free lysine in some cases. There was a good correlation between transformants
expressing higher levels of AKIII protein and those having higher levels of
free
threonine, but this was not the case for lysine. These relatively small
increases of
free threonine or lysine were not sufficient to yield detectable increases in
the
levels of total threonine or lysine in the seeds. Expression of the cts/lysC-
M4
gene in the seeds resulted in a 4 to 23-fold increase in the level of free
threonine in
the seeds and a 2 to 3-fold increase in the level of free lysine in some
cases. There
was a good correlation between transformants expressing higher levels of AKIII
protein and those having higher levels of free threonine, but this was again
not the
case for lysine. The larger increases of free threonine were sufficient to
yield
detectable increases in the levels of total threonine in the seeds. Sixteen to
twenty-five percent increases in total threonine content of the seeds were
observed
in three lines which were sampled multiple times. (Isoleucine to leucine
ratios are
shown for comparison.) The lines that showed increased total threonine were
the
same ones the showed the highest levels of increase in free threonine and high
expression of the AK][II-M4 protein. From these results it can be estimated
that
free threonine represents about I% of the total threonine present in a normal
tobacco seed, but about 18% of the total threonine present in seeds expressing
high levels of AKIII-M4.
TABLE 3
BT548 Transformants: phaseolin 5' :region/cts/IvsC/phaseolin 3'
BT549 Transformants: phaseolin 5' region/cts/lysC-M4/phaseolin 3'
SEED SEED
FREE AMINO ACID TOTAL AMINO ACID
LINE K/L T/L LL K/L T/L I/L WESTERN
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NORMAL 0.49 1.34 0.68 0.35 0.68 0.63 -
548-2A 1.15 2.3 0.78 0.43 0.71 0.67 +
548-4D 0.69 5.3 0.80 0.35 0.69 0.65 +
548-6A 0.39 3.5 0.85 0.35 0.69 0.64 +
548-7A 0.82 4.2 0.83 0.36 0.68 0.65 ++
548-14A 0.41 3.1 0.82 0.32 0.67 0.65 +
548-18A 0.51 1.5 0.69 0.37 0.67 0.63 -
548-22A 1.41 2.9 0.75 0.47 0.74 0.65 +++
548-24A 0.73 3.7 0.81 0.38 0.68 0.65 ++
548-41A 0.40 2.8 0.77 0.37 0.68 0.65 +
548-50A 0.46 4.0 0.81 0.33 0.68 0.65 +
548-57A 0.50 3.8 0.80 0.33 0.67 0.65 ++
549-5A 0.63 5.9 0.69 0.32 0.65 0.65 +
549-7A 0.51 8.3 0.78 0.33 0.67 0.63 ++
549-20A 0.67 30 0.88 0.38* 0.82* 0.65* ++++
549-34A 0.43 1.3 0.69 0.32 0.64 0.63 -
549-39D 0.83 16 0.83 0.35 0.71 0.63 +++
549-40A 0.80 4.9 0.74 0.33 0.63 0.64 +
549-41C 0.99 13 0.80 0.38* 0.79* 0.65* +
549-46A 0.48 7.7 0.84 0.34 0.70 0.64 +
549-52A 0.81 9.2 0.80 0.39 0.70 0.65 ++
549-57A 0.60 15 0.77 0.35* 0.85* 0.64* +++
549-60D 0.85 11 0.79 0.37 0.73 0.65 ++
Normal was calculated as the average of 6 samples for free amino acid and 23
samples for total
amino acids.
* Indicates average of at least 5 samples
Seeds derived from self-pollination of two plants transformed with the
phaseolin 5' region/cts/llysC-M4/phaseolin 3' region, plants 549-5A and 549-
40A,
showed 3 kanamycin resistant to 1 kanamycin sensitive seedlings, indicative of
a
single site of insertion of the transgene. Progeny plants were grown, self-
pollinated and seed was analyzed for segregation of the kanamycin marker gene.
Progeny plants that were homozygous for the transgene insert, thus containing
two copies of the gene cassette, accumulated approximately 2 times as much
threonine in their seed as their sibling heterozygous progeny with one copy of
the
gene cassette and about 8 times as much as seed without the gene. This
demonstrates that the level of expression of the E. coli enzyme controls the
accumulation of free threonine.
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EXAMPLE 9
Transformation of Tobacco with the 35S Promoter/ecodapA Chimeric Genes
The 35S promoter/Cab ]leader/ecodapA/Nos 3' and 35S promoter/Cab
leader/cts/ecodapA/Nos 3', chimeric genes were isolated as 3.1, and 3.3 kb
BamH I-Sal I fragments, respectively and inserted into BamH I-Sal I digested
binary vector pZS971K , (Figure 5), yielding plasmids pBT462 and pBT463,
respectively. The binary vector is described in Example 7.
The binary vectors containing the chimeric ecodapA genes were transferred
by tri-parental matings to Agrobacterium strain LBA4404/pAL4404, the
Agrobacterium transformants used to inoculate tobacco leaf disks and the
resulting transgenic plants regenerated by the methods set out in Example 7.
To assay for expression of the chimeric genes in leaves of the transformed
plants, protein was extracted as described in Example 7, with the following
modifications. The supernatant from the first ammonium sulfate precipitation,
approximately 18 mL, was mixed with an additional 12 mL of cold, saturated
ammonium sulfate. 'The mixture was set on ice for 30 min and centrifuged as
described in Example 7. The supernatant was decanted and the pellet, which
contained the DHDPS protein, was resuspended in 1 mL of THE and desalted by
passage over a Sephadex G-25 M column (Column PD-10, Pharmacia).
The leaf extracts were assayed for DHDPS activity as described in Example
4. E. coli DHDPS could be distinguished from tobacco DHDPS activity by its
increased resistance to lysine.; E. coli DHDPS retained 80-90% of its activity
at
0.1mM lysine, while tobacco DHDPS was completely inhibited at that
concentration of lysine. One of ten transformants containing the chimeric
gene,
35S promoter/Cab leader/ecodapA/Nos 3', showed E. coli DHDPS expression,
while five of ten trarisformants containing the chimeric gene, 35S
promoter/Cab
leader/cts/ecodapA/Nos 3' showed E. coli DHDPS expression.
Free amino acids were extracted from leaves as described in Example 7.
Expression of the chimeric gene, 35S promoter/Cab leader/cts/ecodapA/Nos 3',
but not 35S promoter/Cab leader/ecodapA/Nos 3' resulted in substantial
increases
in the level of free lysine in the leaves. Free lysine levels from two to 90-
fold
higher than untransftrmed tobacco were observed.
The transformed plants were allowed to flower, self-pollinate and go to
seed. Seeds from several lines transformed with the 35S promoter/Cab leader/
cts/ecodapA/Nos 3' gene were surface sterilized and germinated on agar plates
in
the presence of kanamycin. Lines that showed 3 kanamycin resistant to I
kanamycin sensitive seedlings, indicative of a single site of insertion of the
transgenes, were identified. Progeny that were homozygous for the transgene
insert were obtained from these lines using standard genetic analysis. The
SUBSTITUTE SHEET (RULE 26)
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homozygous progeny were then characterized for expression of E. coli DHDPS in
young and mature leaves and for the levels of free amino acids accumulated in
young and mature leaves and in mature seeds.
Expression of active E. coli DHDPS enzyme was clearly evident in both
young and mature leaves of the homozygous progeny of the transformants (Table
4). High levels of free lysine, 50 to 100-fold higher than normal tobacco
plants,
accumulated in the young leaves of the plants, but a much smaller accumulation
of
free lysine (2 to 8-fold) was seen in the larger leaves. Experiments that
measure
lysine in the phloem suggest that lysine is exported from the large leaves.
This
exported lysine may contribute to the accumulation of lysine in the small
growing
leaves, which are known to take up, rather than export nutrients. Since the
larger
leaves make up the major portion of the biomass of the plant, the total
increased
accumulation of lysine in the plant is more influenced by the level of lysine
in the
larger leaves. No effect on the free lysine levels in the seeds of these
plants was
observed (Table 4).
TABLE 4
Progeny of BT463 transformants homozygous for
35S promoter/Cab leader/cts/ecodaaA/Nos 3'
LEAF E. COLI SEED FREE
LEAF FREE AMINO ACID DHDPS AMINO ACID
LINE SIZE K/L K/TOT OD/60'/mg K/L
NORMAL 3 in. 0.5 0.006 0 0.5
463-18C-2 3 in. 47 0.41 7.6 0.4
463-18C-2 12 in. 1 0.02 5.5 ---
463-25A-4 3 in. 58 0.42 6.6 0.4
463-25A-4 12 in. 4 0.02 12.2 ---
463-38C-3 3 in. 28 0.28 6.1 0.5
463-38C-3 12 in. 2 0.04 8.3 ---
EXAMPLE 10
Transformation of Tobacco with the Phaseolin Promoter/ecodapA Chimeric
Genes
The chimeric gene cassettes, phaseolin 5' region/ecodapA/phaseolin 3'
region, and phaseolin 5' region/cts/ecodpAA/phaseolin 3' region (Example 6)
were
isolated as approximately 2.6 and 2.8 kb Hind III fragments, respectively.
These
fragments were inserted into the unique Hind III site of the binary vector
pZS97
(Figure 6), yielding pBT506 and pBT534, respectively. This vector is described
in Example 8.
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The binary vectors containing the chimeric ecodapA genes were transferred
by tri-parental matings to Agrobacterium strain LBA4404/pAL4404, the
Agrobacterium transformants used to inoculate tobacco leaf disks and the
resulting transgenic plants were regenerated by the methods set out in Example
7.
To assay for expression of the chimeric genes, the transformed plants were
allowed to flower, self-pollinate and go to seed. Total seed proteins were
extracted as described in Example 8 and immunologically analyzed as described
in Example 7, with the following modification. The Western blot membranes
were exposed to the DHDPS antibodies prepared in Example 4 at a 1:5000
dilution of the rabbit serum using standard protocol provided by BioRad with
their
Immun-Blot Kit.
Thirteen of fourteen transformants containing the chimeric gene, phaseolin
5' region/ecodapA/phaseolin 3' region and nine of thirteen transformants
containing the chimeric gene, phaseolin 5' region/cts/ecodapAA/phaseolin 3'
region,
produced DHDPS protein detectable via Western blotting (Table 3). Protein
which reacted with the DHDPS antibody was of several sizes. Most of the
protein
was equal in size to DHDPS produced in E. coli, whether or not the chimeric
gene
included the chloroplast transit sequence. This indicated that the chloroplast
targeting signal had been efficiently removed from the precursor protein
synthesized. This further suggests the majority of the protein entered the
chloroplast. In addition, some proteins of lower molecular weight were
observed,
probably representing breakdown products of the DHDPS polypeptide.
To measure free amino acid composition and total amino acid composition
of the seeds, free amino acids and total amino acids were extracted from
mature
seeds and analyzed as described in Example 8. Expression of either the ecodapA
gene or cts/ecodapA had no effect on the total lysine or threonine composition
of
the seeds in any of the transformed lines (Table 5). Several of the lines that
were
transformed with the iphaseolin 5' region/cts/ecodapAA/phaseolin 3' chimeric
gene
were also tested for any effect on the free amino acid composition. Again, not
even a modest effect on the lysine or threonine composition of the seeds was
observed in lines expressing high levels of E. coli DHDPS protein (Table 5).
This
was a surprising result, given the dramatic effect (described in Example 9)
that
expression of this protein has on the free lysine levels in leaves.
One possible explanation for this was that the DHDPS protein observed via
Western blot was not functional. To test this hypothesis, total protein
extracts
were prepared from mature seeds and assayed for DHDPS activity.
Approximately 30-40 mg of seeds were put into a 1.5 mL disposable plastic
microfuge tube and ground in 0.25 mL of 50 mM Tris-HCI, 50 mM NaCl, 1 mM
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EDTA (TNE). The grinding was done using a motorized grinder with disposable
plastic shafts designed to fit into the microfuge tube. The resultant
suspensions
were centrifuged for 5 min at room temperature in a microfuge to remove
particulates. Approximately 0.1 mL of aqueous supernatant was removed
between the pelleted material and the upper oil phase. The seed extracts were
assayed for DHDPS activity as described in Example 4. E. coli DHDPS could be
distinguished from tobacco DHDPS activity by its increased resistance to
lysine;
E. coli DHDPS retained about 50% of its activity at 0.4 mM lysine, while
tobacco
DHDPS was completely inhibited at that concentration of lysine. High levels of
E. coli DHDPS activity were seen in all four seed extracts tested eliminating
this
explanation.
The presence of the cts sequence in the chimeric ecodapA gene was
essential for eliciting accumulation of high levels of lysine in leaves. Thus
another possible explanation was that the cts sequence had somehow been lost
during the insertion of the chimeric phaseolin 5'
region/cts/ecodapAA/phaseolin 3'
gene into the binary vector. PCR analysis of several of the transformed lines
demonstrated the presence of the cts sequence, however, ruling out this
possibility.
A third explanation was that amino acids are not normally synthesized in
seeds, and therefore the other enzymes in the pathway were not present in the
seeds. The results of experiments presented in Example 8, wherein expression
of
phaseolin 5' region/cts/lysC-M4/phaseolin 3' gene resulted in accumulation of
high levels of free threonine in seeds, indicate that this is not the case.
Taken together these results and the results presented in Example 9,
demonstrate that expression of a lysine-insensitive DHDPS in either seeds or
leaves is not sufficient to achieve accumulation of increased free lysine in
seeds.
TABLE 5
BT506 Transformants: phaseolin 5' region/ecodapA/phaseolin 3'
BT534 Transformants: phaseolin 5' region/cts/ecodapAA/phaseolin 3'
SEED: FREE SEED: TOTAL E. COLI
AMINO ACIDS AMINO ACIDS DHDPS
LINE K/L T/L K/L T/L 0D/60'/MG WESTERN
NORMAL 0.49 1.34 0.35 0.68
506-2B 0.34 0.66 +
506-4B 0.33 0.67 +
506-16A 0.34 0.67 +
506-17A 0.36 0.55 7.7 +++
506-19A 0.37 0.45 ++
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506-22A 0.34 0.67 ++
506-23B 0.35 0.67 ++
506-33B 0.34 0.67 ++
506-38B 0.36 0.69 8.7 ++
506-39A 0.37 0.70 ++
506-40A 0.36 0.68 -
506-47A 0.32 0.68 +++
506-48A 0.33 0.69 +++
506-49A 0.33 0.69 +++
534-8A 0.34 0.66 -
534-9A 0.36 0.67 ++
534-22B 0.43 1.32 0.39 0.51 4.9 +++
534-31A 0.34 0.66 -
534-38A 0.35 1.49 0.42 0.33 +++
534-39A 0.38 0.69 +
534-7A 0.34 0.67 +++
534-25B 0.35 0.67 +++
534-34B 0.80 1.13 0.42 0.70 -
534-35A 0.43 1.18 0.33 0.67 +++
534-37B 0.42 1.58 0.37 0.68 -
534-43A 0.35 0.68 +++
534-48A 0.46 1.24 0.35 0.68 6.2 +++
EXAMPLE 11
Transformation of Tobacco with the 35S Promoter/cts/dapA
plus 35S Promoter/cts/lYsC-M4 Chimeric Genes
The 35S promoter/Cab leader/cts/ecodA/Nos 3', and 35S promoter/Cab
leader/cts/lysC-M4/Nos 3' chimeric genes were combined in the binary vector
pZS97K (Figure 5). The binary vector is described in Example 7. An
oligonucleotide adaptor was synthesized to convert the BamH I site at the 5'
end
of the 35S promoter/Cab leader/cts/1ysC-M4/Nos 3' chimeric gene (see Figure
4a)
to an EcoR I site. The 35S promoter/Cab leader/cts/lysC-M4/Nos 3' chimeric
gene was then isolated as a 3.6 kb EcoR I fragment from plasmid pBT540
(Example 6) and inserted into pBT463 (Example 9) digested with EcoR I,
yielding
plasmid pBT564. This vector has both the 35S promoter/Cab
leader/cts/ecodyAA/Nos 3', and 35S promoter/Cab leader/cts/ly C-M4/Nos 3'
chimeric genes inserted in the same orientation.
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The binary vector containing the chimeric ecodapA and 1ysC-M4 genes was
transferred by tri-parental matings to Agrobacterium strain LBA4404/pAL4404,
the Agrobacterium transformants used to inoculate tobacco leaf disks and the
resulting transgenic plants regenerated by the methods set out in Example 7.
To assay for expression of the chimeric genes in leaves of the transformed
plants, protein was extracted as described in Example 7 for AKIII, and as
described in Example 9 for DHDPS. The leaf extracts were assayed for DHDPS
activity as described in Examples 4 and 9. E. coli DHDPS could be
distinguished
from tobacco DHDPS activity by its increased resistance to lysine; E. coli
DHDPS
retained 80-90% of its activity at 0.1 mM lysine, while tobacco DHDPS was
completely inhibited at that concentration of lysine. Extracts were
characterized
immunologically for expression of AKIII and DHDPS proteins via Western blots
as described in Examples 7 and 10.
Ten of twelve transformants expressed E. coli DHDPS enzyme activity
(Table 6). There was a good correlation between the level of enzyme activity
and
the amount of DHDPS protein detected immunologically. As described in
Example 7, the AK assay was not sensitive enough to detect enzyme activity in
these extracts. However, AKIII-M4 protein was detected immunologically in
eight of the twelve extracts. In some transformants, 564-21A and 47A, there
was
a large disparity between the level of expression of DHDPS and AKIII-M4, but
in
of 12 lines there was a good correlation.
Free amino acids were extracted from leaves and analyzed for amino acid
composition as described in Example 7. In the absence of significant AKIII-M4,
the level of expression of the chimeric gene, 35S promoter/Cab
leader/cts/ecodapA/Nos 3' determined the level of lysine accumulation (Table
6).
Compare lines 564-21A, 47A and 39C, none of which expresses significant
AKIII-M4. Line 564-21A accumulates about 10-fold higher levels of lysine than
line 564-47A which expresses a lower level of E. coli DHDPS and 40-fold higher
levels of lysine than 564-39C which expresses no E. coli DHDPS. However, in
transformants that all expressed similar amounts of E. coli DHDPS (564-18A,
56A, 36E, 55B, 47A), the level of expression of the chimeric gene, 35S
promoter/Cab leader/cts/lysC-M4/Nos 3', controlled the level of lysine
accumulation. Thus it is clear that although expression of 35S promoter/Cab
leader/cts/lysC-M4/Nos 3' has no effect on the free amino acid levels of
leaves
when expressed alone (see Example 7), it can increase lysine accumulation when
expressed in concert with the 35S promoter/Cab leader/cts/ecodapAA/Nos 3'
chimeric gene. Expression of these genes together did not effect the level of
any
other free amino acid in the leaves.
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TABLE 6
BT564 Transformants: 3 5S promoter/Cab leader/cts/ecodapAA/Nos 3'
35S promoter/Cab leader/cts/lusC-M4/Nos 3'
E. COLI
FREE AA LEAF DHDPS WESTERN WESTERN
LINE nmoll4mg FREE AA LEAF U/MG/HR DHDPS AK-III
TOT K K/L K/TOT
564-21A 117 57 52 0.49 2.4 +++ +/-
564-18A 99 56 69 0.57 1.1 ++ ++
564-56A 104 58 58 0.56 1.5 ++ ++
564-36E 85 17 17 0.20 1.5 ++ +++
564-55B 54 5 9.1 0.10 1.0 ++ +
564-47A 18 1 4.8 0.06 0.8 ++
564-35A 37 7 13 0.18 0.3 + ++
564-60D 61 3 4.5 0.06 0.2 + ++
564-45A 46 it 8.1 0.09 0.4 + +
564-44B 50 1 1.7 0.02 0.1 +/- -
564-49A 53 1 1.0 0.02 0 +/- -
564-39C 62 1 1.4 0.02 0 - -
Free amino acids were extracted from mature seeds derived from self-
pollinated plants and quantitated as described in Example 8. There was no
significant difference in the free amino acid content of seeds from
untransformed
plants compared to that from the plants showing the highest free lysine
accumulation in leaves, i.e. plants 564-18A, 564-21A, 564-36E, 564-56A.
EXAMPLE 12
Transformation of Tobacco with the Phaseolin Promoter/cts/ecodapA plus
Phaseolin Premoter/cts/lysC-M4 Chimeric Genes
The chimeric gene cassettes, phaseolin 5' region/cts/ecodapAA/phaseolin 3'
region and phaseolin 5' region/cts/lysC-M4/phaseolin 3' (Example 6) were
combined in the binary vector pZS97 (Figure 6). The binary vector is described
in
Example 8. To accomplish this the phaseolin 5' region/cts/ecodapA/phaseolin 3'
chimeric gene was isolated as a 2.7 kb Hind III fragment and inserted into the
Hind III site of vector pUC1318 [Kay et al (1987) Nucleic Acids Res. 6:2778],
yielding pBT568. It was then possible to digest pBT568 with BamH I and isolate
the chimeric gene on a 2.7 kb BamH I fragment. This fragment was inserted into
BamH I digested pBT549 (Example 8), yielding pBT570. This binary vector has
both chimeric genes, phaseolin 5' region/cts/ecodpA/phaseolin 3' gene and
phaseolin 5' region/cts/lysC-114/phaseolin 3' inserted in the same
orientation.
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The binary vector pBT570 was transferred by tri-parental mating to
Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformants used
to inoculate tobacco leaf disks and the resulting transgenic plants
regenerated by
the methods set out in Example 7.
To assay for expression of the chimeric genes in the seeds of the
transformed plants, the plants were allowed to flower, self-pollinate and go
to
seed. Total proteins were extracted from mature seeds and analyzed via western
blots as described in Example 8.
Twenty-one of twenty-five transformants expressed the DHDPS protein and
nineteen of these also expressed the AKIII protein (Table 7). The amounts of
the
proteins expressed were related to the number of gene copies present in the
transformants; the highest expressing lines, 570-4B, 570-12C, 570-59B and
570-23B, all had two or more sites of insertion of the gene cassette based on
segregation of the kanamycin marker gene. Enzymatically active E. coli DHDPS
was observed in mature seeds of all the lines tested wherein the protein was
detected.
To measure free amino acid composition of the seeds, free amino acids were
extracted from mature seeds and analyzed as described in Example 8. There was
a
good correlation between transformants expressing higher levels of both DHDPS
and AKIII protein and those having higher levels of free lysine and threonine.
The highest expressing lines (marked by asterisk in Table 7) showed up to a 2-
fold
increase in free lysine levels and up to a 4-fold increase in the level of
free
threonine in the seeds.
In the highest expressing lines it was possible to detect a high level of
a-aminoadipic acid. This compound is known to be an intermediate in the
catabolism of lysine in cereal seeds, but is normally detected only via
radioactive
tracer experiments due to its low level of accumulation. The build-up of high
levels of this intermediate indicates that a large amount of lysine is being
produced in the seeds of these transformed lines and is passing through the
catabolic pathway. The build-up of a-aminoadipic acid was not observed in
transformants expressing only E. coli DHDPS or only AKIII-M4 in seeds. These
results show that it is necessary to express both enzymes simultaneously to
produce high levels of free lysine.
TABLE 7
BT570 Transformants: phaseolin 5'region/cts/ ysC-M4/phaseolin 3' region
phaseolin 5'region/cts/ecodayA/phaseoiin 3' region
FREE AMINO TOTAL AMINO WESTERN WESTERN E. COLT
ACIDS/SEED ACIDS/SEED E. COLT E. COLI DHDPS Progeny
LINE K/L T/L K/L T/L DHDPS AKIN U/MG/HR Kanr:Kans
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NORMAL 0.49 1.3 0.35 0.68 - -
570-4B 0.31 2.6 0.34 0.64 r-+-t ++ 15:1
570-7C 0.39 2.3 0.34 0.64 ++ +
570-8B 0.29 2.1 0.34 0.63 + -
570-12C* 0.64 5.1 0.36 0.68 ++++ +-H-+ > 4.3 >15:1
570-18A 0.33 3.0 0.35 0.65 ++ ++ 15:1
570-24A 0.33 2.0 0.34 0.65 ++ -
570-37A 0.33 2.1 0.434 0.64 +/- +/-
570-44A 0.29 2.1 034 0.64 ++ +
570-46B 0.41 2.1 0.35 0.65 ++ +
570-51B 0.33 1.5 033 0.64 - - 0
570-59B* 0.46 3.0 0:35 0.65 2.6 >15:1
570-80A 0.31 2.2 0.34 0.64 ++ +
570- I 1 A 0.28 2.3 0.34 0.67 ++ ++ 3:1
570-17B 0.27 1.6 0.34 0.65 - -
570-20A 0.41 2.3 0.35 0.67 ++ +
570-21B 0.26 2.4 0.34 0.68 ++ +
570-23B* 0.40 3.6 0.34 0.68 +++ +++ 3.1 63:1
570-25D 0.30 2.3 0.35 0.66 ++ +/-
570-26A 0.28 1.5 0.34 0.64 - -
570-32A 0.25 2.5 034 0.67 ++ +
570-35A 0.25 2.5 0.34 0.63 ++ ++ 3:1
570-38A-1 0.25 2.6 0.34 0.64 ++ ++ 3:1
570-38A-3 0.33 1.6 0.35 0.63 - -
570-42A 0.27 2.5 0.34 0.62 ++ ++ 3:1
570-45A 0.60 3.4 0.39 0.64 ++ ++ 3:1
* indicates free amino acid sample has rx-aminoadipic acid
EXAMPLE 13
Use of the cts/lysC-M4 Chimeric Gene as a Selectable
Marker for Tobacco Transformation
The 35S promoter/Cab deader/cts/lvsC-M4/Nos 3' chimeric gene in the
binary vector pZS97K (pBT542, see Example 7) was used as a selectable genetic
marker for transformation of tobacco. High concentrations of lysine plus
threonine inhibit growth of shoots from tobacco leaf disks. Expression of
active
lysine and threonine insensitive AKIII-M4 reverses this growth inhibition (see
Example 7).
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The binary vector pBT542 was transferred by tri-parental mating to
Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformants used
to inoculate tobacco leaf disks and the resulting transformed shoots were
selected
on shooting medium containing 3 mM lysine plus 3 mM threonine. Shoots were
transferred to rooting media containing 3 mM lysine plus 3 mM threonine.
Plants
were grown from the rooted shoots. Leaf disks from the plants were placed on
shooting medium containing 3 mM lysine plus 3 mM threonine. Transformed
plants were identified by the shoot proliferation which occurred around the
leaf
disks on this medium.
EXAMPLE 14
Transformation of Tobacco with the 35S Promoter/cts/cordapA Chimeric Gene
The 35S promoter/Cab leader/cts/cordapA/Nos 3' chimeric gene was
isolated as a 3.0 kb BamH I-Sal I fragment and inserted into BamH I-Sal I
digested binary vector pZS97K (Figure 5), yielding plasmid pFS852. The binary
vector is described in Example 7.
The binary vector containing the chimeric cordapA gene was transferred by
tri-parental mating to Agrobacterium strain LBA4404/pAL4404, the
Agrobacterium transformant was used to inoculate tobacco leaf disks and the
resulting transgenic plants were regenerated by the methods set out in Example
7.
To assay for expression of the chimeric gene in leaves of the transformed
plants, protein was extracted as described in Example 7, with the following
modifications. The supernatant from the first ammonium sulfate precipitation,
approximately 18 mL, was mixed with an additional 12 mL of cold, saturated
ammonium sulfate. The mixture was set on ice for 30 min and centrifuged as
described in Example 7. The supernatant was decanted and the pellet, which
contained the DHDPS protein, was resuspended in 1 mL of THE and desalted by
passage over a Sephadex G-25 M column (Column PD-10, Pharmacia).
The leaf extracts were assayed for DHDPS protein and enzyme activity as
described in Example 4. Corynebacteria DHDPS enzyme activity could be
distinguished from tobacco DHDPS activity by its insensitivity to lysine
inhibition. Eight of eleven transformants showed Corynebacteria DHDPS
expression, both as protein detected via western blot and as active enzyme.
Free amino acids were extracted from leaves as described in Example 7.
Expression of Corynebacteria DHDPS resulted in large increases in the level of
free lysine in the leaves (Table 8). However, there was not a good correlation
between the level of expression of DHDPS and the amount of free lysine
accumulated. Free lysine levels from 2 to 50-fold higher than untransformed
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tobacco were observed. There was also a 2 to 2.5-fold increase in the level of
total leaf lysine in the lines that showed high levels of free lysine.
TABLE 8
FS586 transformants: 35S promoter/Cab leader/cts/cordapA/Nos 3'
FREE AMINO TOTAL AMINO WESTERN CORYNE.
ACIDS/LEAF ACIDS/LEAF CORYNE. DHDPS
LINE K/L K/L DHDPS U/MG/HR
NORMAL 0.5 0.8 - -
FS586-2A 1.0 0.8 - -
FS586-4A 0.9 0.8 + 6.1
FS586-11B 3.6 0.8 + 3.4
FS586-11D 26 2.0 + 3.5
FS586-13A 2.4 0.8 + 3.5
FS586-19C 5.1 0.8 + 3.1
FS586-22B >15 1.5 + 2.3
FS586-30B 0.8 - -
FS586-38B 18 1.5 ++ 3.9
FS586-51A 1.3 0.8 - -
FS586-58C 1.2 0.8 + 5.1
The plants were allowed to flower, self-pollinate and go to seed. Mature
seed was harvested and assayed for free amino acid composition as described in
Example 8. There was no difference in the free lysine content of the
transformants compared to untransformed tobacco seed.
EXAMPLE 15
Transformation of Tobacco with the KTI3 promoter/cts/cordapA or
Phaseolin Promoter/cts/cordapA plus
Phaseolin Promoter/cts/lysC-M4 Chimeric Genes
The chimeric gene cassettes, KTI3 5' region/cts/ cord apAA/KTI3 3' region
and phaseolin 5' region/cts/ IL C-M4/phaseolin 3' as well as phaseolin 5'
region/cts/ cordapAA/phaseolin 3' region and phaseolin 5' region/cts/
lysC-M4/phaseolin 3' (Example 6) were combined in the binary vector pZS97
(Figure 6). The binary vector is described in Example 8.
To accomplish this the KTI3 5' region/cts/corda AA/ KTI3 3' region chimeric
gene cassette was isolated as a. 3.3 kb BamH I fragment and inserted into BamH
I
digested pBT549 (Example 8), yielding pFS883. This binary vector has the
chimeric genes, KTI3 5' region/cts/cordapAA/KTI3 3' region and phaseolin 5'
region/cts/l sC-M4/phaseolin 3' region inserted in opposite orientations.
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The phaseolin 5' region/cts/cordapA/phaseolin 3'region chimeric gene
cassette was modified using oligonucleotide adaptors to convert the Hind III
sites
at each end to BamH I sites. The gene cassette was then isolated as a 2.7 kb
BamH I fragment and inserted into BamH I digested pBT549 (Example 8),
yielding pFS903. This binary vector has both chimeric genes, phaseolin 5'
region/cts/cordayAA/phaseolin 3' region and phaseolin 5'
region/cts/IvsC-M4/phaseolin 3' region inserted in the same orientation.
The binary vectors pFS883 and pFS903 were transferred by tri-parental
mating to Agrobacterium strain LBA4404/pAL4404, the Agrobacterium
transformants were used to inoculate tobacco leaf disks and the resulting
transgenic plants were regenerated by the methods set out in Example 7.
To assay for expression of the chimeric genes in the seeds of the
transformed plants, the plants were allowed to flower, self-pollinate and go
to
seed. Total proteins were extracted from mature seeds and analyzed via western
blots as described in Example 8.
Twenty-one of twenty-two transformants tested expressed the DHDPS
protein and eighteen of these also expressed the AKIII protein (Table 8).
Enzymatically active Corynebacteria DHDPS was observed in mature seeds of all
the lines tested wherein the protein was detected except one.
To measure free amino acid composition of the seeds, free amino acids were
extracted from mature seeds and analyzed as described in Example 8. There was
a
good correlation between transformants expressing higher levels of both DHDPS
and AKIII protein and those having higher levels of free lysine and threonine.
The highest expressing lines showed up to a 3-fold increase in free lysine
levels
and up to a 8-fold increase in the level of free threonine in the seeds. As
was
described in Example 12, a high level of a-aminoadipic acid, indicative of
lysine
catabolism, was observed in many of the transformed lines (indicated by
asterisk
in Table 9). There was no major difference in the free amino acid composition
or
level of protein expression between the transformants which had the KTI3 or
Phaseolin regulatory sequences driving expression of the Corynebacteria DHDPS
gene.
TABLE 9
FS883 Transformants: phaseolin 5' region/cts/1ysC-M4/phaseo1in 3'
KTI3 5' region/cts/cordaLA/KTI3 3'
FS903 Transformants: phaseolin 5' region/cts/IvsC-M4/phaseolin 3'
phaseolin 5' region/cts/cor4WA/phaseolin 3'
FREE AMINO WESTERN WESTERN CORYNE.
ACIDS/SEED CORYNE. E. COLT DHDPS Progeny
LINE K/L T/L DHDPS AKIII U/MG/HR Kanr:Kann
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NORMAL 0.5 1.3 - -
FS883-4A 0.9 4.0 + + >15:1
FS883-11A 1.0 3.5 ++ ++ 3.1 3:1
FS883-14B 0.5 2.5 ++ ++
FS883-16A* 0.7 10.5 + +++ p
FS883-17A* 1.0 5.0 +++ +++ 7.0
FS883-18C* 1.2 3.5 ++ + 5.8 3:1
FS883-21A 0.5 1.5 + +/-
FS883-26B* 1.1 3.6 ++ ++ 2.4
FS883-29B 0.5 1.5 + - 0.4
FS883-32B 0.7 2.4 ++ + 1.5 3:1
FS883-38B* 1.1 11.3 + ++ 2.0
FS883-59C* 1.4 6.1 + + 0.5 15:1
FS903-3C 0.5 1.8 + +++
FS903-8A* 0.8 2.1 +++ ++++
FS903-9B 0.6 1.8 ++ ++ 4.3
FS903-IOA 0.5 1.5 - -
FS903-22F 0.5 1.8 ++ ++ 0.9
FS903-35B* 0.8 2.1 ++ ++
FS903-36B 0.7 1.5 + -
FS903-40A 0.6 1.8 + +
FS903-41A* 1.2 2.0 ++ +++
FS903-42A 0.7 2.2 ++ +++ 5.4
FS903-44C 0.5 1.9
FS903-53B 0.6 1.9
* indicates free amino acid sample has a-aminoadipic acid
Free amino acid composition and expression of bacterial DHDPS and AKIII
proteins was also analyzed in developing seeds of two lines that segregated as
single gene cassette insertions (see Table 10). Expression of the DHDPS
protein
under control of the KTI3 promoter was detected at earlier times than that of
the
AKIII protein under control of the Phaseolin promoter, as expected. At 14 days
after flowering both proteins were expressed at a high level and there was
about
an 8-fold increase in the level of free lysine compared to normal seeds. These
results confirm that simultaneous expression of lysine insensitive DHDPS and
lysine-insensitive AK results in the production of high levels of free lysine
in
seeds. Free lysine does not continue to accumulate to even higher levels,
however. In mature seeds free lysine is at a level 2 to 3-fold higher than in
normal
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mature seeds, and the lysine breakdown product a-aminoadipic acid accumulates.
These results provide further evidence that lysine catabolism occurs in seeds
and
prevents accumulation of the high levels of free lysine produced in
transformants
expressing lysine insensitive DHDPS and lysine insensitive AK.
TABLE 10
Developing seeds of FS883 Transformants:
phaseolin 5' region/cts/lysC-M4/phaseolin 3' region
KTI3 5' region/cts/cordanA/KTI3 3' region
FREE AMINO WESTERN WESTERN
DAYS AFTER ACIDS/SEED CORYNE. E. COLI
LINE FLOWERING K/L T/L DHDPS AKIII
FS883-18C 9 1.1 2.1 - -
FS883-18C 10 1.4 3.3 +/- -
FS883-18C 11 1.4 2.5 + -
FS883-18C 14 4.3 1.0 ++ ++
FS883-18C* MATURE 1.2 3.5 +++ ++
FS883-32B 9 1.3 2.9 + -
FS883-32B 10 1.6 2.7 + -
FS883-32B 11 1.4 2.3 + -
FS883-32B* 14 3.9 1.3 ++ ++
FS883-32B* MATURE 0.7 2.4 +++ ++
* indicates free amino acid sample has a-aminoadipic acid
EXAMPLE 16
Transformation of Oilseed Rape with the Phaseolin Promoter/cts/cordapA and
Phaseolin Promoter/cts/1ysC-M4 Chimeric Genes
The chimeric gene cassettes, phaseolin 5' region/ cts/cordapA/phaseolin 3'
region, phaseolin 5' region/ cts/lysC-M4/phaseolin 3', and phaseolin 5'
region/
cts/cordapA/phaseolin 3' region plus phaseolin 5' region/cts/lysC-M4/phaseolin
3'
(Example 6) were inserted into the binary vector pZS 199 (Figure 7A), which is
similar to pSZ97K described in Example 8. In pZS199 the 35S promoter from
Cauliflower Mosaic Virus replaced the Nos promoter driving expression of the
NPT II to provide better expression of the marker gene, and the orientation of
the
polylinker containing the multiple restriction endonuclease sites was
reversed.
To insert the phaseolin 5' region/cts/cordapA/ phaseolin 3' region, the gene
cassette was isolated as a 2.7 kb BamH I fragment (as described in Example 15)
and inserted into BamH I digested pZS 199, yielding plasmid pFS926 (Figure
7B).
This binary vector has the chimeric gene, phaseolin 5'
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region/cts/cordaDA/phaseolin 3' region inserted in the same orientation as the
35S/NPT II/nos 3' marker gene.
To insert the phaseolin 5' region/cts/lysC-M4/phaseolin 3' region, the gene
cassette was isolated as a 3.31kb EcoR Ito Spe I fragment and inserted into
EcoR I
plus Xba I digested pZS 199, yielding plasmid pBT593 (Figure 7C). This binary
vector has the chimeric gene, phaseolin 5' region/cts/lysC-M4/phaseolin 3'
region
inserted in the same orientation as the 35S/NPT 11/nos 3' marker gene.
To combine the two cassettes, the EcoR I site of pBT593 was converted to a
BamH I site using oligonucleotide adaptors, the resulting vector was cut with
BamH I and the phaseolin 5' region/cts/cordap_A/ phaseolin 3' region gene
cassette
was isolated as a 2.7 kb BamH I fragment and inserted, yielding pBT597 (Figure
7D). This binary vector has both chimeric genes, phaseolin 5'
region/cts/cordayAA/phaseolin 3' region and phaseolin 5' region/cts/IysC-
M4/phaseolin 3' region inserted in the same orientation as the 35S/NPT II/nos
3'
marker gene.
Brassica napus cultivar "Westar" was transformed by co-cultivation of
seedling pieces with disarmed Agrobacterium tumefaciens strain LBA4404
carrying the appropriate binary vector.
B. napus seeds were sterilized by stirring in 10% (v/v) Clorox, 0.1% SDS
for thirty min, and then rinsed thoroughly with sterile distilled water. The
seeds
were germinated on sterile medium containing 30 mM CaC12 and 1.5% agar, and
grown for 6 d in the dark at 24
Liquid cultures of Agrobacterium for plant transformation were grown
overnight at 28 C in Minimal A medium containing 100 mg/L kanamycin. The
bacterial cells were pelleted by centrifugation and resuspended at a
concentration
of 108 cells/mL in liquid Murashige and Skoog Minimal Organic medium
containing 100 uM acetosyringone.
B. napus seedling hypocotyls were cut into 5 mm segments which were
immediately placed into the bacterial suspension. After 30 min, the hypocotyl
pieces were removed from the bacterial suspension and placed onto BC-35 callus
medium containing 1 100 uM acetosyringone. The plant tissue and Agrobacteria
were co-cultivated for 3 d at 24 C in dim light.
The co-cultivation was terminated by transferring the hypocotyl pieces to
BC-35 callus medium containing 200 mg/L carbenicillin to kill the
Agrobacteria,
and 25 mg/L kanamycin to select for transformed plant cell growth. The
seedling
pieces were incubated on this medium for three weeks at 24 under continuous
light.
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After three weeks, the segments were transferred to BS-48 regeneration
medium containing 200 mg/L carbenicillin and 25 mg/L kanamycin. Plant tissue
was subcultured every two weeks onto fresh selective regeneration medium,
under
the same culture conditions described for the callus medium. Putatively
transformed calli grew rapidly on regeneration medium; as calli reached a
diameter of about 2 mm, they were removed from the hypocotyl pieces and placed
on the same medium lacking kanamycin
Shoots began to appear within several weeks after transfer to BS-48
regeneration medium. As soon as the shoots formed discernible stems, they were
excised from the calli, transferred to MSV-1A elongation medium, and moved to
a
16:8-h photoperiod at 24 .
Once shoots had elongated several internodes, they were cut above the agar
surface and the cut ends were dipped in Rootone . Treated shoots were planted
directly into wet Metro-Mix 350 soiless potting medium. The pots were covered
with plastic bags which were removed when the plants were clearly growing;
after
about 10 days. Results of the transformation are shown in Table 11.
Transformed
plants were obtained with each of the binary vectors,
Minimal A Bacterial Growth Medium
Dissolve in distilled water
10.5 g potassium phosphate, dibasic
4.5 g potassium phosphate, monobasic
1.0 g ammonium sulfate
0.5 g sodium citrate, dihydrate
Make up to 979 mL with distilled water
Autoclave
Add 20 mL filter-sterilized 10% sucrose
Add I mL filter-sterilized I M MgSO4
Brassica Callus Medium BC-35
Per liter:
Murashige and Skoog Minimal Organic Medium
(MS salts, 100 mg/L i-inositol, 0.4 mg/L thiamine; GIBCO #510-3118)
30 g sucrose
18 g mannitol
0.5 mg/L 2,4-D
0.3 mg/L kinetin
0.6% agarose
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pH 5.8
Brassica Regeneration Medium BS-48
Murashige and Skoog Minimal Organic Medium
Gamborg B5 Vitamins (SIGMA #1019)
g glucose
250 mg xylose
600 mg MES
0.4% agarose
pH 5.7
Filter-sterilize and add after autoclaving:
2.0 mg/L zeatin
0.1 mg/L IAA
Brassica Shoot Elongation Medium MSV-1A
Murashige and Skoog Minimal Organic Medium
Gamborg B5 Vitamins
10 g sucrose
0.6% agarose
pH 5.8
TABLE 11
Canola transformants
NUMBER OF
BINARY NUMBER OF NUMBER OF SHOOTING NUMBER OF
VECTOR CUTE ENDS KANR CALLI CALL! PLANTS
pZS199 120 41 5 2
pFS926 600 278 52 28
pBT593 600 70 10 3
pBT597 600 223 40 23
Plants were grown under a 16:8-h photoperiod, with a daytime temperature
of 23 and a nighttime temperature of 17 . When the primary flowering stem
began to elongate, it was covered with a mesh pollen-containment bag to
prevent
outcrossing. Self-pollination was facilitated by shaking the plants several
times
each day. Mature seeds derived from self-pollinations were harvested about
three
months after planting.
A partially defatted seed meal was prepared as follows: 40 mg of mature dry
seed was ground with a mortar and pestle under liquid nitrogen to a fine
powder.
One milliliter of hexane was added and the mixture was shaken at room
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temperature for 15 min. The meal was pelleted in an eppendorf centrifuge, the
hexane was removed and the hexane extraction was repeated. Then the meal was
dried at 65 for 10 min until the hexane was completely evaporated leaving a
dry
powder. Total proteins were extracted from mature seeds as follows.
Approximately 30-40 mg of seeds were put into a 1.5 mL disposable plastic
microfuge tube and ground in 0.25 mL of 50 mM Tris-HC1 pH 6.8, 2 mM EDTA,
1% SDS, 1% (v/v) P-mercaptoethanol. The grinding was done using a motorized
grinder with disposable plastic shafts designed to fit into the microfuge
tube. The
resultant suspensions were centrifuged for 5 min at room temperature in a
microfuge to remove particulates. Three volumes of extract was mixed with 1
volume of 4 X SDS-gel sample buffer (0.1 M Tris-HC1 pH6.8, 6.7% SDS, 16.7%
(v/v) (3-mercaptoethanol, 33% (v/v) glycerol) and 5 L from each extract were
run
per lane on an SDS polyacrylamide gel, with bacterially produced DHDPS or
AKIII serving as a size standard and protein extracted from untransformed
tobacco seeds serving as a negative control. The proteins were then
electrophoretically blotted onto a nitrocellulose membrane. The membranes were
exposed to the DHDPS or AKIII antibodies at a 1:5000 dilution of the rabbit
serum using standard protocol provided by BioRad with their Immun-Blot Kit.
Following rinsing to remove unbound primary antibody the membranes were
exposed to the secondary antibody, donkey anti-rabbit Ig conjugated to
horseradish peroxidase (Amersham) at a 1:3000 dilution. Following rinsing to
remove unbound secondary antibody, the membranes were exposed to Amersham
chemiluminescence reagent and X-ray film.
Eight of eight FS926 transformants and seven of seven BT597 transformants
expressed the DHDPS protein. The single BT593 transformant and five of seven
BT597 transformants expressed the AKIII-M4 protein (Table 12). Thus it is
straightforward to express these proteins in oilseed rape seeds.
To measure free amino acid composition of the seeds, free amino acids were
extracted from 40 mg of the defatted meal in 0.6 mL of
methanol/chloroform/water mixed in ratio of 12v/5v/3v (MCW) at room
temperature. The mixture was vortexed and then centrifuged in an eppendorf
microcentrifuge for about 3 min. Approximately 0.6 mL of supernatant was
decanted and an additional 0.2 mL of MCW was added to the pellet which was
then vortexed and centrifuged as above. The second supernatant, about 0.2 mL,
was added to the first. To this, 0.2 mL of chloroform was added followed by
0.3 mL of water. The mixture was vortexed and then centrifuged in an eppendorf
microcentrifuge for about 3 min, the upper aqueous phase, approximately 1.0
mL,
was removed, and was dried down in a Savant Speed Vac Concentrator. The
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samples were hydrolyzed in 6 N hydrochloric acid, 0.4% (v/v) (3-
mercaptoethanol
under nitrogen for 24 h at 110-120 ; 1/4 of the sample was run on a Beckman
Model 6300 amino acid analyzer using post-column ninhydrin detection. Relative
free amino acid levels in the seeds were compared as ratios of lysine or
threonine
to leucine, thus using leucine as an internal standard.
In contrast to tobacco seeds, expression of Corynebacterium DHDPS lead to
large increases in accumulation of free lysine in rapeseed transformants. The
highest expressing lines showed a greater than 100-fold increase in free
lysine
level in the seeds. The transformant that expressed AKIII-M4 in the absence of
Corynebacteria DHDPS showed a 5-fold increase in the level of free threonine
in
the seeds. Concomitant expression of both enzymes resulted in accumulation of
high levels of free lysine, but not threonine.
A high level of a-aminoadipic acid, indicative of lysine catabolism, was
observed in many of the transformed lines. Thus, prevention of lysine
catabolism
by inactivation of lysine ketoglutarate reductase should further increase the
accumulation of free lysine in the seeds. Alternatively, incorporation of
lysine
into a peptide or lysine-rich protein would prevent catabolism and lead to an
increase in the accumulation of lysine in the seeds.
To measure the total amino acid composition of mature seeds, 2 mg of the
defatted meal were hydrolyzed in 6 N hydrochloric acid, 0.4% (v/v) P-mercapto-
ethanol under nitrogen for 24h at 110-120 ; 1/100 of the sample was run on a
Beckman Model 6300 amino acid analyzer using post-column ninhydrin
detection. Relative amino acid levels in the seeds were compared as
percentages
of lysine, threonine or a-aminoadipic acid to total amino acids.
There was a good correlation between expression of DHDPS protein and
accumulation of high levels of lysine in the seeds of transformants. Seeds
with a
5-100% increase in the lysine level, compared to the untransformed control,
were
observed. In the transformant with the highest level, lysine makes up about
13%
of the total seed amino acids, considerably higher than any previously known
rapeseed seed. This transforrnant expresses high levels of both E. coli AKIII-
M4
and Corynebacterium DHDPS.
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TABLE 12
FS926 Transformants: phaseolin 5' region/cts/cordapA/phaseolin 3'
BT593 Transformants: phaseolin 5' region/cts/ivsC-M4/phaseo1in 3'
BT597 Transformants: phaseolin 5' region/cts/1ysC-M4/phaseolin 3'
phaseolin 5' region/cts/cordapAA/phaseolin 3'
WESTERN WESTERN % TOTAL AMINO
FREE AMINO ACIDS CORYNE. E. COLI ACIDS
LINE K/L T/L AA/L DHDPS AKIII-M4 K T AA
WESTAR 0.8 2.0 0 - - 6.5 5.6 0
ZS199 1.3 3.2 0 - - 6.3 5.4 0
FS926-3 140 2.0 16 ++++ - 12 5.1 1.0
FS926-9 110 1.7 12 ++++ - 11 5.0 0.8
FS926-11 7.9 2.0 5.2 ++ - 7.7 5.2 0
FS926-6 14 1.8 4.6 +++ - 8.2 5.9 0
FS926-22 3.1 1.3 0.3 + - 6.9 5.7 0
FS926-27 4.2 1.9 1.1 ++ - 7.1 5.6 0
FS926-29 38 1.8 4.7 ++++ - 12 5.2 1.6
FS926-68 4.2 1.8 0.9 ++ - 8.3 5.5 0
BT593-42 1.4 11 0 - ++ 6.3 6.0 0
BT597-14 6.0 2.6 4.3 ++ +/- 7.0 5.3 0
BT597-145 1.3 2.9 0 + -
BT597-4 38 3.7 4.5 ++++ i-+++ 13 5.6 1.6
BT597-68 4.7 2.7 1.5 ++ + 6.9 5.8 0
BT597-100 9.1 1.9 1.7 +++ ++ 6.6 5.7 0
BT597-148 7.6 2.3 0.9 +++ + 7.3 5.7 0
BT597-169 5.6 2.6 1.7 +++ +++ 6.6 5.7 0
AA is a-amino adipic acid
EXAMPLE 17
Transformation of Maize Using a Chimeric lysC-M4 Gene
as a Selectable Marker
Embryogenic callus cultures were initiated from immature embryos (about
1.0 to 1.5 mm) dissected from kernels of a corn line bred for giving a "type
II
callus" tissue culture response. The embryos were dissected 10 to 12 d after
pollination and were placed with the axis-side down and in contact with
agarose-
solidified N6 medium [Chu et al. (1974) Sci Sin 18:659-6681 supplemented with
0.5 mg/L 2,4-D (N6-0.5). The embryos were kept in the dark at 27 C. Friable
embryogenic callus consisting of undifferentiated masses of cells with somatic
proembryos and somatic embryos borne on suspensor structures proliferated from
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the scutellum of the immature embryos. Clonal embryogenic calli isolated from
individual embryos were identified and sub-cultured on N6-0.5 medium every 2
to
3 weeks.
The particle bombardment method was used to transfer genes to the callus
culture cells. A BiolisticTM PDS-1000/He (BioRAD Laboratories, Hercules, CA)
was used for these experiments.
The plasmid pBT573, containing the chimeric gene HH534 5' region/
mcts/1vsC-M4/HH2-1 3' region (see Example 6) designed for constitutive gene
expression in corn, was precipitated onto the surface of gold particles. To
accomplish this 2.5 tg of pBT:573 (in water at a concentration of about 1
mg/mL)
was added to 25 mL of gold particles (average diameter of 1.5 m) suspended in
water (60 mg of gold per mL). Calcium chloride (25 mL of a 2.5 M solution) and
spermidine (10 mL of a 1.0 M solution) were then added to the gold-DNA
suspension as the tube was vortexing. The gold particles were centrifuged in a
microfuge for 10 s and the supernatant removed. The gold particles were then
resuspended in 200 m]L of absolute ethanol, were centrifuged again and the
supernatant removed. Finally, the gold particles were resuspended in 25 mL of
absolute ethanol and sonicated twice for one sec. Five 4L of the DNA-coated
gold particles were then loaded on each macro carrier disk and the ethanol was
allowed to evaporate away leaving the DNA-covered gold particles dried onto
the
disk.
Embryogenic callus (from the callus line designated #132.2.2) was arranged
in a circular area of about 6 cm. in diameter in the center of a 100 X 20 mm
petri
dish containing N6-0.5 medium supplemented with 0.25M sorbitol and 0.25M
mannitol. The tissue was placed on this medium for 2 h prior to bombardment as
a pretreatment and remained on the medium during the bombardment procedure.
At the end of the 2 h pretreatment period, the petri dish containing the
tissue was
placed in the chamber of the PDS-1000/He. The air in the chamber was then
evacuated to a vacuum of 28 inch of Hg. The macrocarrier was accelerated with
a
helium shock wave using a rupture membrane that bursts when the He pressure in
the shock tube reaches 1100 psi. The tissue was placed approximately 8 cm from
the stopping screen. Four plates of tissue were bombarded with the DNA-coated
gold particles. Immediately following bombardment, the callus tissue was
transferred to N6-0.5 medium without supplemental sorbitol or mannitol.
Seven d after bombardment small (2-4 mM diameter) clumps of callus
tissue were transferred to N6-0.5 medium lacking casein or proline, but
supplemented with 2mM each of lysine and threonine (LT). The tissue continued
to grow slowly on this medium and was transferred to fresh N6-0.5 medium
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supplemented with LT every 2 weeks. After 12 weeks two clones of actively
growing callus was identified on two separate plates containing LT-
supplemented
medium. These clones continued to grow when sub-cultured on the selective
medium. The presence of the l-M4 gene in the selected clones was confirmed
by PCR analysis. Callus was transferred to medium that promotes plant
regeneration.
EXAMPLE 18
Transformation of Corn with the
d
Constitutive Corn Promoter/cts/ecodapA an
Constitutive Corn Promoter/cts/lysC-M4
The chimeric gene cassettes, HH534 5' region/ mcts/ecodapA/HH2-1 3'
region plus HH534 5' region/ mcts/lysC-M4/HH2-1 3' region, (Example 6) were
inserted into the vector pGem9z to generate a corn transformation vector.
Plasmid
pBT583 (Example 6) was digested with Sal I and an 1850 bp fragment containing
the HH534 5' region/mcts/ecodapA/HH2-1 3' region gene cassette was isolated.
This DNA fragment was inserted into pBT573 (Example 6), which carries the
HH534 5' region/mcts/ lysC-M4/HH2-1 3' region, digested with Xho I. The
resulting vector with both chimeric genes in the same orientation was
designated
pBT586.
Vector pBT586 was introduced into embryogenic corn callus tissue using
the particle bombardment method. The establishment of the embryogenic callus
cultures and the parameters for particle bombardment were as described in
Example 17.
Either one of two plasmid vectors containing selectable markers were used
in the transformations. One plasmid, pALSLUC [Fromm et al. (1990)
Biotechnology 8:833-839], contained a cDNA of the maize acetolactate synthase
(ALS) gene. The ALS cDNA had been mutated in vitro so that the enzyme coded
by the gene would be resistant to chlorsulfuron. This plasmid also contains a
gene
that uses the 35S promoter from Cauliflower Mosaic Virus and the 3' region of
the
nopaline synthase gene to express a firefly luciferase coding region [de Wet
et al.
(1987) Molec. Cell Biol. 7:725-737]. The other plasmid, pDETRIC, contained the
bar gene from Streptomyices hygroscopicus that confers resistance to the
herbicide
glufosinate [Thompson et al. (1987 The EMBO Journal 6:2519-2523]. The
bacterial gene had its translation codon changed from GTG to ATG for proper
translation initiation in plants [De Block et al. (1987) The EMBO Journal
6:2513-2518]. The bar gene was driven by the 35S promoter from Cauliflower
Mosaic Virus and uses the termination and polyadenylation signal from the
octopine synthase gene from Agrobacterium tumefaciens.
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For bombardment, 2.5 g of each plasmid, pBT586 and one of the two
selectable marker plasmids, was co-precipitated onto the surface of gold
particles
as described in Example 17. Bombardment of the embryogenic tissue cultures
was also as described in Example 17.
Seven days after bombardment the tissue was transferred to selective
medium. The tissue bombarded with the selectable marker pALSLUC was
transferred to N6-0.5 medium that contained chlorsulfuron (30 ng/L) and lacked
casein or proline. The tissue bombarded with the selectable marker, pDETRIC,
was transferred to N6-0.5 medium that contained 2 mg/L glufosinate and lacked
casein or proline. The tissue continued to grow slowly on these selective
media.
After an additional 2 weeks the tissue was transferred to fresh N6-0.5 medium
containing the selective agents.
Chlorsulfuron- and glufosinate-resistance callus clones could be identified
after an additional 6-8 weeks. These clones continued to grow when transferred
to
the selective media.
The presence of pBT586 in the transformed clones has been confirmed by
PCR analysis. Functionality of the introduced AK enzyme was tested by plating
out transformed clones on N6-0.5 media containing 2 mM each of lysine and
threonine (LT selection; see Example 13). All of the clones were capable of
growing on LT medium indicating that the E. coli aspartate kinase was
expressed
and was functioning properly. To test that the E. coli DHDPS enzyme was
functional, transformed callus was plated on N6-0.5 media containing 2 M
2-aminoethylcysteine (AEC), a lysine analog and potent inhibitor of plant
DHDPS. The transformed callus tissue was resistant to AEC indicating that the
introduced DHDPS, which is about 16-fold less sensitive to AEC than the plant
enzyme, was being produced and was functional. Plants have been regenerated
from several transformed clones and are being grown to maturity.
EXAMPLE 19
Transformation of Soybean with the Phaseolin Promoter/cts/cordapA and
Phaseolin Promoter/cts/lysC-M4 Chimeric Genes
The chimeric gene cassettes, phaseolin 5' region/ cts/cordaaA/phaseolin 3'
region plus phaseolin 5' region/cts/1vsC-M4/phaseolin 3', (Example 6) were
inserted into the soybean transformation vector pBT603 (Figure 8A). This
vector
has a soybean transformation marker gene consisting of the 35S promoter from
Cauliflower Mosaic Virus driving expression of the E. coli (3-glucuronidase
gene
[Jefferson et al. (1986) Proc. Natl. Acad. Sci. USA 83:8447-8451] with the Nos
3'
region in a modified pGEM9Z: plasmid.
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To insert the phaseolin 5' region/cts/1ysC-M4/ phaseolin 3' region, the gene
cassette was isolated as a 3.3 kb Hind III fragment and inserted into Hind III
digested pBT603, yielding plasmid pBT609. This binary vector has the chimeric
gene, phaseolin 5' region/ cts/sC-M4/phaseolin 3' region inserted in the
opposite
orientation from the 35S/GUS/Nos 3' marker gene.
To insert the phaseolin 5' region/cts/cordapA/ phaseolin 3'region 3', the gene
cassette was isolated as a 2.7 kb BamH I fragment (as described in Example 15)
and inserted into BamH I digested pBT609, yielding plasmid pBT614 (Figure
8B). This vector has both chimeric genes,
phaseolin 5' region/cts/lysC-M4/phaseolin 3' region and
phaseolin 5' region/cts/cordapA/phaseolin 3' region inserted in the same
orientation, and both are in the opposite orientation from the 35S/GUS/Nos 3'
marker gene.
Soybean was transformed with plasmid pBT614 according to the procedure
described in United States Patent No. 5,015,580. Soybean transformation was
performed by Agracetus Company (Middleton, WI). Seeds from five transformed
lines were obtained and analyzed.
It was expected that the transgenes would be segregating in the RI seeds of
the transformed plants. To identify seeds that carried the transformation
marker
gene, a small chip of the seed was cut off with a razor and put into a well in
a
disposable plastic microtiter plate. A GUS assay mix consisting of 100 mM
NaH2PO4, 10 mM EDTA, 0.5 mM K4Fe(CN)6, 0.1% Triton X-100, 0.5 mg/mL
5-Bromo-4-chloro-3-indolyl (3-D-glucuronic acid was prepared and 0.15 mL was
added to each microtiter well. The microtiter plate was incubated at 37 for
45 min. The development of blue color indicated the expression of GUS in the
seed.
Five of seven transformed lines showed approximately 3:1 segregation for
GUS expression indicating that the GUS gene was inserted at a single site in
the
soybean genome. The other transformants showed 9:1 and 15:1 segregation,
suggesting that the GUS gene was inserted at two sites.
A meal was prepared from a fragment of individual seeds by grinding into a
fine powder. Total proteins were extracted from the meal by adding 1 mg to
0.1 mL of 43 mM Tris-HC1 pH 6.8, 1.7% SDS, 4.2% (v/v) R-mercaptoethanol, 8%
(v/v) glycerol, vortexing the suspension, boiling for 2-3 min and vortexing
again.
The resultant suspensions were centrifuged for 5 min at room temperature in a
microfuge to remove particulates and 10 L from each extract were run per lane
on an SDS polyacrylamide gel, with bacterially produced DHDPS or AKIII
serving as a size standard. The proteins were then electrophoretically blotted
onto
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a nitrocellulose membrane. The membranes were exposed to the DHDPS or
AKIII antibodies, at a 1:5000 or 1:1000 dilution, respectively, of the rabbit
serum
using standard protocol provided by BioRad with their Immun-Blot Kit.
Following rinsing to remove unbound primary antibody the membranes were
exposed to the secondary antibody, donkey anti-rabbit Ig conjugated to
horseradish peroxidase (Amersham) at a 1:3000 dilution. Following rinsing to
remove unbound secondary antibody, the membranes were exposed to Amersham
chemiluminescence reagent and X-ray film.
Six of seven transformants expressed the DHDPS protein. In the six
transformants that expressed DHDPS, there was excellent correlation between
expression of GUS and DHDPS in individual seeds (Table 13). Therefore, the
GUS and DHDPS genes are integrated at the same site in the soybean genome.
Four of seven transformants expressed the AKIII protein, and again there was
excellent correlation between expression of AKIII, GUS and DHDPS in
individual seeds (Table 13). Thus, in these four transformants the GUS, AKIII
and DHDPS genes are integrated at the same site in the soybean genome. One
transformant expressed only GUS in its seeds.
To measure free amino acid composition of the seeds, free amino acids were
extracted from 8-10 milligrams of the meal in 1.0 mL of methanol/chloro-
form/water mixed in ratio of 12v/5v/3v (MCW) at room temperature. The
mixture was vortexed and then centrifuged in an eppendorf microcentrifuge for
about 3 min; approximately 0.8 mL of supernatant was decanted. To this
supernatant, 0.2 mL of chloroform was added followed by 0.3 mL of water. The
mixture was vortexed and then centrifuged in an eppendorf microcentrifuge for
about 3 min, the upper aqueous phase, approximately 1.0 mL, was removed, and
was dried down in a Savant Speed Vac Concentrator. The samples were
hydrolyzed in 6 N hydrochloric acid, 0.4% (v/v) P-mercaptoethanol under
nitrogen for 24 h at 110-120 ; 1/10 of the sample was run on a Beckman Model
6300 amino acid analyzer using post-column ninhydrin detection. Relative free
amino acid levels in the seeds were compared as ratios of lysine to leucine,
thus
using leucine as an internal standard.
Soybean transformants expressing Corynebacteria DHDPS alone and in
concert with E. coli AKIII-M4 accumulated high levels of free lysine in their
seeds. From 20 fold to 120-fold increases in free lysine levels were observed
(Table 13). A high level of saccharopine, indicative of lysine catabolism, was
also
observed in seeds that contained high levels of lysine. Thus, prevention of
lysine
catabolism by inactivation of lysine ketoglutarate reductase should further
increase the accumulation of fee lysine in the seeds. Alternatively,
incorporation
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of lysine into a peptide or lysine-rich protein would prevent catabolism and
lead to
an increase in the accumulation of lysine in the seeds.
To measure the total amino acid composition of mature seeds, 1-1.4
milligrams of the seed meal was hydrolyzed in 6 N hydrochloric acid, 0.4%
(v/v)
P-mercaptoethanol under nitrogen for 24 h at 110-120 ; 1/50 of the sample was
run on a Beckman Model 6300 amino acid analyzer using post-column ninhydrin
detection. Lysine (and other amino acid) levels in the seeds were compared as
percentages of the total amino acids.
The soybean seeds expressing Corynebacteria DHDPS showed substantial
increases in accumulation of total seed lysine. Seeds with a 5-35% increase in
total lysine content, compared to the untransformed control, were observed. In
these seeds lysine makes up 7.5-7.7% of the total seed amino acids.
Soybean seeds expressing Corynebacteria DHDPS in concert with E. coli
AKIII-M4 showed much greater accumulation of total seed lysine than those
expressing Corynebacteria DHDPS alone. Seeds with a more than four-fold
increase in total lysine content were observed. In these seeds lysine makes up
20-25% of the total seed amino acids, considerably higher than any previously
known soybean seed.
TABLE 13
% TOTAL
LINE-SEED GUS Free LYS/LEU DHDPS AKIII SEED LYS
A2396-145-4 - 0.9 - - 5.8
A2396-145-8 - 1.0 - -
A2396-145-5 - 0.8 5.9
A2396-145-3 - 1.0
A2396-145-9 + 2.0
A2396-145-6 + 4.6
A2396-145-1 + 8.7
A2396-145-10 + 18.4 7.5
A2396-145-7 + 21.7 + - 6.7
A2396-145-2 + 45.5 + - 7.2
A5403-175-9 - 1.3
A5403-175-4 - 1.2 - - 6.0
A5403-175-3 - 1.0 - - 6.0
A5403-175-7 + 1.5
A5403-175-5 + 1.8
A5403-175-1 + 6.2
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A5403-175-2 + 6.5 6.3
A5403-175-6 + 14.4
A5403-175-8 + 47.8 + - 7.7
A5403-175-10 + 124.3 + - 7.5
A5403-181-9 + 1.4
A5403-181-10 + 1.4 - - 5.7
A5403-181-8 + 0.9
A5403-181-6 + 1.5
A5403-181-4 - 0.7 - - 5.9
A5403-181-5 + 1.1
A5403-181-2 - 1.8 - - 5.6
A5403-181-3 + 2.7 - - 5.5
A5403-181-7 + 1.9
A5403-181-1 - 2.3
A5403-183-9 - 0.8
A5403-183-6 - 0.7 - - 6.0
A5403-183-8 - 1.3
A5403-183-4 - 1.3 - - 6.0
A5403-183-5 + 0.9
A5403-183-3 + 3.1
A5403-183-1 + 3.3
A5403-183-7 + 9.9
A5403-183-10 + 22.3 + + 6.7
A5403-183-2 + 23.1 + + 7.3
A5403-196-8 - 0.9 - - 5.9
A5403-196-6 + 8.3
A5403-196-1 + 16.1 + + 6.8
A5403-196-7 + 27.9
A5403-196-3 + 52.8
A5403-196-5 + 26
A5403-196-2 + 16.2 + +
A5403-196-10 + 29 + + 7.5
A5403-196-4 + 58.2 + + 7.6
A5403-196-9 + 47.1
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A2396-233-1 + + + 25
A2396-233-2 + 18
A2396-233-3 + 23
A2396-233-4 + 20
A2396-233-5 - +l- - 6.0
A2396-233-6 + 16
A2396-233-13 + + + 18
A2396-234-1 + + + 8.3
A2396-2342 + + + 13
A2396-234-3 + 10
A2396-234-4 + 19
A2396-234-9 + 15
A2396-234-16 - - - 5.9
wild type - 0.9 - - 5.6
control
EXAMPLE 20
Isolation of a Plant
Lysine Ketogl~t rate Reductase Gene
Lysine Ketoglutarate Reductase (LKR) enzyme activity has been observed
in immature endosperm of developing maize seeds [Arruda et al. (1982) Plant
Physiol. 69:988-989]. LKR activity increases sharply from the onset of
endosperm development, reaches a peak level at about 20 d after pollination,
and
then declines [Arruda et al. (1983) Phytochemistry 22:2687-2689].
In order to clone the corn LKR gene, RNA was isolated from developing
seeds 19 days after pollination. This RNA was sent to Clontech Laboratories,
Inc., (Palo Alto, CA) for the custom synthesis of a cDNA library in the vector
Lambda Zap II. The conversion of Lambda Zap II library into a phagemid
library, then into a plasmid library was accomplished following the protocol
provided by Clontech. Once converted into a plasmid library the ampicillin-
resistant clones obtained carry the cDNA insert in the vector pBluescript SK(-
).
Expression of the cDNA is under control of the lacZ promoter on the vector.
Two phagemid libraries were generated using the mixtures of the Lambda
Zap II phase and the filamentous helper phage of 100 L to 1 .tL. Two
additional
libraries were generated using mixtures of 100 L Lambda Zap II to 10 L
helper
phage and 20 L Lambda Zap 11 to 10 L helper phase. The titers of the
phagemid preparations were similar regardless of the mixture used and were
about
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2 x 103 ampicillin-resistant-transfectants per mL with E. coli strain XL I -
Blue as
the host and about 1 x 103 with DE126 (see below) as host.
To select clones that carried the LKR gene a specially designed E. coli host,
DE126 was constructed. Construction of DE126 occurred in several stages. (1) A
generalized transducing stock of coliphage P 1 vir was produced by infection
of a
culture ofTST1 [F-,;rraDl39. A(argF-lac)205, flb5301, ptsF25, relAl, rpsL150,
malE52::Tn10, deoC 1, X-] (E. coli Genetic Stock Center #6137) using a
standard
method (for Methods see J. Miller, Experiments in Molecular Genetics).
(2) This phage stock was used as a donor in a transductional cross (for
Method see J. Miller, Experiments in Molecular Genetics) with strain GIF 106M
1
[F-, arg-, ii1vA296, j;C1001, ithrA1101, metL1000, X-, rrpsL9, malT1, y-7,
rntl-2, thi l (?), supE44(?)] (E. soli Genetic Stock Center #5074) as the
recipient.
Recombinants were selected on rich medium [L supplemented with DAP]
containing the antibiotic tetracycline. The transposon Tn10, conferring
tetracycline resistance, is inserted in the malE gene of strain TST1.
Tetracycline-
resistant transductants derived from this cross are likely to contain up to 2
min of
the E. coli chromosome in the vicinity of malE. The genes malE and 1ysC are
separated by less than 0.5 minutes, well within cotransduction distance.
(3) 200 tetracycline-resistant transductants were thoroughly phenotyped;
appropriate fermentation and nutritional traits were scored. The recipient
strain
GIF106M1 is completely devoid of aspartokinase isozymes due to mutations in
thrA, metL and 1ysC, and therefore requires the presence of threonine,
methionine,
lysine and meso-diarninopimelic acid (DAP) for growth. Transductants that had
inherited 1ysC+ with malE::Tn10 from TSTI would be expected to grow on a
minimal medium that. contains vitamin B 1, L-arginine, L-isoleucine and L-
valine
in addition to glucose: which serves as a carbon and energy source. Moreover
strains having the genetic constitution of 1ysC+, metL- and thrA- will only
express
the lysine sensitive aspartokinase. Hence addition of lysine to the minimal
medium should prevent the growth of the lysC+ recombinant by leading to
starvation for threonine, methionine and DAP. Of the 200 tetracycline
resistant
transductants examined, 49 grew on the minimal medium devoid of threonine,
methionine and DAP.. Moreover, all 49 were inhibited by the addition of L-
lysine
to the minimal medium. One of these transductants was designated DE125.
DE125 has the phenotype of tetracycline resistance, growth requirements for
arginine, isoleucine and valine, and sensitivity to lysine. The genotype of
this
strain is F- malE52::TnlO arg- ijvA296 thrAl 101 metL1000 lambda- rpsL9
malT 1 j-7 mtl-2 thi IM E44(?).
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(4) This step involves production of a male derivative of strain DE 125.
Strain DE 125 was mated with the male strain AB 1528 [F' 16/delta(gpt- roA 62,
lacYI or lacZ4, glnV44, galK2 rac (?), hisG4, rfbdl, mgl-51, kdgK51(?), ilvC7,
aruE3, thi-1] (E. coli Genetic Stock Center #1528) by the method of
conjugation.
F' 16 carries the ilvGMEDAYC gene cluster. The two strains were cross streaked
on rich medium permissive for the growth of each strain. After incubation, the
plate was replica plated to a synthetic medium containing tetracycline,
arginine,
vitamin B 1 and glucose. DE 125 cannot grow on this medium because it cannot
synthesize isoleucine. Growth of AB1528 is prevented by the inclusion of the
antibiotic tetracycline and the omission of proline and histidine from the
synthetic
medium. A patch of cells grew on this selective medium. These recombinant
cells underwent single colony isolation on the same medium. The phenotype of
one clone was determined to be Ilv+, Arg-, TetR, Lysine-sensitive, male
specific
phage (MS2)-sensitive, consistent with the simple transfer of F'1 6 from AB
1528
to DE125. This clone was designated DE126 and has the genotype
F'16/malE52::Tn10, arg-, i1vA296, thrA1101, metL100, lysC+, ?c,1 sL9, ma1T1,
y-7, mtl-2, thi-l?, supE44?. It is inhibited by 20 4g/mL of L-lysine in a
synthetic medium.
To select for clones from the corn cDNA library that carried the LKR gene,
100 L of the phagemid library was mixed with 100 L of an overnight culture
of
DE126 grown in L broth and the cells were plated on synthetic media containing
vitamin B 1, L-arginine, glucose as a carbon and energy source, 100 g/mL
ampicillin and L-lysine at 20, 30 or 40 g/mL. Four plates at each of the
three
different lysine concentrations were prepared. The amount of phagemid and
DE 126 cells was expected to yield about I x 105 ampicillin-resistant
transfectants
per plate. Ten to thirty lysine-resistant colonies grew per plate (about 1
lysine-
resistant per 5000 ampicillin-resistant colonies).
Plasmid DNA was isolated from 10 independent clones and retransformed
into DE 126. Seven of the ten DNAs yielded lysine-resistant clones
demonstrating
that the lysine-resistance trait was carried on the plasmid. Several of the
cloned
DNAs were sequenced and biochemically characterized. The inserted DNA
fragments were found to be derived from the E. coli genome, rather than a corn
cDNA indicating that the cDNA library provided by Clontech was contaminated.
Another method was used to identify plant cDNAs that encode LKR. This
method was based upon expected homology between plant LKR and fungal genes
encoding saccharopine dehydrogenase. Fungal saccharopine dehydrogenase
(glutamate-forming) and saccharopine dehydrogenase (lysine-forming) catalyze
the final two steps in the fungal lysine biosynthetic pathway. Plant LKR and
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fungal saccharopine dehydrogenase (lysine-forming) catalyze both forward and
reverse reactions, use identical substrates and use similar co-factors.
Similarly,
plant saccharopine dehydrogenase (glutamate-forming), which catalyzes the
second step in the lysine catabolic pathway, works in both forward and reverse
reactions, uses identical substrates and uses similar co-factors as fungal
saccharopine dehydrogenase (glutamate-forming).
Biochemical and genetic evidence derived from human and bovine studies
has demonstrated that mammalian LKR and saccharopine dehydrogenase
(glutamate-forming) enzyme activities are present on a single protein with a
monomer molecular weight of about 117,000. This contrasts with the fungal
enzymes which are carried on separate proteins, saccharopine dehydrogenase
(lysine-forming) with a molecular weight of about 44,000 and saccharopine
dehydrogenase (glutamate-forming) with a molecular weight of about 51,000.
Plant LKR has been reported to have a molecular weight of about 140,000
indicating that it is like the animal catabolic protein wherein both LKR and
saccharopine dehydrogenase (glutamate-forming) enzyme activities are present
on
a single protein.
Several genes for fungal saccharopine dehydrogenases have been isolated
and sequenced [Xuan et al. (1990) Mol. Cell. Biol. 10:4795-4806, Feller et al.
(1994) Mol. Cell. Biol. 14:6411-6418]. The fungal protein sequences, deduced
from these gene sequences, were used to search plant cDNA databases for DNA
fragments that encoded plant proteins homologous to the fungal saccharopine
dehydrogenases. We discovered two plant cDNA fragments from Arabidopsis
thaliana, SEQ ID NO: 102: and SEQ ID NO: 103:, that encoded polypeptides SEQ
ID NO:104: and SEQ ID NO:105:, respectively, that are homologous to fungal
saccharopine dehydrogenase (glutamate-forming). The sequence similarity
between the fungal and plant polypeptides (see Figure 9) demonstrate that
these
cDNAs encode Arabidopsis saccharopine dehydrogenase. Oligonucleotides SEQ
ID NO:108: and SEQ ID NO:1.09 were synthesized and used for PCR
amplification of a 2.24 kb DNA fragment from genomic Arabidopsis. DNA.
DNA sequencing of the fragment confirmed that it encoded LKR/SDH. The
fragment was labeled with digoxigenin (DIG) using Boehringer Mannheim's Dig-
High Prime kit and protocol. This probe was used to screen a CD4-8 Landsberg
erecta genomic library by plaque hybridization. Approximately 2.7 X 105
recombinant phage were plated on the host E. coli LE392, grown overnight at
37 . The protocol was as described in the DIG Wash and Block Set (Boehringer
Mannheim) with the hybridization temperature set at 55 . Five positive clones
were isolated; one was subcloned into plasmid vector pBluescript SK +1-
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(Stratagene), transformed into DH5a TM competent cells (GibcoBRL) and
sequenced.
The complete genomic sequence of the Arabidopsis LKR/SDH gene is
shown in SEQ ID NO:110. The sequence includes approximately 2 kb of 5'
noncoding sequence and 500 bp of 3' noncoding sequence and 23 introns.
Overlapping fragments of the corresponding cDNA were isolated from total
Arabidopsis RNA by RT-PCR. Sequence analysis of the LKR-SDH cDNA
revealed an ORF of 3.16 kb, which predicts a protein of 117 kd, and confirms
that
the LKR and SDH enzymes reside on one polypeptide. The complete protein
coding sequence of Arabidopsis LKR/SDH gene, derived from the cDNA, is
shown in SEQ ID NO: 111. The deduced amino acid sequence of Arabidopsis
LKR/SDH protein is shown in SEQ ID NO:112. The protein lacks an N-terminal
targeting sequence implying that the lysine degradative pathway is located in
the
plant cell cytosol.
Degenerate oligonucleotides, SEQ ID NO: 113 and SEQ ID NO: 114, were
designed based upon comparison of the Arabidopsis LKR/SDH amino acid
sequence with that of other LKR proteins. These were used to amplify soybean
and corn LKR/SDH cDNA fragments using PCR from mRNA, or cDNA
synthesized from mRNA, isolated from developing soybean or corn seeds. The
soybean and corn PCR-generated cDNA fragments were cloned and sequenced.
The sequence of the soybean LKR/SDH cDNA fragment is shown in SEQ ID
NO: 115, and the sequence of the corn cDNA fragment is shown in SEQ ID
NO: 116. The deduced partial amino acid sequence of soybean LKR/SDH protein
is shown in SEQ ID NO:1 17 and the deduced partial amino acid sequence of corn
LKR/SDH protein is shown in SEQ ID NO:118. The partial cDNAs encoding
corn and soybean LKR/SDH obtained by PCR, above, were used in protocols that
extended the sequence information for these functions. These protocols, which
included RACE and direct DNA:DNA hybridization to cDNA libraries for the
identification of overlapping clones, are well known to persons skilled in the
art.
From these efforts, more complete sequences for the corn and soybean cDNAs for
LKR/SDH were obtained. SEQ ID NOS: 119 and 120 list, respectively, near full-
length sequences for the LKR/SDH coding regions from soybean and corn. The
deduced protein sequences encoded by these soybean and corn cDNAs are shown
in SEQ ID NOS:121 and 122, respectively.
Partial cDNA clones for LKR/SDH from rice and wheat were identifid in
libraries prepared from rice roots and leafs and from wheat seedlings. cDNA
libraries were prepared in Uni-ZAPTM XR vectors according to the
manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA).
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Conversion of the Uni-ZAPT?4 XR libraries into plasmid libraries was
accomplished according to the protocol provided by Stratagene. Upon
conversion, cDNA inserts were contained in the plasmid vector pBluescript.
cDNA inserts from randomly picked bacterial colonies containing recombinant
pBluescript plasmids were amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA sequences or
plasmid DNA was prepared from cultured bacterial cells. Amplified insert
DNAs or plasmid DNAs were sequenced in dye-primer sequencing reactions to
generate partial cDNA sequences (expressed sequence tags or "ESTs"; see
Adams, M. D. et al., (1991) Science 252:1651). The resulting ESTs were
analyzed using a Perkin Elmer Model 377 fluorescent sequencer. Possible
protein products encoded by the ESTs were compared to the full-length
sequence of Arabidopsis LKRISDH (SEQ ID NO:112). A contig for a partial
cDNA from rice was constructed and is presented in SEQ ID: 125. The
predicted prtein fragment from the cDNA contig is shown in SEQ ID NO: 126.
Another cDNA from rice was identified which corresponds to the 3' end of a
LKR/SDH coding region and this sequence is set forth in SEQ ID NO: 127. The
predicted protein fragment is shown in SEQ ID NO: 128. A partial wheat clone
was identified and possesses the sequence presented in SEQ ID NO: 129. The
predicted protein fragment encoded by this cDNA is set forth isn SEQ ID
NO: 130.
The SDH coding region encompasses 1.4 kb on 3' end of the Arabidopsis
cDNA clone (SEQ ID NO:131), and encodes a protein of about 52 kD (SEQ ID
NO:132). A DNA fragment encoding SDH was generated using PCR primers,
which added desired restriction enzyme sites, and ligated into prokaryotic
expression vector pBT430 (see Example 2). Addition of the restriction enzyme
cleavage site resulted in a change from thr to ala encoded by the second
codon.
High level expression of Arabidopsis SDH was achieved in E. coli
BL21(DE3)LysS host which expressed T7 RNA polymerase. Extracts from
IPTG-induced cells that were transformed with the vector carrying the 1.4 kb
insert were analyzed by SDS-PAGE and a protein of the expected size was
overproduced in these cells. Separation of the cell extracts into its
supernatant
(soluble) and pellet (insoluble) fractions showed that substantial amounts of
protein were present in both. SDH activity was measured in the soluble
fraction
of the bacterial extracts. No SDH activity was observed in extracts from cells
transformed with an unmodified vector. Extracts from cells containing the SDH
cDNA insert converted substantial amounts of NAD+ to NADH. The reaction
was specific for SDH because no significant activity was observed in the
absence
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of the SDH substrate saccharopine. The SDH protein has been purified from
these
bacterial extracts and used to raise rabbit antibodies to the protein.
In order to block expression of the LKR gene in transformed plants, a
chimeric gene designed for cosuppression of LKR is constructed by linking the
LKR gene or gene fragment to any of the plant promoter sequences described
above. (See U.S. Patent No. 5,231,020 for methodology to block plant gene
expression via cosuppression.) The corn LKR gene, SEQ ID NO: 120, was
modified by introducing an Nco I site at position 7 and a Kpn I site at
position
1265 using PCR. This Nco I and Kpn I DNA fragment containing the corn LKR
gene fragment was inserted into a plasmid containing the glutelin 2 promoter
and
kD zein 3' region (see Example 25) to create a chimeric gene for suppression
of
LKR expression in corn endosperm. The soybean LKR gene, SEQ ID NO: 119,
was modified by introducing an Nco I site at position 2 and a Kpn I site at
position
690 using PCR. This Nco I and Kpn I DNA fragment containing the soybean
LKR gene fragment was inserted into a plasmid containing the KTI3 promoter and
the KTI3 3' region (see Example 6) to create a chimeric gene for suppression
of
LKR expression in soybean seeds. Alternatively, a chimeric gene designed to
express antisense RNA for all or part of the LKR is constructed by linking the
LKR gene or gene fragment in reverse orientation to any of the plant promoter
sequences described above. (See U.S. patent 5,107,065 for methodology to block
plant gene expression via antisense RNA.) Either the cosuppression or
antisense
chimeric gene is introduced into plants via transformation as described in
other
Examples, e.g. Example 18 or Example 19. Transformants wherein expression of
the endogenous LKR gene is reduced or eliminated are selected.
EXAMPLE 21
Construction of Synthetic Genes in Expression Vector pSK5
To facilitate the construction and expression of the synthetic genes
described below, it was necessary to construct a plasmid vector with the
following
attributes:
1. No Ear I restriction endonuclease sites such that insertion of
sequences would produce a unique site.
2. Containing a tetracycline resistance gene to avoid loss of plasmid
during growth and expression of toxic proteins.
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3. Containing approximately 290 bp from plasmid pBT430 including
the T7 promoter and terminator segment for expression of inserted sequences in
E. coli.
4. Containing unique EcoR I and Nco I restriction endonuclease
recognition sites in proper location behind the T7 promoter to allow insertion
of
the oligonucleotide sequences.
To obtain attributes 1 and 2 Applicants used plasmid pSKI which was a
spontaneous mutant of pBR322 where the ampicillin gene and the Ear I site near
that gene had been deleted. Plasmid pSKI retained the tetracycline resistance
gene, the unique EcoR I restriction sites at base 1 and a single Ear I site at
base
2353. To remove the Ear I site at base 2353 of pSK1 a polymerase chain
reaction
(PCR) was performed using pSKI as the template. Approximately 10 femtomoles
of pSK1 were mixed with 1 ,g each of oligonucleotides SM70 and SM71 which
had been synthesized on an ABI1306B DNA synthesizer using the manufacturer's
procedures.
SM70 5'-CTGACTCGCTGCGCTCGGTC 3' SEQ ID NO:16
SM71 5'-TATTTTCTCCTTACGCATCTGTGC-3' SEQ ID NO:17
The priming sites of these oligonucleotides on the pSKI template are
depicted in Figure 10. The PCR was performed using a Perkin-Elmer Cetus kit
(Emeryville, CA) according to the instructions of the vendor on a thermocycler
manufactured by the same company. The 25 cycles were I min at 95 , 2 min at
42 and 12 min at 72". The olligonucleotides were designed to prime
replication
of the entire pSKI plasmid excluding a 30 b fragment around the Ear I site
(see
Figure 10). Ten microliters of the 100 L reaction product were run on a 1 %
agarose gel and stained with ethidium bromide to reveal a band of about 3.0 kb
corresponding to the predicted size of the replicated plasmid.
The remainder of the PCR reaction mix (90 L) was mixed with 20 L of
2.5 mM deoxynucleotide triphosphates (dATP, dTTP, dGTP, and dCTP), 30 units
of Klenow enzyme added and the mixture incubated at 37 for 30 min followed by
65 for 10 min. The Klenow enzyme was used to fill in ragged ends generated by
the PCR. The DNA was ethanol precipitated, washed with 70% ethanol, dried
under vacuum and resuspended in water. The DNA was then treated with T4
DNA kinase in the presence of 1 mM ATP in kinase buffer. This mixture was
incubated for 30 min at 37 followed by 10 min at 65 . To 10 L of the kinase-
treated preparation, 2 L of 5:X ligation buffer and 10 units of T4 DNA ligase
were added. The ligation was carried out at 15 for 16 h. Following ligation,
the
DNA was divided in half and one half digested with Ear I enzyme. The Klenow,
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kinase, ligation and restriction endonuclease reactions were performed as
described in Sambrook et al., [Molecular Cloning, A Laboratory Manual, 2nd ed.
(1989) Cold Spring Harbor Laboratory Press]. Klenow, kinase, ligase and most
restriction endonucleases were purchased from BRL. Some restriction
endonucleases were purchased from NEN Biolabs (Beverly, MA) or Boehringer
Mannheim (Indianapolis, IN). Both the ligated DNA samples were transformed
separately into competent JM103 [supE thi A(lac-proAB) F' [traD36 proAB, lacl4
lacZ OM 15] restriction minus] cells using the CaC12 method as described in
Sambrook et al., [Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold
Spring Harbor Laboratory Press] and plated onto media containing 12.5 g/mL
tetracycline. With or without Ear I digestion the same number of transformants
were recovered suggesting that the Ear I site had been removed from these
constructs. Clones were screened by preparing DNA by the alkaline lysis
miniprep procedure as described in Sambrook et al., [Molecular Cloning, A
Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press]
followed by restriction endonuclease digest analysis. A single clone was
chosen
which was tetracycline-resistant and did not contain any Ear I sites. This
vector
was designated pSK2. The remaining EcoR I site of pSK2 was destroyed by
digesting the plasmid with EcoR I to completion, filling in the ends with
Klenow
and ligating. A clone which did not contain an EcoR I site was designated
pSK3.
To obtain attributes 3 and 4 above, the bacteriophage T7 RNA polymerase
promoter/terminator segment from plasmid pBT430 (see Example 2) was
amplified by PCR. Oligonucleotide primers SM78 (SEQ ID NO: 18) and SM79
(SEQ ID NO: 19) were designed to prime a 300b fragment from pBT430 spanning
the T7 promoter/terminator sequences (see Figure 10).
SM78 5'-TTCATCGATAGGCGACCACACCCGTCC-3' SEQ ID NO: 18
SM79 5'-AATATCGATGCCACGATGCGTCCGGCG-3' SEQ ID NO:19
The PCR reaction was carried out as described previously using pBT430 as
the template and a 300 bp fragment was generated. The ends of the fragment
were
filled in using Klenow enzyme and phosphorylated as described above. DNA
from plasmid pSK3 was digested to completion with PvuII enzyme and then
treated with calf intestinal alkaline phosphatase (Boehringer Mannheim) to
remove the 5' phosphate. The procedure was as described in Sambrook et al.,
[Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor
Laboratory Press]. The cut and dephosphorylated pSK3 DNA was purified by
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ethanol precipitation and a portion used in a ligation reaction with the PCR
generated fragment containing the T7 promoter sequence. The ligation mix was
transformed into JM103 [supE thi A(lac-proAB) F' [traD36 proAB, laclq lacZ
AM 15] restriction minus] and tetracycline-resistant colonies were screened.
Plasmid DNA was prepared via the alkaline lysis mini prep method and
restriction
endonuclease analysis. was performed to detect insertion and orientation of
the
PCR product. Two clones were chosen for sequence analysis: Plasmid pSK5 had
the fragment in the orientation shown in Figure 10. Sequence analysis
performed
on alkaline denatured double-stranded DNA using Sequenase T7 DNA
polymerase (US Biochemical Corp.) and manufacturer's suggested protocol
revealed that pSK5 had no PCR replication errors within the T7
promoter/terminator sequence.
The strategy for the construction of repeated synthetic gene sequences based
on the Ear I site is depicted in :Figure 11. The first step was the insertion
of an
oligonucleotide sequence encoding a base gene of 14 amino acids. This
oligonucleotide insert contained a unique Ear I restriction site for
subsequent
insertion of oligonucleotides encoding one or more heptad repeats and added an
unique Asp 718 restriction site for use in transfer of gene sequences to plant
vectors. The overhanging ends of the oligonucleotide set allowed insertion
into
the unique Nco I and EcoR I sites of vector pSK5.
M E E K M K A M E E K
SM81 5'-CAT(;GAGGAGAAGATGAAGGCGATGGAAGAGAAG
SM80 3'--CTCCTCTTCTACTTCCGCTACCTTCTCTTC
NCO I EAR I
M K A (SEQ ID NO:22)
SM81 ATGAAG(;CGTGATAGGTACCG-3' (SEQ ID NO:20)
SM80 TACTTCCGCACTATCCATGGCTTAA-5' (SEQ ID NO:21)
ASP718 ECOR I
DNA from plasmid pSK5 was digested to completion with Nco I and
EcoR I restriction endonucleases and purified by agarose gel electrophoresis.
Purified DNA (0.1 g) was mixed with 1 g of each oligonucleotide SM80 (SEQ
ID NO:14) and SM81 (SEQ ID NO:13) and ligated. The ligation mixture was
transformed into E. coli strain JM103 [supE thi A(lac-proAB) F' [traD36 proAB,
laclq lacZ AM 151 restriction minus] and tetracycline resistant transformants
screened by rapid plasmid DNA preps followed by restriction digest analysis. A
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clone was chosen which had one each of Ear I, Nco I, Asp 718 and EcoR I sites
indicating proper insertion of the oligonucleotides. This clone was designated
pSK6 (Figure 12). Sequencing of the region of DNA following the T7 promoter
confirmed insertion of oligonucleotides of the expected sequence.
Repetitive heptad coding sequences were added to the base gene construct
of described above by generating oligonucleotide pairs which could be directly
ligated into the unique Ear I site of the base gene. Oligonucleotides SM84
(SEQ
ID NO:23) and SM85 (SEQ ID NO:24) code for repeats of the SSP5 heptad.
Oligonucleotides SM82 (SEQ ID NO:25) and SM83 (SEQ ID NO:26) code for
repeats of the SSP7 heptad.
SSP5 M E E K M K A (SEQ ID NO:28)
SM84 5'-GATGGAGGAGAAGATGAAGGC-3' (SEQ ID NO:23)
SM85 3'- CCTCCTCTTCTACTTCCGCTA-5' (SEQ ID NO:24)
SSP7 M E E K L K A (SEQ ID NO:27)
SM82 5'-GATGGAGGAGAAGCTGAAGGC-3' (SEQ ID NO:25)
SM83 3'- CCTCCTCTTCGACTTCCGCTA-5' (SEQ ID NO:26)
Oligonucleotide sets were ligated and purified to obtain DNA fragments
encoding multiple heptad repeats for insertion into the expression vector.
Oligonucleotides from each set totaling about 2 g were phosphorylated, and
ligated for 2 h at room temperature. The ligated multimers of the
oligonucleotide
sets were separated on an 18% non-denaturing 20 X 20 X 0.015 cm
polyacrylamide gel (Acrylamide: bis-acrylamide = 19:1). Multimeric forms
which separated on the gel as 168 bp (8n) or larger were purified by cutting a
small piece of polyacrylamide containing the band into fine pieces, adding 1.0
mL
of 0.5 M ammonium acetate, 1 mM EDTA (pH 7.5) and rotating the tube at 37
overnight. The polyacrylamide was spun down by centrifugation, 1 g of tRNA
was added to the supernatant, the DNA fragments were precipitated with 2
volumes of ethanol at -70 , washed with 70% (v/v) ethanol, dried, and
resuspended in 10 L of water.
Ten micrograms of pSK6 DNA were digested to completion with Ear I
enzyme and treated with calf intestinal alkaline phosphatase. The cut and
dephosphorylated vector DNA was isolated following electrophoresis in a low
melting point agarose gel by cutting out the banded DNA, liquefying the
agarose
at 55 , and purifying over NACS PREPAC columns (BRL) following
manufacturer's suggested procedures. Approximately 0.1 g of purified Ear I
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digested and phosphatase treated pSK6 DNA was mixed with 5 L of the gel
purified multimeric oliigonucleotide sets and ligated. The ligated mixture was
transformed into E. co 1i strain JM103 [supE thi 0(lac-proAB) F' [traD36
proAB,
lacP lacZ AM15] restriction minus] and tetracycline-resistant colonies
selected.
Clones were screened by restriction digests of rapid plasmid prep DNA to
determine the length of the inserted DNA. Restriction endonuclease analyses
were usually carried out by digesting the plasmid DNAs with Asp 718 and Bgl
II,
followed by separation of fragments on 18% non-denaturing polyacrylamide gels.
Visualization of fragments with ethidium bromide, showed that a 150 bp
fragment
was generated when only the base gene segment was present. Inserts of the
oligonucleotide fragments increased this size by multiples of 21 bases. From
this
screening several clones were chosen for DNA sequence analysis and expression
of coded sequences in E. coli.
Table 14
Sequence by Heptad
Clone # SEO ID INTO: Amino Acid Repeat (SSP) SEQ ID NO:
C15 29 5.7.7.7.7.7.5 30
C20 31 5.7.7.7.7.7.5 32
C30 33 5.7.7.7.7.5 34
D16 35 5.5.5.5 36
D20 37 5.5.5.5.5 38
D33 39 5.5.5.5 40
The first and last SSP`i heptads; flanking the sequence of each construct are
from
the base gene described above. Inserts are designated by underlining.
Because the gel purification of the oligomeric forms of the oligonucleotides
did not give the expected enrichment of longer (i.e., >8n) inserts, Applicants
used
a different procedure for a subsequent round of insertion constructions. For
this
series of constructs four more sets of oligonucleotides were generated which
code
for SSP 8,9,10 and 11 amino acid sequences respectively:
SSPB M E E K L K K (SEQ ID NO:49)
SM86 5'-GATGGAGGAGAAGCTGAAGAA-3' (SEQ ID NO:41)
SM87 3'- CCTCCTCTTCGACTTCTTCTA-5' (SEQ ID NO:42)
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SSP9 M E E K L K W (SEQ ID NO:50)
SM88 5'-GATGGAGGAGAAGCTGAAGTG-3' (SEQ ID NO:43)
SM89 3'- CCTCCTCTTCGACTTCACCTA-5' (SEQ ID N0:44)
SSP10 M E E K M K K (SEQ ID NO:51)
SM90 5'-GATGGAGGAGAAGATGAAGAA-3' (SEQ ID NO:45)
SM91 3'- CCTCCTCTTCTACTTCTTCTA-5' (SEQ ID NO:46)
SSP11 M E E K M K W (SEQ ID NO:52)
SM92 5'-GATGGAGGAGAAGATGAAGTG-3' (SEQ ID NO:47)
SM93 3'- CCTCCTCTTCTACTTCACCTA-5' (SEQ ID NO:48)
The following HPLC procedure was used to purify multimeric forms of the
oligonucleotide sets after phosphorylating and ligating the oligonucleotides
as
described above. Chromatography was performed on a Hewlett Packard Liquid
Chromatograph instrument, Model 1090M. Effluent absorbance was monitored at
260 rim. Ligated oligonucleotides were centrifuged at 12,000xg for 5 min and
injected onto a 2.5 micron TSK DEAE-NPR ion exchange column (35 cm x
4.6 mm i.d.) fitted with a 0.5 micron in-line filter (Supelco). The
oligonucleotides
were separated on the basis of length using a gradient elution and a two
buffer
mobile phase [Buffer A: 25 mM Tris-Cl, pH 9.0, and Buffer B: Buffer A + 1 M
NaCI]. Both Buffers A and B were passed through 0.2 micron filters before use.
The following gradient program was used with a flow rate of 1 mL per min at 30
:
Time %A %B
initial 75 25
0.5 min 55 45
min 50 50
20 min 38 62
23 min 0 100
30 min 0 100
31 min 75 25
Fractions (500 L) were collected between 3 min and 9 min. Fractions
corresponding to lengths between 120 bp and 2000 bp were pooled as determined
from control separations of restriction digests of plasmid DNAs.
The 4.5 mL of pooled fractions for each oligonucleotide set were
precipitated by adding 10 g of tRNA and 9.0 mL of ethanol, rinsed twice with
70% ethanol and resuspended in 50 gL of water. Ten L of the resuspended
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HPLC purified oligonucleotides were added to 0.1 g of the Ear I cut,
dephosphorylated pSK6 DNA described above and ligated overnight at 15 . All
six oligonucleotide sets described above which had been phosphorylated and
self-
ligated but not purified by gel or HPLC were also used in separate ligation
reactions with the pSK6 vector. The ligation mixtures were transformed into
E. coli strain DH5a [supE44 OlacU169 (b80 lacZ AM 15) hsdR17 recAI endAI
gyr196 thi 1 relAI ] and tetracycline-resistant colonies selected. Applicants
chose
to use the DH5a [supE44 OlacU169 (D80 lacZ DM15) hsdR17 recAl endAl
gyr196 thiI relAI] strain for alt subsequent work because this strain has a
very
high transformation rate and is recA-. The recA- phenotype eliminates concerns
that these repetitive DNA structures may be substrates for homologous
recombination leading to deletion of multimeric sequences.
Clones were screened as described above. Several clones were chosen to
represent insertions of each of the six oligonucleotide sets.
Table 15
Sequence by Heptad
Clone SEO ID NO: Amino Acid Repeat (SSP) SEO ID NO:
82-4 53 7.7.7.7.7.7.5 54
84-H3 55 5.5.5.5 56
86-1123 57 5.8.8.5 58
88-2 59 5.9.9.9.5 60
90-H8 61 5.10.10.10.5 62
92-2 63 5.11.11.5 64
The first and last SSP5 heptads flanking the sequence represent the base gene
sequence. Insert sequences are underlined. Clone numbers including the letter
"H" designate HPLC-purified oligonucleotides. The loss of the first base gene
repeat in clone 82-4 may have resulted from homologous recombination between
the base gene repeats 5.5 before the vector pSK6 was transferred to the recA-
strain. The HPLC procedure did not enhance insertion of longer multimeric
forms
of the oligonucleotide sets into the base gene but did serve as an efficient
purification of the ligated oligonucleotides.
Oligonucleotides were designed which coded for mixtures of the SSP
sequences and which varied codon usage as much as possible. This was done to
reduce the possibility of deletion of repetitive inserts by recombination once
the
synthetic genes were transformed into plants and to extend the length of the
constructed gene segments. These oligonucleotides encode four repeats of
heptad
coding units (28 amino acid residues) and can be inserted at the unique Ear I
site
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in any of the previously constructed clones. SM96 and SM97 code for SSP(5)4,
SM98 and SM99 code for SSP(7)4 and SM100 plus SM101 code for SSP8.9.8.9.
M E E K M K A M E E K M K
SM96 5'-GATGGAGGAAAAGATGAAGGCGATGGAGGAGAAAATGAAA
SM97 3' CCTCCTTTTCTACTTCCGCTACCTCCTCTTTTACTTT
A M E E K M K A M E E K M K A (SEQ ID NO:67)
GCTATGGAGGAAAAGATGAAAGCGATGGAGGAGAAAATGAAGGC-3' (SEQ ID NO:65)
CGATACCTCCTTTTCTACTTTCGCTACCTCCTCTTTTACTTCCGCTA-5' (SEQ ID NO:66)
M E E K L K A M E E K L K
SM98 5'-GATGGAGGAAAAGCTGAAAGCGATGGAGGAGAAACTCAAG
SM99 3' CCTCCTTTTCGACTTTCGCTACCTCCTCTTTGAGTTC
A M E E K L K A M E E K L K A (SEQ ID NO:70)
GCTATGGAAGAAAAGCTTAAAGCGATGGAGGAGAAACTGAAGGC-3' (SEQ ID NO:68)
CGATACCTTCTTTTCGAATTTCGCATCCTCCTCTTTGACTTCCGCTA-5' (SEQ ID NO:69)
M E E K L K K M E E K L K
SM100 5'-GATGGAGGAAAAGCTTAAGAAGATGGAAGAAAAGCTGAAA
SM101 3' CCTCCTTTTCGAATTCTTCTACCTTCTTTTCGACTTT
W M E E K L K K M E E K L K W (SEQ ID NO:73)
TGGATGGAGGAGAAACTCAAAAAGATGGAGGAAAAGCTTAAATG-3' (SEQ ID NO:71)
ACCTACCTCCTCTTTGAGTTTTTCATCCTCCTTTTCGAATTTACCTA-5' (SEQ ID NO:72)
DNA from clones 82-4 and 84-H3 were digested to completion with Ear I
enzyme, treated with phosphatase and gel purified. About 0.2 .tg of this DNA
were mixed with 1.0 g of each of the oligonucleotide sets SM96 and SM97,
SM98 and SM99 or SM100 and SM101 which had been previously
phosphorylated. The DNA and oligonucleotides were ligated overnight and then
the ligation mixes transformed into E. coli strain DH5a. Tetracycline-
resistant
colonies were screened as described above for the presence of the
oligonucleotide
inserts. Clones were chosen for sequence analysis based on their restriction
endonuclease digestion patterns.
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Table 16
Sequence by Heptad
Clone # SEO ID NO: Amino Acid Repeat (SSP) SEO ID NO:
2-9 74 7.7.7.7.7.7.8.9.8.9.5 75
3-5 78 7.7.7.7.7.7.5.5 79
5-1 76 5.5.5.7.7.7.7.5 77
Inserted oligonucleotide segments are underlined
Clone 2-9 was derived from oligonucleotides SM 100 (SEQ ID NO:71) and
SM101 (SEQ ID NO:72) ligated into the Ear I site of clone 82-4 (see above).
Clone 3-5 (SEQ ID NO:78) was derived from the insertion of the first 22 bases
of
the oligonucleotide set SM96 (SEQ ID NO:65) and SM97 (SEQ ID NO:66) into
the Ear I site of clone 82-4 (SEQ ID NO:53). This partial insertion may
reflect
improper annealing of these highly repetitive oligos. Clone 5-1 (SEQ ID NO:76)
was derived from oligonucleotides SM98 (SEQ ID NO:68) and SM99 (SEQ ID
NO:69) ligated into the Ear I site of clone 84-H3 (SEQ ID NO:55).
Strategy II.
A second strategy for construction of synthetic gene sequences was
implemented to allow more flexibility in both DNA and amino acid sequence.
This strategy is depicted in Figure 13 and Figure 14. The first step was the
insertion of an oligonucleotide sequence encoding a base gene of 16 amino
acids
into the original vector pSK5. This oligonucleotide insert contained an unique
Ear I site as in the previous base gene construct for use in subsequent
insertion of
oligonucleotides encoding one or more heptad repeats. The base gene also
included a BspH I site at the 3' terminus. The overhanging ends of this
cleavage
site are designed to allow "in frame" protein fusions using Nco I overhanging
ends. Therefore, gene segments can be multiplied using the duplication scheme
described in Figure 14. The overhanging ends of the oligonucleotide set
allowed
insertion into the unique Nco I and EcoR I sites of vector pSK5.
M E E K M K K L E E K
SM107 5'-CATGGAGGAGAAGATGAAAAAGCTCGAAGAGAAG
SM106 :3'-CTCC'.TCTTCTACTTTTTCGAGCTTCTCTTC
NCO I EAR I
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M K V M K (SEQ ID NO:82)
ATGAAGGTCATGAAGTGATAGGTACCG-3' (SEQ ID NO:80)
TACTTCCAGTACTTCACTATCCATGGCTTAA-5' (SEQ ID NO:81)
BSPH I ASP 718
The oligonucleotide set was inserted into pSK5 vector as described in Strategy
I
above. The resultant plasmid was designated pSK34.
Oligonucleotide sets encoding 35 amino acid "segments" were ligated into
the unique Ear I site of the pSK34 base gene using procedures as described
above.
In this case, the oligonucleotides were not gel or HPLC purified but simply
annealed and used in the ligation reactions. The following oligonucleotide
sets
were used:
SEG 3 L E E K M K A M E D K M K W
SM110 5'-GCTGGAAGAAAAGATGAAGGCTATGGAGGACAAGATGAAATGG
SM111 3'-CCTTCTTTTCTACTTCCGATACCTCCTGTTCTACTTTACC
L E E K M K K (SEQ ID NO:85)
(amino acids 8-28)
CTTGAGGAAAAGATGAAGAA-3' (SEQ ID NO:83)
GAACTCCTTTTCTACTTCTTCGA-5' (SEQ ID NO:84)
SEG 4 L E E K M K A M E D K M K W
SM112 5'-GCTCGAAGAAAGATGAAGGCAATGGAAGACAAAATGAAGTGG
SM113 3'-GCTTCTTTCTACTTCCGTTACCTTCTGTTTTACTTCACC
L E E K M K K (SEQ ID NO:86)
(amino acids 8-28)
CTTGAGGAGAAAATGAAGAA-3' (SEQ ID NO:87)
GAACTCCTCTTTTACTTCTTCGA-5' (SEQ ID NO:88)
SEG 5 L K E E M A K M K D E M W K
SM114 5'-GCTCAAGGAGGAAATGGCTAAGATGAAAGACGAAATGTGGAAA
SM115 3'-GTTCCTCCTTTACCGATTCTACTTTCTGCTTTACACCTTT
L K E E M K K (SEQ ID NO:89)
(amino acids 8-28)
CTGAAAGAGGAAATGAAGAA (SEQ ID NO:90)
GACTTTCTCCTTTACTTCTTCGA (SEQ ID NO:91)
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Clones were screened for the presence of the inserted segments by restriction
digestion followed by separation of fragments on 6% acrylamide gels. Correct
insertion of oligonucleotides was confirmed by DNA sequence analyses. Clones
containing segments 3., 4 and 5 respectively were designated pSKseg3, pSKseg4,
and pSKseg5.
These "segment" clones were used in a duplication scheme as shown in
Figure 14. Ten gg of plasmid pSKseg3 were digested to completion with Nhe I
and BspH I and the 1503 bp fragment isolated from an agarose gel using the
Whatman paper technique. Ten g of plasmid pSKseg4 were digested to
completion with Nhe I and Nco I and the 2109 bp band gel isolated. Equal
amounts of these fragments were ligated and recombinants selected on
tetracycline. Clones were screened by restriction digestions and their
sequences
confirmed. The resultant plasmid was designated pSKseg34.
pSKseg34 and pSKseg5 plasmid DNAs were digested, fragments isolated
and ligated in a similar manner as above to create a plasmid containing DNA
sequences encoding segment 5 fused to segments 3 and 4. This construct was
designated pSKseg534 and encodes the following amino acid sequence:
SSP534 NH2-MEEKMKKLKEEMAKMKDEMWKLKEEMKKLEEKMKVMEEKMKKLEEKMKA
MEDKMKWLEEKMKKLEEKMKVMEEKMKKLEEKMKAMEDKMKWLEEKMKK
LEEKMKVMK-COOH (SEQ ID NO:92)
EXAMPLE 22
Construction of SSP Chimeric Genes for Expression in the Seeds of Plants
To express the synthetic gene products described in Example 21 in plant
seeds, the sequences were transferred to the seed promoter vectors pCW108,
pC W 109 or pML 113 (Figure 15). The vectors pC W 108 and pML 113 contain the
bean phaseolin promoter (from base +1 to base -494),and 1191 bases of the 3'
sequences from bean phaseolin gene. Plasmid pCW109 contains the soybean
(3-conglycinin promoter (from base +l to base -619) and the same 1191 bases of
3'
sequences from the bean phaseolin gene. These vectors were designed to allow
direct cloning of coding sequences into unique Nco I and Asp 718 sites. These
vectors also provide sites (Hind III or Sal I) at the 5' and 3' ends to allow
transfer
of the promoter/coding region/3' sequences directly to appropriate binary
vectors.
To insert the synthetic storage protein gene sequences, 10 g of vector DNA
were digested to completion with Asp 718 and Nco I restriction endonucleases.
The linearized vector was purified via electrophoresis on a 1.0% agarose gel
overnight electrophoresis at 15 volts. The fragment was collected by cutting
the
agarose in front of the band, inserting a 10 X 5 mm piece of Whatman 3MM paper
into the agarose and electrophoresing the fragment into the paper [Errington,
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(1990) Nucleic Acids Research, 18:17]. The fragment and buffer were spun out
of
the paper by centrifugation and the DNA in the -100 p.L was precipitated by
adding 10 mg of tRNA, 10 p.L of 3 M sodium acetate and 200 L of ethanol. The
precipitated DNA was washed twice with 70% ethanol and dried under vacuum.
The fragment DNA was resuspended in 20 L of water and a portion diluted
10-fold for use in ligation reactions.
Plasmid DNA (10 mg) from clone 3-5 (carrying the SSP3-5 coding
sequence) and pSK534 (carrying the SSP534 coding sequence) was digested to
completion with Asp 718 and Nco I restriction endonucleases. The digestion
products were separated on an 18% polyacrylamide non-denaturing gel. Gel
slices containing the desired fragments were cut from the gel and purified by
inserting the gel slices into a I% agarose gel and electrophoresing for 20 min
at
100 volts. DNA fragments were collected on 10 X 5 mm pieces of Whatman
3MM paper, the buffer and fragments spun out by centrifugation and the DNA
precipitated with ethanol. The fragments were resuspended in 6 .iL water. One
microliter of the diluted vector fragment described above, 2 L of 5X ligation
buffer and 1 L of T4 DNA ligase were added. The mixture was ligated overnight
at 15 .
The ligation mixes were transformed into E. coli strain DH5a [supE44
MlacU169 (080 lacZ AM15) hsdRl7 recAI endAl gyr196 thiI relAI] and
ampicillin-resistant colonies selected. The clones were screened by
restriction
endonuclease digestion analyses of rapid plasmid DNAs and by DNA sequencing.
EXAMPLE 23
Tobacco Plants Containing the Chimeric Genes Phaseolin
Promoter/cts/lysC-M4 and f3-conglycinin promoter/SSP3-5
The binary vector pZS97 was used to transfer the chimeric SSP3-5 gene of
Example 22 and the chimeric E. coli dapA and lysC-M4 genes of Example 4 to
tobacco plants. Binary vector pZS97 (Figure 6) is part of a binary Ti plasmid
vector system [Bevan, (1984) Nucl. Acids. Res. 12:8711-8720] of Agrobacterium
tumefaciens. The vector contains: (1) the chimeric gene nopaline
synthase::neomycin phosphotransferase (nos::NPTII) as a selectable marker for
transformed plant cells [Bevan et al., (1983) Nature 304:184-186], (2) the
left and
right borders of the T-DNA of the Ti plasmid [Bevan, (1984) Nucl. Acids. Res.
12:8711-8720], (3) the E. coli lacZ a-complementing segment [Viera et al.,
(1982) Gene 19:259-267] with a unique Sal I site(pSK97K) or unique Hind III
site
(pZS97) in the polylinker region, (4) the bacterial replication origin from
the
Pseudomonas plasmid pVSI [Itoh et al., (1984) Plasmid 11:206-220], and (5) the
bacterial (3-lactamase gene as a selectable marker for transformed A.
tumefaciens.
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Plasmid pZS97 DNA was digested to completion with Hind III enzyme and
the digested plasmid was gel purified. The Hind III digested pZS97 DNA was
mixed with the Hind III digested and gel isolated chimeric SSP3-5 gene of
Example 22, ligated, transformed and colonies selected on ampicillin.
The binary vector containing the chimeric gene was transferred by tri-
parental mating [Ruvkin et al., (1981) Nature 289:85-88] to Agrobacterium
strain
LBA4404/pAL4404 [Hockema et al., (1983), Nature 303:179-180] selecting for
carbenicillin resistance. Cultures of Agrobacterium containing the binary
vector
was used to transform tobacco leaf disks [Horsch et al., (1985) Science
227:1229-1231]. Transgenic plants were regenerated in selective medium
containing kanamycin.
Transformed tobacco plants containing the chimeric gene, [i-conglycinin
promoter/SSP3-5/phaseolin 3' region, were thus obtained. Two transformed
lines,
pSK44-3A and pSK44=-9A, which carried a single site insertion of the SSP3-5
gene were identified based upon 3:1 segregation of the marker gene for
kanamycin resistance. Progeny of the primary transformants, which were
homozygous for the transgene, pSK44-3A-6 and pSK44-9A-5, were then
identified based upon 4:0 segregation of the kanamycin resistance in seeds of
these plants.
Similarly, transformed tobacco plants with the chimeric genes phaseolin 5'
region/cts/lysC-M4/phaseolin 3' region and phaseolin 5'
region/cts/ecodaA/phaseolin 3' region were obtained as described in Example
12.
A transformed line, BT570-45A, which carried a single site insertion of the
DHDPS and AK genes was identified based upon 3:1 segregation of the marker
gene for kanamycin resistance. Progeny from the primary transformant which
were homozygous for the transgene, BT570-45A-3 and BT570-45A-4, were then
identified based upon 4:0 segregation of the kanamycin resistance in seeds of
these plants.
To generate plants carrying all three chimeric genes genetic crosses were
performed using the homozygous parents. Plants were grown to maturity in
greenhouse conditions. Flowers to be used as male and female were selected one
day before opening and older flowers on the inflorescence removed. For
crossing,
female flowers were chosen at the point just before opening when the anthers
were
not dehiscent. The corolla was opened on one side and the anthers removed.
Male flowers were chosen as flowers which had opened on the same day and had
dehiscent anthers shedding mature pollen. The anthers were removed and used to
pollinate the pistils of the anther-stripped female flowers. The pistils were
then
covered with plastic tubing to prevent further pollination. The seed pods were
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allowed to develop and dry for 4-6 weeks and harvested. Two to three separate
pods were recovered from each cross. The following crosses were performed:
Male X Female
BT570-45A-3 pSK44-3A-6
BT570-45A-4 pSK44-3A-6
pSK44-3A-6 BT570-45A-4
BT570-45A-5 pSK44-9A-5
pSk44-9A-5 BT570-45A-5
Dried seed pods were broken open and seeds collected and pooled from each
cross. Thirty seeds were counted out for each cross and for controls seeds
from
selfed flowers of each parent were used. Duplicate seed samples were
hydrolyzed
and assayed for total amino acid content as described in Example 8. The amount
of increase in lysine as a percent of total seeds amino acids over wild type
seeds,
which contain 2.56% lysine, is presented in Table 16 along the copy number of
each gene in the endosperm of the seed.
TABLE 17
copy number
AK & DHDPS copy number lysine
male X female genes SSP gene increase
BT570-45A X BT570-45A 1 * 0 0
pSK44-9A X pSK44-9A 0 1 * 0.12
pSK44-9A-5 X pSK44-9A-5 0 2 0.29
pSK44-9A-5 X BT570-45A-5 1 1 0.6
BT570-45A-5 X pSK44-9A-5 1 1 0.29
pSK44-3A X pSK44-3A 0 1 * 0.28
pSK44-3A-6 X pSK44-3A-6 0 2 0.5
pSK44-3A-6 X BT570-45A-4 1 1 0.62
BT570-45A-3 X pSK44-3A-6 1 1 0.27
BT570-45A-4 X pSK44-3A-6 1 1 0.29
* copy number is average in population of seeds
The results of these crosses demonstrate that the total lysine levels in seeds
can be increased by the coordinate expression of the lysine biosynthesis genes
and
the high lysine protein SSP3-5. In seeds derived from hybrid tobacco plants,
this
synergism is strongest when the biosynthesis genes are derived from the female
parent. It is expected that the lysine level would be further increased if the
biosynthesis genes and the lysine-rich protein genes were all homozygous.
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EXAMPLE 24
Soybean Plants Containing the Chimeric Genes Phaseolin Promoter/cts/cordaDA
Phaseolin Promoter/cts/lysC-M4 and Phaseolin Promoter/SSP3-5
Transformed soybean plants that express the chimeric gene, phaseolin
promoter/cts/cordapAl phaseolin 3' region and phaseolin promoter/ets/lysC-M4/
phaseolin 3' region have been described in Example 19. Transformed soybean
plants that express the chimeric gene, phaseolin promoter/SSP3-5/phaseolin 3'
region, were obtained by inserting the chimeric gene as an isolated Hind III
fragment into an equivalent soybean transformation vector plasmid pML63
(Figure 16) and carrying out transformation as described in Example 19.
Seeds from primary transformants were sampled by cutting small chips from
the sides of the seeds away from the embryonic axis. The chips were assayed
for
GUS activity as described in Example 19 to determine which of the segregating
seeds carried the transgenes. Half seeds were ground to meal and assayed for
expression of SSP3-5 protein by Enzyme Linked ImmunoSorbent Assay (ELISA).
was performed as follows:
A fusion protein of glutathione-S-transferase and the SSP3-5 gene product
was generated through the use of the Pharmacia pGEX GST Gene Fusion
System (Current Protocols in Molecular Biology, Vol. 2, pp 16.7.1-8, (1989)
John
Wiley and Sons). The fusion protein was purified by affinity chromatography on
glutathione agarose (Sigma) or glutathione Sepharose (Pharmacia) beads,
concentrated using Centricon 10 (Amicon) filters, and then subjected to SDS
polyacrylamide electrophoresis (15% acrylamide, 19:1 acrylamide:bisacrylamide)
for further purification. The gel was stained with Coomassie Blue for 30 min,
destained in 50% (v/v) methanol, 10% (v/v) acetic acid and the protein bands
electroeluted using an. Amicon Centriluter Microelectroeluter (Paul T.
Matsudaira
ed., A Practical Guide to Protein and Peptide Purification for
Microsequencing,
Academic Press, Inc. New York, 1989). A second gel prepared and run in the
same manner was stained in a non acetic acid containing stain [9 parts 0.1 %
Coomassie Blue G250 (Bio-Rad) in 50% (v/v) methanol and 1 part Serva Blue
(Serva, Westbury, NY) in distilled water] for 1-2 h. The gel was briefly
destained
in 20%(v/v) methanol, 3%(v/v) glycerol for 0.5-1 h until the GST-SSP3-5 band
was just barely visible. This band was excised from the gel and sent with the
electroeluted material to Hazelton Laboratories for use as an antigen in
immunizing a New Zealand Rabbit. A total of I mg of antigen was used (0.8 mg
in gel, 0.2 mg in solution). Test bleeds were provided by Hazelton
Laboratories
every three weeks. The approximate titer was tested by western blotting of E.
coli
extracts from cells containing the SSP-3-5 gene under the control of the T7
promoter at different dilutions of protein and of serum.
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IgG was isolated from the serum using a Protein A Sepharose column. The
IgG was coated onto microtiter plates at 5 g per well. A separate portion of
the
IgG was biotinylated.
Aqueous extracts from transgenic plants were diluted and loaded into the
wells usually starting with a sample containing I g of total protein. The
sample
was diluted several more times to insure that at least one of the dilutions
gave a
result that was within the range of a standard curve generated on the same
plate.
The standard curve was generated using chemically synthesized SSP3-5 protein.
The samples were incubated for 1 h at 37 and the plates washed. The
biotinylated IgG was then added to the wells. The plate was incubated at 370
for
1 h and washed. Alkaline phosphatase conjugated to streptavidin was added to
the
wells, incubated at 37 for 1 h and washed. A substrate consisting of 1 mg/mL
p
nitrophenylphosphate in 1 M diethanolamine was added to the wells and the
plates
incubated at 37 for 1 h. A 5% EDTA stop solution was added to the wells and
the absorbance read at 405 nm minus 650 nm reading. Transgenic soybean seeds
contained 0.5 to 2.0% of water extractable protein as SSP3-5.
The remaining half seeds positive for GUS and SSP3-5 protein were planted
and grown to maturity in greenhouse conditions. To determine homozygotes for
the GUS phenotype, seed from these R1 plants were screened for segregation of
GUS activity as above. Plants homozygous for the phaseolin/SSP3-5 gene are
then crossed with homozygous transgenic soybeans expressing the
Corynebacterium dapA gene product or expressing the Corynebacterium dapA
gene product plus the E. coli lsC-M4 gene product.
As an preferred alternative to bringing the chimeric SSP gene and chimeric
cordapA gene plus the E. coli lysC-M4 gene together via genetic crossing, a
single
soybean transformation vector carrying all the genes can be constructed from
the
gene fragments described above and transformed into soybean as described in
Example 19.
EXAMPLE 25
Construction of Chimeric Genes for Expression of Corynebacterium DHDPS.
lysr-Corn DHDPS. E. coliAKIII-M4 and SSP3-5 proteins in the Embryo and
Endosperm of Transformed Corn
The following chimeric genes were made for transformation into corn:
globulin 1 promoter/mcts/lysC-M4/NOS 3' region
globulin 1 promoter/mcts/cordapA/NOS 3 region
glutelin 2 promoter/mcts/1vsC-M4/NOS 3' region
glutelin 2 promoter/mcts/cordapA/NOS 3' region
globulin I promoter/SSP3-5/globulin 1 3' region
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glutelin 2 promoter/SSP3-5/10 kD 3' region
globulin 1 promoter/corn lysr-mutant DHDPS gene/globulin 1 3' region
glutelin 2 promoter/corn lysr-mutant DHDPS gene/10 kD 3' region
The glutelin 2 promoter was cloned from corn genomic DNA using PCR
with primers based on the published sequence [Reina et al. (1990) Nucleic
Acids
Res. 18:6426-6426]. The promoter fragment includes 1020 nucleotides upstream
from the ATG translation start codon. An Nco I site was introduced via PCR at
the ATG start site to allow for direct translational fusions. A BamH I site
was
introduced on the 5' end of the promoter. The 1.02 kb BamH Ito Nco I promoter
fragment was cloned into the BamH Ito Nco I sites of the plant expression
vector
pML63 (see Example 24) replacing the 35S promoter to create vector pML90.
This vector contains the glutelin 2 promoter linked to the GUS coding region
and
the NOS 3'.
The 10 kD zein 3' region was derived from a 10 kD zein gene clone
generated by PCR from genomic DNA using oligonucleotide primers based on the
published sequence [Kirihara et al. (1988) Gene 71:359-370]. The 3' region
extends 940 nucleotides from the stop codon. Restriction endonuclease sites
for
Kpn I, Sma I and Xba l[ sites were added immediately following the TAG stop
codon by oligonucleotide insertion to facilitate cloning. A Sma Ito Hind III
segment containing the 10 kD 3'region was isolated and ligated into Sma I and
Hind III digested pML90 to replace the NOS 3' sequence with the 10 kD
3'region,
thus creating plasmid pML 103. pML 103 contains the glutelin 2 promoter, an
Nco I site at the ATG start codon of the GUS gene, Sma I and Xba I sites after
the
stop codon, and 940 nucleotides of the 10 kD zein 3' sequence.
The globulin 1 promoter and 3' sequences were isolated from a Clontech
corn genomic DNA library using oligonucleotide probes based on the published
sequence of the globulin 1 gene [Kriz et al. (1989) Plant Physiol. 91:636].
The
cloned segment includes the promoter fragment extending 1078 nucleotides
upstream from the ATG translation start codon, the entire globulin coding
sequence including introns and the 3' sequence extending 803 bases from the
translational stop. To allow replacement of the globulin 1 coding sequence
with
other coding sequences an Nco I site was introduced at the ATG start codon,
and
Kpn I and Xba I sites were introduced following the translational stop codon
via
PCR to create vector pCC50. There is a second Nco I site within the globulin I
promoter fragment. The globulin 1 gene cassette is flanked by Hind III sites.
The plant amino acid biosynthetic enzymes are known to be localized in the
chloroplasts and therefore are synthesized with a chloroplast targeting
signal.
Bacterial proteins such as DHDPS and AKIII have no such signal. A chloroplast
transit sequence (cts) was therefore fused to the cordapA and 1vsC-M4 coding
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sequence in the chimeric genes described below. For corn the cts used was
based
on the cts of the small subunit of ribulose 1,5-bisphosphate carboxylase from
corn
[Lebrun et al. (1987) Nucleic Acids Res. 15:4360] and is designated mcts to
distinguish it from the soybean cts. The oligonucleotides SEQ ID NOS:94-99
were synthesized and used as described in Example 6.
To construct the chimeric gene:
globulin 1 promoter/mcts/lysC-M4/NOS 3' region
an Nco I to Hpa I fragment containing the mcts/lysC-M4 coding sequence was
isolated from plasmid pBT558 (see Example 6) and inserted into Nco I plus Sma
I
digested pCC50 creating plasmid pBT663.
To construct the chimeric gene:
globulin 1 promoter/mcts/cordspA/NOS 3 region
an Nco I to Kpn I fragment containing the mcts/ecodapA coding sequence was
isolated from plasmid pBT576 (see Example 6) and inserted into Nco I plus Kpn
I
digested pCC50 creating plasmid pBT662. Then the ecodapA coding sequence
was replaced with the cordapA coding sequence as follows. An Aft II to Kpn I
fragment containing the distal two thirds of the mcts fused to the cordapA
coding
sequence was inserted into Aft II to Kpn I digested pBT662 creating plasmid
pBT677.
To construct the chimeric gene:
glutelin 2 promoter/mcts/lysC-M4/NOS 3' region
an Nco Ito Hpa I fragment containing the mcts/lysC-M4 coding sequence was
isolated from plasmid pBT558 (see Example 6) and inserted into Nco I plus Sma
I
digested pML90 creating plasmid pBT580.
To construct the chimeric gene:
glutelin 2 promoter/mcts/cordpAA/NOS 3' region
an Nco Ito Kpn I fragment containing the mcts/cordapA coding sequence was
isolated from plasmid pBT677 and inserted into Nco I to Kpn I digested pML90,
creating plasmid pBT679.
The chimeric genes:
globulin 1 promoter/mcts/lysC-M4/NOS 3' region and
globulin I promoter/mcts/cordapA/NOS 3 region
were linked on one plasmid as follows. pBT677 was partially digested with
Hind III and full-length linearized plasmid DNA was isolated. A Hind III
fragment carrying the globulin 1 promoter/mcts/lysC-M4/NOS 3' region was
isolated from pBT663 and ligated to the linearized pBT677 plasmid creating
pBT680 (Figure 17).
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The chimeric genes:
glutelin 2 promoter/rrmcts/IYSC-M4/NOS 3' region and
glutelin 2 promoter/rncts/cordapA/NOS 3' region
were linked on one plasmid as follows. pBT580 was partially digested with Sal
I
and full-length linearized plasmid DNA was isolated. A Sal I fragment carrying
the glutelin 2 promoter/mcts/cordapA/NOS 3' region was
isolated from pBT679 and ligated to the linearized pBT580 plasmid creating
pBT681 (Figure 18).
To construct the chimeric gene:
glutelin 2 promoter/SSP3-5/10 kD 3' region
the plasmid pML103 (above) containing the glutelin 2 promoter and 10 kD zein
3'
region was cleaved at the Nco I and Sma I sites. The SSP3-5 coding region
(Example 22) was isolated as an Nco I to blunt end fragment by cleaving with
Xba I followed by filling in the sticky end using Klenow fragment of DNA
polymerase, then cleaving with Nco I. The 193 base pair Nco I to blunt end
fragment was ligated into the Nco I and Sma I cut pML 103 to create pLH 104
(Figure 19).
To construct the chimeric gene:
globulin 1 promoter/SSP3-5/globulin 1 3'region
the 193 base pair Nco I and Xba I fragment containing the SSP3-5 coding region
(Example 22) was inserted into plasmid pCC50 (above) between the globulin 1 5'
and 3' regions creating pLH105 (Figure 20).
The corn DHDPS cDNA gene was cloned and sequenced previously [Frisch
et al. (1991) Mol Gen Genet 228:287-293]. A mutation that rendered the protein
insensitive to feedback inhibition by lysine was introduced into the gene.
This
mutation is a single nucleotide change that results in a single amino acid
substitution in the protein; alal.66 is changed to val. The lysr corn DHDPS
gene
was obtained from Dr. Burle Gengenbach at the University of Minnesota. An
Nco I site was introduced at the translation start codon of the gene and a Kpn
I site
was introduced immediately following the translation stop codon of the gene
via
PCR using the following primers:
SEQ ID NO:106: 5'-ATT(:000ATG GTTTCGCCGA CGAAT
SEQ ID NO:107: 5'-CTCTCGGTAC CTAGTACCTA CTGATCAAC
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To construct the chimeric gene:
globulin 1 promoter/lysr corn DHDPS gene/globulin 1 3'region the 1144 base
pair
Nco I and Kpn I fragment containing the lysr corn DHDPS gene was inserted into
plasmid pCC50 (above) between the globulin 1 5' and 3' regions creating pBT739
(Figure 21).
To construct the chimeric gene:
glutelin 2 promoter/lysr corn DHDPS gene/10 kD 3' region
the 1144 base pair Nco I and Kpn I fragment containing the lysr corn DHDPS
gene was inserted into a plasmid containing the glutelin 2 promoter and 10 kD
zein 3' region creating plasmid pBT756 (Figure 22).
Corn transformations were done as described in Examples 17 and 18 with
the following exceptions:
1) Embryogenic cell culture development was as described in Example 17
except the exact culture used for bombardment was designated LH132.5.X, or
LH132.6.X.
2) The selectable marker used for these experiments was either the 35S/bar
gene from pDETRIC as described in Example 18 or 35S/Ac, a synthetic
phosphinothricin-N-acetyltransferase (pat) gene under the control of the 35S
promoter and 3' terminator/ polyadenylation signal from Cauliflower Mosaic
Virus [Eckes et al., (1989) J Cell Biochem Suppl 13 D]
3) The bombardment parameters were as described for Example 17 and 18
except that the bombardments were performed as "tribombardments" by co-
precipitating 1.5 gg of each of the DNAs (35S/bar or 35S/Ac, pBT681 and
pLH104 or 35S/Ac, pbt680 and pLH105) onto the gold particles.
4) Selection of transgenic cell lines was as described for glufosinate
selection as in Example 18 except that the tissue was placed on the selection
media within 24 h after bombardment.
EXAMPLE 26
Corn Plants Containing Chimeric Genes for Expression of Corvnebacterium
DHDPS and E. coliAKlll-M4 or lysr-Corn DHDPS in the Embryo and
Endosperm
Corn was transformed as described in Example 25 with the chimeric genes:
= globulin 1 promoter/mcts/cordpA/NOS 3 region along with or without
globulin 1 promoter/mcts/l rsC-M4/NOS 3' region; or
= glutelin 2 promoter/mcts/cordapAA/NOS 3' region along with or without
glutelin 2 promoter/mcts/1vsC-M4/NOS 3' region.
Plants regenerated from transformed callus were analyzed for the presence
of the intact transgenes via Southern blot or PCR. The plants were either
selfed or
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outcrossed to an elite line to generate F 1 seeds. Six to eight seeds were
pooled
and assayed for expression of the Corynebacterium DHDPS protein and the E.
coli AKIII-M4 protein by western blot analysis. The free amino acid
composition
and total amino acid composition of the seeds were determined as described in
previous examples.
Expression of the Corynebacterium DHDPS protein, driven by either the
globulin 1 or glutelin 2 promoter, was observed in the corn seeds (Table 12).
Expression of the E. coli AKIII-M4 protein, driven by the glutelin promoter
was
also observed in the corn seeds. Free lysine levels in the seeds increased
from
about 1.4% of free amino acids in control seeds to 15-27% in seeds of three
different transformants expressing Corynebacterium DHDPS from the globulin 1
promoter. The increased free lysine, and a high level of saccharopine,
indicative
of lysine catabolism, were both localized to the embryo in seeds expressing
Corynebacterium DHDPS from the globulin 1 promoter. No increase in free
lysine was observed in seeds expressing Corynebacterium DHDPS from the
glutelin 2 promoter with or without E. coli AKIII-M4. Lysine catabolism is
expected to be much greater in the endosperm than the embryo and this probably
prevents the accumulation of increased levels of lysine in seeds expressing
Corynebacterium DHDPS plus E. coli AKIII-M4 from the glutelin 2 promoter.
Lysine normally represents about 2.3% of the seed amino acid content. It is
therefore apparent from Table 12 that a 130% increase in lysine as a percent
of
total seed amino acids was found in seeds expressing Corynebacterium DHDPS
from the globulin 1 promoter.
TABLE 12
WESTERN WESTERN % LYS OF % LYS OF
TRANSGENIC CORYNE. E. COLT FREE SEED TOTAL SEED
LINE PROMOTER DHDPS AKIII-M4 AMINO ACIDS AMINO ACIDS
1088.1.2 x elite globulins I + - 15 3.6
1089.4.2 x elite globulin 1 + - 21 5.1
1099.2.1 x self globulin 1 + - 27 5.3
1090.2.1 x elite glutelin 2 + - 1.2 1.7
1092.2.1 x elite glutelin 2 + + 1.1 2.2
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) ADDRESSEE: E. I. DU PONT DE NEMOURS AND COMPANY
(B) STREET: 1007 MARKET STREET
(C) CITY: WILMINGTON
(D) STATE: DELAWARE
(E) COUNTRY: U.S.A.
(F) ZIP: 19898
(A) TELEPHONE: 302-992-5481
(B) TELEFAX: 302-892-7949
(C) TELEX: 835420
(ii) TITLE OF INVENTION: CHIMERIC GENES AND METHODS FOR
INCREASING THE LYSINE CONTENT OF
THE SEEDS OF PLANTS
(iii) NUMBER OF SEQUENCES: 132
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: DISKETTE, 3.50 INCH
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MICROSOFT WINDOWS 95
(D) SOFTWARE: MICROSOFT WORD FOR WINDOWS 95 (7.0)
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/824,627
(B) FILING DATE: MARCH 27, 1997
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: CHRISTENBURY, LYNNE M.
(B) REGISTRATION NUMBER: 30,971
(C) REFERENCE/DOCKET NUMBER: BB-1037-F
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(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1350 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1350
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATG GCT GAA ATT GTT GTC TCC AAA TTT GGC GGT ACC AGC GTA GCT GAT 48
Met Ala Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
TTT GAC GCC ATG AAC CGC AGC GCT GAT ATT GTG CTT TCT GAT GCC AAC 96
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
GTG CGT TTA GTT GTC CTC TCG GCT TCT GCT GGT ATC ACT AAT CTG CTG 144
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
GTC GCT TTA GCT GAA GGA CTG GAA CCT GGC GAG CGA TTC GAA AAA CTC 192
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
GAC GCT ATC CGC AAC ATC CAG TTT GCC ATT CTG GAA CGT CTG CGT TAC 240
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
CCG AAC GTT ATC CGT GAA GAG ATT GAA CGT CTG CTG GAG AAC ATT ACT 288
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
GTT CTG GCA GAA GCG GCG GCG CTG GCA ACG TCT CCG GCG CTG ACA GAT 336
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
GAG CTG GTC AGC CAC GGC GAG CTG ATG TCG ACC CTG CTG TTT GTT GAG 384
Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
ATC CTG CGC GAA CGC GAT GTT CAG GCA CAG TGG TTT GAT GTA CGT AAA 432
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
GTG ATG CGT ACC AAC GAC CGA TTT GGT CGT GCA GAG CCA GAT ATA GCC 480
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
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GCG CTG GCG GAA CTG GCC GCG CTG CAG CTG CTC CCA CGT CTC AAT GAA 528
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
GGC TTA GTG ATC ACC CAG GGA TTT ATC GGT AGC GAA AAT AAA GGT CGT 576
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
ACA ACG ACG CTT GGC CGT GGA GGC AGC GAT TAT ACG GCA GCC TTG CTG 624
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
GCG GAG GCT TTA CAC GCA TCT CGT GTT GAT ATC TGG ACC GAC GTC CCG 672
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
GGC ATC TAC ACC ACC GAT CCA CGC GTA GTT TCC GCA GCA AAA CGC ATT 720
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
GAT GAA ATC GCG TTT GCC GAA GCG GCA GAG ATG GCA ACT TTT GGT GCA 768
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
AAA GTA CTG CAT CCG GCA ACG TTG CTA CCC GCA GTA CGC AGC GAT ATC 816
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
CCG GTC TTT GTC GGC TCC AGC AAA GAC CCA CGC GCA GGT GGT ACG CTG 864
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
GTG TGC AAT AAA ACT GAA AAT CCG CCG CTG TTC CGC GCT CTG GCG CTT 912
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
CGT CGC AAT CAG ACT CTG CTC ACT TTG CAC AGC CTG AAT ATG CTG CAT 960
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
TCT CGC GGT TTC CTC GCG GAA GTT TTC GGC ATC CTC GCG CGG CAT AAT 1008
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
ATT TCG GTA GAC TTA ATC ACC ACG TCA GAA GTG AGC GTG GCA TTA ACC 1056
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
CTT GAT ACC ACC GGT TCA ACC TCC ACT GGC GAT ACG TTG CTG ACG CAA 1104
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
TCT CTG CTG ATG GAG CTT TCC GCA CTG TGT CGG GTG GAG GTG GAA GAA 1152
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
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GGT CTG GCG CTG GTC GCG TTG ATT GGC AAT GAC CTG TCA AAA GCC TGC 1200
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
GCC GTT GGC AAA GAG GTA TTC GGC GTA CTG GAA CCG TTC AAC ATT CGC 1248
Ala Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
ATG ATT TGT TAT GGC GCA TCC AGC CAT AAC CTG TGC TTC CTG GTG CCC 1296
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
GGC GAA GAT GCC GAG CAG GTG GTG CAA AAA CTG CAT AGT AAT TTG TTT 1344
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
GAG TAA 1350
Glu *
450
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GATCCATGGC TGAAATTGTT GTCTCCAAAT TTGGCG 36
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GTACCGCCAA ATTTGGAGAC AACAATTTCA GCCATG 36
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
123
CA 02280196 2000-06-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCCGGGCCAT GGCTACAGGT TTAACAGCTA AGACCGGAGT AGAGCACT 48
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GATATCGAAT TCTCATTATA GAACTCCAGC TTTTTTC 37
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 917 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..911
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CC ATG GCT ACA GGT TTA ACA GCT AAG ACC GGA GTA GAG CAC TTC GGC 47
Met Ala Thr Gly Leu Thr Ala Lys Thr Gly Val Glu His Phe Gly
1 5 10 15
ACC GTT GGA GTA GCA ATG GTT ACT CCA TTC ACG GAA TCC GGA GAC ATC 95
Thr Val Gly Val Ala Met Val Thr Pro Phe Thr Glu Ser Gly Asp Ile
20 25 30
GAT ATC GCT GCT GGC CGC GAA GTC GCG GCT TAT TTG GTT GAT AAG GGC 143
Asp Ile Ala Ala Gly Arg Glu Val Ala Ala Tyr Leu Val Asp Lys Gly
35 40 45
TTG GAT TCT TTG GTT CTC GCG GGC ACC ACT GGT GAA TCC CCA ACG ACA 191
Leu Asp Ser Leu Val Leu Ala Gly Thr Thr Gly Glu Ser Pro Thr Thr
50 55 60
ACC GCC GCT GAA AAA CTA GAA CTG CTC AAG GCC GTT CGT GAG GAA GTT 239
Thr Ala Ala Glu Lys Leu Glu Leu Leu Lys Ala Val Arg Glu Glu Val
65 70 75
GGG GAT CGG GCG AAG CTC ATC GCC GGT GTC GGA ACC AAC AAC ACG CGG 287
Gly Asp Arg Ala Lys Leu Ile Ala Gly Val Gly Thr Asn Asn Thr Arg
80 85 90 95
124
CA 02280196 2000-06-02
ACA TCT GTG GAA CTT GCG GAA GCT GCT GCT TCT GCT GGC GCA GAC GGC 335
Thr Ser Val Glu Leu Ala Glu Ala Ala Ala Ser Ala Gly Ala Asp Gly
100 105 110
CTT TTA GTT GTA ACT CCT TAT TAC TCC AAG CCG AGC CAA GAG GGA TTG 383
Leu Leu Val Val Thr Pro Tyr Tyr Ser Lys Pro Ser Gln Glu Gly Leu
115 120 125
CTG GCG CAC TTC GGT GCA ATT GCT GCA GCA ACA GAG GTT CCA ATT TGT 431
Leu Ala His Phe Gly Ala Ile Ala Ala Ala Thr Glu Val Pro Ile Cys
130 135 140
CTC TAT GAC ATT CCT GGT CGG TCA GGT ATT CCA ATT GAG TCT GAT ACC 479
Leu Tyr Asp Ile Pro Gly Arg Ser Gly Ile Pro Ile Glu Ser Asp Thr
145 150 155
ATG AGA CGC CTG AGT GAA TTA CCT ACG ATT TTG GCG GTC AAG GAC GCC 527
Met Arg Arg Leu Ser Glu Leu Pro Thr Ile Leu Ala Val Lys Asp Ala
160 165 170 175
AAG GGT GAC CTC GTT GCA GCC ACG TCA TTG ATC AAA GAA ACG GGA CTT 575
Lys Gly Asp Leu Val Ala Ala Thr Ser Leu Ile Lys Glu Thr Gly Leu
180 185 190
GCC TGG TAT TCA GGC GAT GAC CCA CTA AAC CTT GTT TGG CTT GCT TTG 623
Ala Trp Tyr Ser Gly Asp Asp Pro Leu Asn Leu Val Trp Leu Ala Leu
195 200 205
GGC GGA TCA GGT TTC ATT TCC GTA ATT GGA CAT GCA GCC CCC ACA GCA 671
Gly Gly Ser Gly Phe Ile Ser Val Ile Gly His Ala Ala Pro Thr Ala
210 215 220
TTA CGT GAG TTG TAC ACA AGC TTC GAG GAA GGC GAC CTC GTC CGT GCG 719
Leu Arg Glu Leu Tyr Thr Ser Phe Glu Glu Gly Asp Leu Val Arg Ala
225 230 235
CGG GAA ATC AAC GCC AAA CTA TCA CCG CTG GTA GCT GCC CAA GGT CGC 767
Arg Glu Ile Asn Ala Lys Leu Ser Pro Leu Val Ala Ala Gln Gly Arg
240 245 250 255
TTG GGT GGA GTC AGC TTG GCA AAA GCT GCT CTG CGT CTG CAG GGC ATC 815
Leu Gly Gly Val Ser Leu Ala Lys Ala Ala Leu Arg Leu Gln Gly Ile
260 265 270
AAC GTA GGA GAT CCT CGA CTT CCA ATT ATG GCT CCA AAT GAG CAG GAA 863
Asn Val Gly Asp Pro Arg Leu Pro Ile Met Ala Pro Asn Glu Gln Glu
275 280 285
CTT GAG GCT CTC CGA GAA GAC ATG AAA AAA GCT GGA GTT CTA TAA TGAGAATTC 917
Leu Glu Ala Leu Arg Glu Asp Met Lys Lys Ala Gly Val Leu
290 295 300
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
125
CA 02280196 2000-06-02
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CTTCCCGTGA CCATGGGCCA TC 22
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CATGGCTGGC TTCCCCACGA GGAAGACCAA CAATGACATT ACCTCCATTG CTAGCAACGG 60
TGGAAGAGTA CAATG 75
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CATGCATTGT ACTCTTCCAC CGTTGCTAGC AATGGAGGTA ATGTCATTGT TGGTCTTCCT 60
CGTGGGGAAG CCAGC 75
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CATGGCTTCC TCAATGATCT CCTCCCCAGC TGTTACCACC GTCAACCGTG CCGGTGCCGG 60
CATGGTTGCT CCATTCACCG GCCTCAAAAG 90
126
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CATGCTTTTG AGGCCGGTGA ATGGAGCAAC CATGCCGGCA CCGGCACGGT TGACGGTGGT 60
AACAGCTGGG GAGGAGATCA TTGAGGAAGC 90
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CCGGTTTGCT GTAATAGGTA CCA 23
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
AGCTTGGTAC CTATTACAGC AAACCGGCAT G 31
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GCTTCCTCAA TGATCTCCTC CCCAGCT 27
127
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CATTGTACTC TTCCACCGTT GCTAGCAA 28
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
7011
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CTGACTCGCT GCGCTCGGTC 20
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
71"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TATTTTCTCC TTACGCATCT GTGC 24
128
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..27
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
78"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TTCATCGATA GGCGACCACA CCCGTCC 27
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..27
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
79"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
AATATCGATG CCACGATGCG TCCGGCG 27
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..55
129
CA 02280196 2000-06-02
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
81"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CATGGAGGAG AAGATGAAGG CGATGGAAGA GAAGATGAAG GCGTGATAGG TACCG 55
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..55
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
80"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
AATTCGGTAC CTATCACGCC TTCATCTTCT CTTCCATCGC CTTCATCTTC TCCTC 55
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= name
/note= "base gene
[(SSP5)2]"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
130
CA 02280196 2000-06-02
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product=
"synthetic
oligonucleotide"
/standard name= "SM
84"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GATGGAGGAG AAGATGAAGG C 21
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
85"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ATCGCCTTCA TCTTCTCCTC C 21
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
82"
131
CA 02280196 2000-06-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GATGGAGGAG AAGCTGAAGG C 21
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
83"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ATCGCCTTCA GCTTCTCCTC C 21
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Met Glu Glu Lys Leu Lys Ala
1 5
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Met Glu Glu Lys Met Lys Ala
1 5
132
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: C15
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..151
(D) OTHER INFORMATION: /function= "synthetic
storage protein"
/product= "protein"
/gene= "ssp"
/standard name=
"5.7.7.7.7.7.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met
1 5 10 15
GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG 94
Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu
20 25 30
AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAA GAG AAG ATG 142
Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met
35 40 45
AAG GCG TGATAGGTAC CG 160
Lys Ala
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu
1 5 10 15
133
CA 02280196 2000-06-02
Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys
20 25 30
Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met Lys
35 40 45
Ala
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: C20
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..151
(D) OTHER INFORMATION: /function= "synthetic
storage protein"
/product= "protein"
/gene= "ssp"
/standard name=
"5.7.7.7.7.7.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met
1 5 10 15
GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG 94
Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu
20 25 30
AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAA GAG AAG ATG 142
Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met
35 40 45
AAG GCG TGATAGGTAC CG 160
Lys Ala
134
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu
1 5 10 15
Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys
20 25 30
Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met Lys
35 40 45
Ala
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 139 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: C30
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..130
(D) OTHER INFORMATION: /function= "synthetic
storage protein"
/product= "protein"
/gene= "ssp"
/standard name=
"5.7.7.7.7.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met
1 5 10 15
GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG 94
Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu
20 25 30
135
CA 02280196 2000-06-02
AAG CTG AAG GCG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC CG 139
Lys Leu Lys Ala Met Glu Glu Lys Met Lys Ala
35 40
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu
1 5 10 15
Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys
20 25 30
Leu Lys Ala Met Glu Glu Lys Met Lys Ala
35 40
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: D16
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..88
(D) OTHER INFORMATION: /function= "synthetic
storage protein"
/product= "protein"
/gene= "ssp"
/standard name=
"5.5.5.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met
1 5 10 15
136
CA 02280196 2000-06-02
GAG GAG AAG ATG AAG GCG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC 95
Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
20 25
CG 97
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
20 25
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: D20
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..109
(D) OTHER INFORMATION: /function= "synthetic
storage protein"
/product= "protein"
/gene= "ssp"
/standard name=
"5.5.5.5.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met
1 5 10 15
137
CA 02280196 2000-06-02
GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG GCG ATG GAA GAG 94
Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu Glu
20 25 30
AAG ATG AAG GCG TGATAGGTAC CG 118
Lys Met Lys Ala
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys
20 25 30
Met Lys Ala
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: D33
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..88
(D) OTHER INFORMATION: /function= "synthetic
storage protein"
/product= "protein"
/gene= "ssp"
/standard name=
"5.5.5.5"
138
CA 02280196 2000-06-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met
1 5 10 15
GAG GAG AAG ATG AAG GCG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC 95
Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
20 25
CG 97
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
20 25
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
86"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
GATGGAGGAG AAGCTGAAGA A 21
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
139
CA 02280196 2000-06-02
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard-name= "SM
8711
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
ATCTTCTTCA GCTTCTCCTC C 21
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard-name= "SM
88"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GATGGAGGAG AAGCTGAAGT G 21
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard-name= "SM
89"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
ATCCACTTCA GCTTCTCCTC C 21
140
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
90"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
GATGGAGGAG AAGATGAAGA A 21
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
9111
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
ATCTTCTTCA TCTTCTCCTC C 21
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
141
CA 02280196 2000-06-02
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
92"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
GATGGAGGAG AAGATGAAGT G 21
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
93"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
ATCCACTTCA TCTTCTCCTC C 21
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Met Glu Glu Lys Leu Lys Lys
1 5
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
142
CA 02280196 2000-06-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Met Glu Glu Lys Leu Lys Trp
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Met Glu Glu Lys Met Lys Lys
5
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Met Glu Glu Lys Met Lys Trp
5
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: 82-4
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..151
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
143
CA 02280196 2000-06-02
/gene= "ssp"
/standard name=
117.7.7.7.7.7.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
C ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG 46
Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met
1 5 10 15
GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG 94
Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu
20 25 30
AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAA GAG AAG ATG 142
Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met
35 40 45
AAG GCG TGATAGGTAC CG 160
Lys Ala
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu
1 5 10 15
Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys
20 25 30
Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met Lys
35 40 45
Ala
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
144
CA 02280196 2000-06-02
(vii) IMMEDIATE SOURCE:
(B) CLONE: 84-H3
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..88
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"5.5.5.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met
1 5 10 15
GAG GAG AAG ATG AAG GCG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC 95
Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
20 25
CG 97
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
20 25
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: 86-H23
145
CA 02280196 2000-06-02
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..88
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"5.8.8.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG CTG AAG AAG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Lys Met
1 5 10 15
GAG GAG AAG CTG AAG AAG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC 95
Glu Glu Lys Leu Lys Lys Met Glu Glu Lys Met Lys Ala
20 25
CG 97
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Lys Met Glu
1 5 10 15
Glu Lys Leu Lys Lys Met Glu Glu Lys Met Lys Ala
20 25
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: 88-2
146
CA 02280196 2000-06-02
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..103
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"5.9.9.9.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
C ATG GAG GAG AAG ATG AAG GCG AAG AAG CTG AAG TGG ATG GAG GAG 46
Met Glu Glu Lys Met Lys Ala Lys Lys Leu Lys Trp Met Glu Glu
1 5 10 15
AAG CTG AAG TGG ATG GAG GAG AAG CTG AAG TGG ATG GAA GAG AAG ATG 94
Lys Leu Lys Trp Met Glu Glu Lys Leu Lys Trp Met Glu Glu Lys Met
20 25 30
AAG GCG TGATAGGTAC CG 112
Lys Ala
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Met Glu Glu Lys Met Lys Ala Lys Lys Leu Lys Trp Met Glu Glu Lys
1 5 10 15
Leu Lys Trp Met Glu Glu Lys Leu Lys Trp Met Glu Glu Lys Met Lys
20 25 30
Ala
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: 90-H8
147
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(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..109
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"5.10.10.10.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG AAG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Lys Met
1 5 10 15
GAG GAG AAG ATG AAG AAG ATG GAG GAG AAG ATG AAG AAG ATG GAA GAG 94
Glu Glu Lys Met Lys Lys Met Glu Glu Lys Met Lys Lys Met Glu Glu
20 25 30
AAG ATG AAG GCG TGATAGGTAC CG 118
Lys Met Lys Ala
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Lys Met Glu
1 5 10 15
Glu Lys Met Lys Lys Met Glu Glu Lys Met Lys Lys Met Glu Glu Lys
20 25 30
Met Lys Ala
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
148
CA 02280196 2000-06-02
(vii) IMMEDIATE SOURCE:
(B) CLONE: 92-2
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..88
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"5.11.11.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG TGG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Trp Met
1 5 10 15
GAG GAG AAG ATG AAG TGG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC 95
Glu Glu Lys Met Lys Trp Met Glu Glu Lys Met Lys Ala
20 25
CG 97
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Trp Met Glu
1 5 10 15
Glu Lys Met Lys Trp Met Glu Glu Lys Met Lys Ala
20 25
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..84
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
149
CA 02280196 2000-06-02
/standard name= "SM
96"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
GATGGAGGAA AAGATGAAGG CGATGGAGGA GAAAATGAAA GCTATGGAGG AAAAGATGAA 60
AGCGATGGAG GAGAAAATGA AGGC 84
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..84
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
9711
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
ATCGCCTTCA TTTTCTCCTC CATCGCTTTC ATCTTTTCCT CCATAGCTTT CATTTTCTCC 60
TCCATCGCCT TCATCTTTTC CTCC 84
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..28
(D) OTHER INFORMATION: /label= name
/note= "(SSP 5)4"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala
20 25
150
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..84
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
98"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
GATGGAGGAA AAGCTGAAAG CGATGGAGGA GAAACTCAAG GCTATGGAAG AAAAGCTTAA 60
AGCGATGGAG GAGAAACTGA AGGC 84
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..84
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
99"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
ATCGCCTTCA GTTTCTCCTC CTACGCTTTA AGCTTTTCTT CCATAGCCTT GAGTTTCTCC 60
TCCATCGCTT TCAGCTTTTC CTCC 84
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
151
CA 02280196 2000-06-02
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..28
(D) OTHER INFORMATION: /label= name
/note= "(SSP 7)4"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu
1 5 10 15
Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala
20 25
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..84
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
100"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
GATGGAGGAA AAGCTTAAGA AGATGGAAGA AAAGCTGAAA TGGATGGAGG AGAAACTCAA 60
AAAGATGGAG GAAAAGCTTA AATG 84
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..84
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
101"
152
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
ATCCATTTAA GCTTTTCCTC CTACTTTTTG AGTTTCTCCT CCATCCATTT CAGCTTTTCT 60
TCCATCTTCT TAAGCTTTTC CTCC 84
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
Met Glu Glu Lys Leu Lys Lys Met Glu Glu Lys Leu Lys Trp Met Glu
1 5 10 15
Glu Lys Leu Lys Lys Met Glu Glu Lys Leu Lys Trp
20 25
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(vii) IMMEDIATE SOURCE:
(B) CLONE: 2-9
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..235
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"7.7.7.7.7.7.8.9.8.9.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
C ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG 46
Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met
1 5 10 15
153
CA 02280196 2000-06-02
GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG 94
Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu
20 25 30
AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAA AAG CTT 142
Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu
35 40 45
AAG AAG ATG GAA GAA AAG CTG AAA TGG ATG GAG GAG AAA CTC AAA AAG 190
Lys Lys Met Glu Glu Lys Leu Lys Trp Met Glu Glu Lys Leu Lys Lys
50 55 60
ATG GAG GAA AAG CTT AAA TGG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC 242
Met Glu Glu Lys Leu Lys Trp Met Glu Glu Lys Met Lys Ala
65 70 75
C 243
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu
1 5 10 15
Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys
20 25 30
Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys
35 40 45
Lys Met Glu Glu Lys Leu Lys Trp Met Glu Glu Lys Leu Lys Lys Met
50 55 60
Glu Glu Lys Leu Lys Trp Met Glu Glu Lys Met Lys Ala
65 70 75
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 175 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
154
CA 02280196 2000-06-02
(vii) IMMEDIATE SOURCE:
(B) CLONE: 5-1
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..172
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"5.5.5.7.7.7.7.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
C ATG GAG GAG AAG ATG AAG GCG ATG GAG GAG AAG ATG AAG GCG ATG 46
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met
1 5 10 15
GAG GAG AAG ATG AAG GCG ATG GAG GAA AAG CTG AAA GCG ATG GAG GAG 94
Glu Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu
20 25 30
AAA CTC AAG GCT ATG GAA GAA AAG CTT AAA GCG ATG GAG GAG AAA CTG 142
Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu
35 40 45
AAG GCC ATG GAA GAG AAG ATG AAG GCG TGATAG 175
Lys Ala Met Glu Glu Lys Met Lys Ala
50 55
(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
Met Glu Glu Lys Met Lys Ala Met Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Glu Lys Met Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys
20 25 30
Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys
35 40 45
Ala Met Glu Glu Lys Met Lys Ala
50 55
155
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 187 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: E. coli
(G) CELL TYPE: DH5 alpha
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..173
(D) OTHER INFORMATION: /function= "synthetic
storage protein
/product= "protein"
/gene= "ssp"
/standard name=
"SSP-3-5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
CC ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG 47
Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met
1 5 10 15
GAG GAG AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAG 95
Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu
20 25 30
AAG CTG AAG GCG ATG GAG GAG AAG CTG AAG GCG ATG GAG GAA AAG ATG 143
Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met
35 40 45
AAG GCG ATG GAA GAG AAG ATG AAG GCG TGATAGGTAC CGAATTC 187
Lys Ala Met Glu Glu Lys Met Lys Ala
50 55
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu
1 5 10 15
Glu Lys Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys
20 25 30
156
CA 02280196 2000-06-02
Leu Lys Ala Met Glu Glu Lys Leu Lys Ala Met Glu Glu Lys Met Lys
35 40 45
Ala Met Glu Glu Lys Met Lys Ala
50 55
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
107"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
CATGGAGGAG AAGATGAAAA AGCTCGAAGA GAAGATGAAG GTCATGAAGT GATAGGTACC 60
G 61
(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..61
(D) OTHER INFORMATION: /product= "synthetic
ligonucleotide"
/standard-name= "SM
106"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
AATTCGGTAC CTATCACTTC ATGACCTTCA TCTTCTCTTC GAGCTTTTTC ATCTTCTCCT 60
C 61
157
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= name
/note= "pSK34 base
gene"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
Met Glu Glu Lys Met Lys Lys Leu Glu Glu Lys Met Lys Val Met Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..63
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard-name= "SM
110"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
GCTGGAAGAA AAGATGAAGG CTATGGAGGA CAAGATGAAA TGGCTTGAGG AAAAGATGAA 60
GAA 63
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
158
CA 02280196 2000-06-02
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..63
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
111"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
AGCTTCTTCA TCTTTTCCTC AAGCCATTTC ATCTTGTCCT CCATAGCCTT CATCTTTTCT 60
TCC 63
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
Met Glu Glu Lys Met Lys Lys Leu Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Asp Lys Met Lys Trp Leu Glu Glu Lys Met Lys Lys Leu Glu Glu Lys
20 25 30
Met Lys Val Met Lys
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
Met Glu Glu Lys Met Lys Lys Leu Glu Glu Lys Met Lys Ala Met Glu
1 5 10 15
Asp Lys Met Lys Trp Leu Glu Glu Lys Met Lys Lys Leu Glu Glu Lys
20 25 30
Met Lys Val Met Lys
159
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..62
(D) OTHER INFORMATION: /product= "synthetic
oligonucletide"
/standard name= "SM
112"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
GCTCGAAGAA AGATGAAGGC AATGGAAGAC AAAATGAAGT GGCTTGAGGA GAAAATGAAG 60
AA 62
(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..62
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
113"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
AGCTTCTTCA TTTTCTCCTC AAGCCACTTC ATTTTGTCTT CCATTGCCTT CATCTTTCTT 60
CG 62
(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
160
CA 02280196 2000-06-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
Met Glu Glu Lys Met Lys Lys Leu Lys Glu Glu Met Ala Lys Met Lys
1 5 10 15
Asp Glu Met Trp Lys Leu Lys Glu Glu Met Lys Lys Leu Glu Glu Lys
20 25 30
Met Lys Val Met Lys
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..63
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
114"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
GCTCAAGGAG GAAATGGCTA AGATGAAAGA CGAAATCTGG AAACTGAAAG AGGAAATGAA 60
GAA 63
(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..63
(D) OTHER INFORMATION: /product= "synthetic
oligonucleotide"
/standard name= "SM
115"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
AGCTTCTTCA TTTCCTCTTT CAGTTTCCAC ATTTCGTCTT TCATCTTAGC CATTTCCTCC 60
TTG 63
161
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
Met Glu Glu Lys Met Lys Lys Leu Lys Glu Glu Met Ala Lys Met Lys
1 5 10 15
Asp Glu Met Trp Lys Leu Lys Glu Glu Met Lys Lys Leu Glu Glu Lys
20 25 30
Met Lys Val Met Glu Glu Lys Met Lys Lys Leu Glu Glu Lys Met Lys
35 40 45
Ala Met Glu Asp Lys Met Lys Trp Leu Glu Glu Lys Met Lys Lys Leu
50 55 60
Glu Glu Lys Met Lys Val Met Glu Glu Lys Met Lys Lys Leu Glu Glu
65 70 75 80
Lys Met Lys Ala Met Glu Asp Lys Met Lys Trp Leu Glu Glu Lys Met
85 90 95
Lys Lys Leu Glu Glu Lys Met Lys Val Met Lys
100 105
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 839 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
GGATCCCCCG GGCTGCAGGA ATTCTACGTA CCATATAGTA AGACTTTGTA TATAAGACGT 60
CACCTCTTAC GTGCATGGTT ATATGTGACA TGTGCAGTGA CGTTGTACCA TATAGTAAGA 120
CTTTGTATAT AAGACGTCAC CTCTTACGTG CATGGTTATA TGTGACATGT GCAGTGACGT 180
TAACCGCACC CTCCTTCCCG TCGTTTCCCA TCTCTTCCTC CTTTAGAGCT ACCACTATAT 240
AAATCAGGGC TCATTTTCTC GCTCCTCACA GGCTCATCAG CACCCCGGCA GTGCCACCCC 300
GACTCCCTGC ACCTGCCATG GGTACGCTAG CCCGGGAGAT CTGACAAAGC AGCATTAGTC 360
CGTTGATCGG TGGAAGACCA CTCGTCAGTG TTGAGTTGAA TGTTTGATCA ATAAAATACG 420
162
CA 02280196 2000-06-02
GCAATGCTGT AAGGGTTGTT TTTTATGCCA TTGATAATAC ACTGTACTGT TCAGTTGTTG 480
AACTCTATTT CTTAGCCATG CCAGTGCTTT TCTTATTTTG AATAACATTA CAGCAAAAAG 540
TTGAAAGACA AAAAAANNNN NCCCCGAACA GAGTGCTTTG GGTCCCAAGC TTCTTTAGAC 600
TGTGTTCGGC GTTCCCCCTA AATTTCTCCC CTATATCTCA CTCACTTGTC ACATCAGCGT 660
TCTCTTTCCC CTATATCTCC ACGCTCTACA GCAGTTCCAC CTATATCAAA CCTCTATACC 720
CCACCACAAC AATATTATAT ACTTTCATCT TCACCTAACT CATGTACCTT CCAATTTTTT 780
TCTACTAATA ATTATTTACG TGCACAGAAA CTTAGGCAAG GGAGAGAGAG AGCGGTACC 839
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
CTAGAAGCCT CGGCAACGTC AGCAACGGCG GAAGAATCCG GTG 43
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
CATGCACCGG ATTCTTCCGC CGTTGCTGAC GTTGCCGAGG CTT 43
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
GATCCCATGG CGCCCCTTAA GTCCACCGCC AGCCTCCCCG TCGCCCGCCG CTCCT 55
163
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
CTAGAGGAGC GGCGGGCGAC GGGGAGGCTG GCGGTGGACT TAAGGGGCGC CATGG 55
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
CATGGCGCCC ACCGTGATGA TGGCCTCGTC GGCCACCGCC GTCGCTCCGT TCCAGGGGC 59
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
TTAAGCCCCT GGAACGGAGC GACGGCGGTG GCCGACGAGG CCATCATCAC GGTGGGCGC 59
(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
GCGCCCACCG TGATGA 16
164
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
CACCGGATTC TTCCGC 16
(2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
GTAAGATTGG TAAAGTCCAG CAAGAAAATG AGATAAAAGA GAAGCCTGAA ATGACGAAAA 60
AATCAGGTGT TTTGATTCTT GGTGCTGGAC GTGTGTNTCG CCCAGCTGCT GATTTCCTAG 120
CTTCAGTTAG AACCATTTCG TCACAGCAAT GGTACAAAAC ATATTTCGGA GCAGACTCTG 180
AAGAGAAAAC AGATGTTCAT GTGATTGTCG CGTCTCTGTA TCTTAAGGAT GCCAAAGAGA 240
CGGTTGAAGG TATTTCAGAT GTAGAAGCAG TTCGGCTAGA TGTATCTGAT AGTGAAAGTC 300
TCCTTAAGTA TGTTTCTCAG GTTGATGTTG TCCTAAGTTT ATTACCTGCA AGTTGTCATG 360
CTTGTTGTAG CA 372
(2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 323 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
GGAAGCACAC TGCGACTCTT TTGGAATTCG GGGACATCAA GAATGGACAA ACAACAACCG 60
CTATGGCCAA GACTGTTGGG ATCCCTGCAG CCATTGGAGC TCTGCTGTTA ATTGAAGACA 120
AGATCAAGAC AAGAGGAGTC TTAAGGCCTC TCGAAGCAGA GGTGTATTTG CCAGCTTTGG 180
165
CA 02280196 2000-06-02
ATATATTGCA AGCATATGGT ATAAAGCTGA TGGAGAAGGC AGAATGATCA AAGAACTCTG 240
TATATTGTTT CTNTCTATAA CTTGGAGTTG GAGACAAAGC TGAAGGAGNC AGNGCCATTA 300
GACCAGCAAA AAAAGGAGGA GGA 323
(2) INFORMATION FOR SEQ ID NO:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
Lys Ile Gly Lys Val Gln Gln Glu Asn Glu Ile Lys Glu Lys Pro Glu
1 5 10 15
Met Thr Lys Lys Ser Gly Val Leu Ile Leu Gly Ala Gly Arg Val Xaa
20 25 30
Arg Pro Ala Ala Asp Phe Leu Ala Ser Val Arg Thr Ile Ser Ser Gin
35 40 45
Gln Trp Tyr Lys Thr Tyr Phe Gly Ala Asp Ser Glu Glu Lys Thr Asp
50 55 60
Val His Val Ile Val Ala Ser Leu Tyr Leu Lys Asp Ala Lys Glu Thr
65 70 75 80
Val Glu Gly Ile Ser Asp Val Glu Ala Val Arg Leu Asp Val Ser Asp
85 90 95
Ser Glu Ser Leu Leu Lys Tyr Val Ser Gln Val Asp Val Val Leu Ser
100 105 110
Leu Leu Pro Ala Ser Cys His Ala Cys Cys Ser
115 120
(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 74 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
Lys His Thr Ala Thr Leu Leu Glu Phe Gly Asp Ile Lys Asn Gly Gln
1 5 10 15
166
CA 02280196 2000-06-02
Thr Thr Thr Ala Met Ala Lys Thr Val Gly Ile Pro Ala Ala Ile Gly
20 25 30
Ala Leu Leu Leu Ile Glu Asp Lys Ile Lys Thr Arg Gly Val Leu Arg
35 40 45
Pro Leu Glu Ala Glu Val Tyr Leu Pro Ala Leu Asp Ile Leu Gln Ala
50 55 60
Tyr Gly Ile Lys Leu Met Glu Lys Ala Glu
65 70
(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:
ATTCCCCATG GTTTCGCCGA CGAAT 25
(2) INFORMATION FOR SEQ ID NO:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:
CTCTCGGTAC CTAGTACCTA CTGATCAAC 29
(2) INFORMATION FOR SEQ ID NO:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:
AGAGAAGCCT GAAATGACGA AAAA 24
167
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:
GTCTTGGCCA TAGCGGTTGT TGTT 24
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8160 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
TCTAGATGCA CATTCAACTC GAGGTTGTTG CATGATGTTT CATTTACCAA AAAAATCATA 60
GTCAAATTAT GTAAGCAAAT GATATTACAG AAAAGTTTTA CTAGAGAGTT TCAGATTTAC 120
ACATGCACAA CGTTAAAAAA AATAGCAGAA AAAAGAAAGA AGAAAAGTTC TTTATTTGTG 180
AGAAAAATGT ATGAAAAAAA AAGAGATGGG TGTAAAAAGC AAAAGGATAG GACCACTGTT 240
ACTTTGTAGC CTCGTTGAGG AATCTCTTCT CGCATCTCGA CTTTTGTGCC ATTGCAAAGT 300
CAATGCCCAG AACTTGTTCC CAGGCCATCT CCAATTAACT ACGTCTATTT AATTAAACTT 360
TTAAAAGAAA ACCTAATAAA TTAAACAAAA GAAAAGCCGT CAACGAAATC TAAGCTTGCA 420
GCGATATCGA TGAACTGATA CCAAAACAAT GTTCAAGTTT CACTTTCAAA TTGTTTTTTC 480
TTGAAATAGT TTATTGGGTA AGGCCCATAG ATATTTCATA AGAAGAACAC TTGTCGAGGT 540
TGAATCGTAT GTCTGCCCAC CGCGGCCCAT GCATCCTCTG TTGGTAGCAT AATCGTTTTA 600
GGCCATACTA TTGTTCGTAC ACACTGATTT TGAAGTCACC TTTGTGCACT CCTTAATTCC 660
TAAATTGAAG AAGCTTGTTC TCATTCTTCT TTGGGTTACA AATGCCAAGG CAAAAGGAAC 720
TTGGGCCAAA TTAAGACAAC AACTCAAGCC CACTCTCTGC AAATAATACT TGGGAATTTT 780
TACTAAAACG GTGCGTTTCA TCCAAGAATC TATTAATATC CCTAACTTGA AATCATCATA 840
TACGTAACCC AACATATTAA AGAGTTAATA ATGTTAAAAA AAGTCTCAGA AGAGAGAGAC 900
GTAGAGAACA CGGAAAGTGG TAACTGGTAA GCGTCGTCAT CGAGGATATA GTAGCTACGT 960
168
CA 02280196 2000-06-02
GAGCAAACGT CTTCACTCAT CTCTGTCTAT TTCTCTTCGA ATACACGTAA TACATTTTCG 1020
ATTGGATTGA TCCTCCCTCG GTCCTATCCA AGTATCCATC CACGTAAACA AGAGCTTGTT 1080
CCTTTCTTGT TTTTTCTTTC TTTAAATAGT AAAAATACTT ATTTCATTTG TTTCGTTTGA 1140
TTTCATTATT ATTGTCTATG GCATTATATA CTATATATAT TATTTCTACA ACATTGGCTG 1200
GCTCACGTTG TTCTCGTGTA TACAACAAAC TTAATTAATG TCTCTCTATT GCATTAGATA 1260
GTTTCGGAGC ATATCCATTA TGTGAAAGCC ACATTAAGTT ATAACTAAAA GTAGTTTTCG 1320
AAAGAGCTTA ATTAAGTTAT GTTCTGTTTC AAATAAAAAT GAACACGAGG GATTTTTTTT 1380
TTTTTTGACA GATCATTATT AACAAAAATG ATTACCTGAA GAAAGGGGAA AATAATTATA 1440
GCTGATTACA GATCATTATT AACAAAAAGA ATTCTTGTCA CATCATTCAT TATAACAAGA 1500
AATATTATAT TATATTAATT TAATCTTTCG CTAACACGCC CACAATATAT TAATCATATA 1560
CGTAATTTAG CTTATAAAAA GGACGGAAAG AGATTATTAC TGCGCCTAAA AAACTCACTA 1620
ATTCCAAAGA AAAAAAAAAG CTTGTATTTT TTCTTGACAA ACCAGCTCAC AGGCATTGCA 1680
TGATCAAACT CATCAGGTAC GTTTTGATTC CTTCTTCCAT AATTTTCCCA TCTTGAGGAA 1740
TGCAAATTTG GAGAGCGCTT TAGCTAAATC ACTGCCTTCA TTTTTTCACT TTGGATTTAA 1800
TAATTTGCAT TCCTCTCTTC CTCTCTGCTC TGTTCTGTTC TGTTCTGTTC TGATTTGAGT 1860
TTTCAATTAA TCGCTCGAGC AAAAGCTATT TCTCAACTCG TTAAATTTCT GTTCCCAGTT 1920
TGTTCGATTT TCAACAGTTT CACATTAAAG TTTGGGTTTT TGATGTTTGG TTGATGAAAC 1980
TCGAAATATG AAATGTTTGT GAATCTATTC CAGGGTGTTT AAAATAAGGG TTTGTTGTTC 2040
ATCTGCAGAG ATTATATGTT TTTACATGAA AGATGAATTC AAATGGCCAT GAGGAGGAGA 2100
AGAAGTTGGG GAATGGAGTT GTGGGGATTC TAGCTGAAAC AGTTAACAAA TGGGAGAGAC 2160
GAACACCATT GACGCCATCG CATTGCGCTC GCCTTTTACA CGGTGGGAAA GACAGAACCG 2220
GCATTTCCCG CATTGTGGTT CAGCCATCTG CTAAGCGTAT CCATCATGAT GCCTTGTATG 2280
AAGATGTTGG GTGTGAAATT TCTGATGATT TGTCTGATTG TGGGCTTATA CTTGGAATCA 2340
AACAACCTGA GGTGTGGGAA TTTGCATTAA AAAGAGTTCC TTTTTTTCTT CTATATATAT 2400
ATCAGTTTAT GAGATTTGAT TCTGTTTGCA GCTAGAAATG ATTCTTCCAG AGAGAGCATA 2460
CGCTTTCTTT TCACATACTC ATAAGGCACA GAAAGAGAAC ATGCCTTTGT TGGATAAAGT 2520
ATTACACTTT TCATTTATCC TTTTAGTCCT ATCTAAGATA CTGAGGAATG TTGACAAAAG 2580
GGGTATCCAA TTGCAGATTC TTTCTGAGAG AGTGACTTTG TGTGATTATG AGCTCATTGT 2640
TGGGGATCAT GGGAAACGAT TATTGGCGTT TGGTAAATAT GCAGGCAGAG CTGGTCTTGT 2700
169
CA 02280196 2000-06-02
TGACTTCTTA CACGGACTTG GACAGCGTAA GCTCATGTTA TAATTCTGAT GATCAGGACA 2760
TGTTTCTGTG CAGAACAAGA TGAGATGTAA TTTTCCATGT TTGATGCAGG ATATCTAAGT 2820
CTAGGATACT CAACACCTTT CCTCTCGCTC GGTGCATCGT ATATGTATTC CTCATTGGCT 2880
GCTGCAAAAG CCGCTGTAAT TTCTGTTGGT GAAGAAATTG CAAGCCAGGG ACTGCCATTA 2940
GGAATCTGCC CTCTTGTATT TGTCTTCACC GGAACAGGAA ATGGTATCTT CTTTAGTTCT 3000
ACTGCGAGTT CTTTGAATCC TTCTGCATAT GTTTCATCTC ATTAAAAAAT TTCTCATCCG 3060
CAGTTTCTCT GGGGGCGCAA GAAATTTTCA AGCTTCTTCC TCACACTTTT GTTGAACCAA 3120
GCAAACTTCC TGAACTATTT GTAAAAGTAA GTCACGCTTT GCTTTTTATT TGGTTTCAGA 3180
GTTTTGAAGA TTCTGAAATG TATATTTCTC ACAGGACAAA GGAATTAGTC AAAATGGGAT 3240
TTCAACAAAG CGAGTCTATC AAGTATATGG TTGTATTATT ACCAGCCAAG ACATGGTTGA 3300
ACACAAAGAT CCATCAAAGT CATTCGACAA AGTAACACTT ACCTTCTTAG CTCCTTGGCT 3360
GTGACTTTTG TTCCACTACG CTAAAGTAGA ATACCTATTA ATTCTTCAAG CTTATGATGT 3420
TTAGGCCGAC TATTATGCAC ACCCGGAACA TTACAATCCA GTTTTCCACG AAAAGATATC 3480
GCCATATACG TCTGTTCTTG GTAGATCCTG ATCACTGTTT TACCTTTAAA GCTCAAGAGT 3540
TTACATATAA GCAAATCCTC TGTCCACTCC GTGACTGTGA CCATCTCATT TTGGTTAGTT 3600
CCAGTGTGTA ACCCCTATGA CTTTCTGTGC AGTAAACTGT ATGTACTGGG AGAAGAGGTT 3660
TCCCTGTCTT CTGAGCACAA AACAGCTTCA AGATTTAACA AAAAAAGGAC TCCCACTAGT 3720
AGGCATATGT GATATAACTT GTGACATCGG TGGCTCCATT GAATTTGTTA ACCGAGCTAC 3780
TTTAATCGAT TCCCCTTTCT TCAGGTAATA TATACTTAGG AAGAGCTTTC TTTTGAGTCA 3840
TCTACGTTTA CTATGATGAA ACTCGTCGAG CTAAACACTA TCTCTAGGTT TAATCCCTCG 3900
AACAATTCAT ACTACGATGA CATGGATGGG GATGGCGTAC TATGCATGGC TGTTGACATT 3960
TTACCCACAG AATTTGCAAA AGAGGTATGT ATGAAGGTTA CAGTTATAGT ACTTAAGATT 4020
AAATCTAAAG TTAAAAACCT TGTATTGAGT GGGAGTTCTT GTGTCCTGAA AAAGGCATCC 4080
CAGCATTTTG GAGATATTCT TTCCGGATTT GTCGGTAGTT TGGCTTCAAT GACTGAAATT 4140
TCAGATCTAC CAGCACATCT GAAGAGGGCT TGCATAAGCT ATAGGGGAGA ATTGACATCT 4200
TTGTATGAGT ATATTCCACG TATGAGGAAG TCAAATCCAG AGTATGTTCT GCTTCGAGCG 4260
TTACTTCATC TGAAATATTT AGGCCTCTTC TCTAAACTAT GTTTTCATCT TTACCCACTT 4320
TAACTGCAGA GAGGCACAAG ATAATATTAT CGCCAACGGG GTTTCCAGCC AGAGAACATT 4380
CAACATATTG GTTAGTTTTG ATGAAGAAAG TATATATAAC TAGTTTCCGA ATCATATGAT 4440
170
CA 02280196 2000-06-02
TTAAGCTAAT GAATTAAGAA AATATATAGT TCAAGACTTA TGATTCATAT CTCTATCAAC 4500
TTTTTGACCA AAGATTGATA CTTTTTCGAC ATCTGTCACA GCATTTTGTG ATGATTTTGA 4560
TTGAGACAAA TCATTTGTAG GTATCTCTGA GCGGACACCT ATTTGATAAG TTTCTGATAA 4620
ACGAAGCTCT TGATATGATC GAAGCGGCTG GTGGCTCATT TCATTTGGCT AAATGTGAAC 4680
TGGGGCAGAG CGCTGATGCT GAATCGTACT CAGAACTTGA AGTAAGTTTC TTTCTGGATA 4740
AAACCTAATC ATTCACATGG AACAACTGTC AAGAGTTTTT AATGTCACGT TTAGGTTCAA 4800
TGTCCTTTTC ACTAAGTCTC GTAAGTTTTT AAAACAAGTA AACAAACTAC AAGCCAAAAA 4860
CATTCTGGCC CCACATTAAC CTATTCCCAC TTGTTAAAGA ACCCATCTTG CATTATCTTG 4920
GTAGGTTGGT GCGGATGATA AGAGAGTATT GGATCAAATC ATTGATTCAT TAACTCGGTT 4980
AGCTAATCCA AATGAAGATT ATATATCCCC ACATAGAGAA GCAAATAAGA TCTCACTGAA 5040
GATTGGTAAA GTCCAGCAAG AAAATGAGAT AAAAGAGAAG CCTGAAATGA CGAAAAAATC 5100
AGGCGTTTTG ATTCTTGGTG CTGGACGTGT GTGTCGCCCA GCTGCTGATT TCCTAGCTTC 5160
AGTTAGAACC ATTTCGTCAC AGCAATGGTA CAAAACATAT TTCGGAGCAG ACTCTGAAGA 5220
GAAAACAGAT GTTCATGTGA TTGTCGCGTC TCTGTATCTT AAGGATGCCA AAGAGGTAGG 5280
AGAAGCCTTT GGGCTTCATC TGAGTAATTC AGTGTATACG ATGAACTATC AATCTTTTAA 5340
AGTTTTACTG ATGATCAAAT TTTCCGCAGA CGGTTGAAGG TATTTCAGAT GTAGAAGCAG 5400
TTCGGCTAGA TGTATCTGAT AGTGAAAGTC TCCTTAAGTA TGTTTCTCAG GTATTTTCCT 5460
AACTTCTCTG TTCTTAGATC ACCTTTACTT CAAACTCCAC TGTTCAAATC CATGATCTTA 5520
TATTTTTTTT TCATTGCACG CAGGTTGATG TTGTCCTAAG TTTATTACCT GCAAGTTGTC 5580
ATGCTGTTGT AGCAAAGACA TGCATTGAGG TAAATTCCTA ACGTTTAATG CGTTTTCCGA 5640
GTGAAGTTAT GAAATTTGCA AATGTTATTC GACATAGAGG TTAAACTTCC TCTGCATAAC 5700
ACATTCTTTC AGTAGTTTCC GGTTCCTAAA TGTCTCTGTT TCTTCTTTCT GATTCACTCA 5760
GCTGAAGAAG CATCTCGTCA CTGCTAGCTA TGTTGATGAT GAAACGTCCA TGTTACATGA 5820
GAAGGCTAAG AGTGCTGGGA TAACGATTCT AGGCGAAATG GGACTGGACC CTGGAATCGG 5880
TATGATATCT CACAACATAG TATCTCTTAA GATCATTTGT TCACTTGATT TAACTTAAGT 5940
GCATTTATCT TCAAAATATT TCCCGGATAA CTGAGAAGGT GATCCTACAA TGAATCTTTC 6000
AGATCACATG ATGGCGATGA AAATGATCAA CGATGCTCAT ATCAAAAAAG GGAAAGTGAA 6060
GTCTTTTACC TCTTATTGTG GAGGGCTTCC CTCTCCTGCT GCAGCAAATA ATCCATTAGC 6120
ATATAAATTT AGGTACGGTA GTCCTTTACG CCATTAACAT ATTTTGTTTT GTTTAACTCA 6180
171
CA 02280196 2000-06-02
TTTAGACATC CTTTCAGAAT TTCGCTTACT CAATTACATC TCGGTATTTT CAGCTGGAAC 6240
CCTGCTGGAG CAATTCGAGC TGGTCAAAAC CCCGCCAAAT ACAAAAGCAA CGGCGACATA 6300
ATACATGTTG ATGGTATGAA AAACAAAATA TGTCTACATG CAGGAGAGGT TGGAGTAGTT 6360
TAGCTTCACT ACACATCATT TTTGTTTAAC CGAGCAATGT AAATCGCAGG GAAGAATCTC 6420
TATGATTCCG CGGCAAGATT CCGAGTACCT AATCTTCCAG CTTTTGCATT GGAGTGTCTT 6480
CCAAATCGTG ACTCCTTGGT TTACGGGGAA CATTATGGCA TCGAGAGCGA AGCAACAACG 6540
ATATTTCGTG GAACACTCAG ATATGAAGGC ATGAATTCCA TAATCACAAC TCACGACTCA 6600
CTTCTCCATA TCTGAAGGCT TAACACTTGT TTTCTTTTGG CTTGTACAGG GTTTAGTATG 6660
ATAATGGCAA CACTTTCGAA ACTTGGATTC TTTGACAGTG AAGCAAATCA AGTACTCTCC 6720
ACTGGAAAGA GGATTACGTT TGGTGCTCTT TTAAGTAACA TTCTAAATAA GGATGCCGAC 6780
AATGAATCAG AGCCCCTAGC GGGAGAAGAA GAGATAAGCA AGAGAATTAT CAAGCTTGGA 6840
CATTCCAAGG AGACTGCAGC CAAAGCTGCC ATATCAATTG TGTAAGCTTC TCCATGAAGA 6900
TATATAATCT GAATGTTGCA GTGTGATTCC AATTCTTCTA CGAAACTCCT AACCCCAATT 6960
CTTTTGTGGT GTCTTAGATT CTTGGGGTTC AACGAAGAGA GGGAGGTTCC ATCACTGTGT 7020
AAAAGCGTAT TTGATGCAAC TTGTTACCTA ATGGAAGAGA AACTAGCTTA TTCCGGAAAT 7080
GAACAGGTCT CTGTTTCATG TGAAAGCATT AGTTTTCTTC TCTCACTTGT ATTTGGTGTT 7140
ACTTACTGAC ATAAACTTTG GACAATCTTT TGCATTATGT TTTCAGGACA TGGTGCTTTT 7200
GCATCACGAA GTAGAAGTGG AATTCCTTGA AAGCAAACGT ATAGAGAAGC ACACTGCGAC 7260
TCTTTTGGAA TTCGGGGACA TCAAGAATGG GCAAACAACA ACCGCTATGG CCAAGACTGT 7320
TGGGATCCCT GCAGCCATTG GAGCTCTGGT CCTTACTAAG ACTTTGATCA CCACTTTTTC 7380
CTGTCTATAT TTCTCTAAAA TGAAAGTTTT AAGCGTTTGT TTTATGATGT TGTGTGTTGC 7440
AGCTGTTAAT TGAAGACAAG ATCAAGACAA GAGGAGTCTT AAGGCCTTTC GAAGCAGAGG 7500
TGTATTTGCC AGGTAAATTA GAATTCCGCT TCAAAAGGAT GTGTGTTGCA GATAAAGACA 7560
ATGATGTTGA TTTGTTGTGT GTTTGGGATA TGTGGTGTTA TACATACAGC TTTGGATATA 7620
TTGCAAGCAT ATGGTATAAA GCTGATGGAG AAGGCAGAAT GATCAAAGAA CTCTGTATAT 7680
TGTTTCTCTC TATAACTTGG AGTTGGAGAC AAAGCTGAAG AAGACAGAGA CATTAGACCA 7740
GCAAAAAAAG AAGAAGAAGG AAGAAGATAA GCCTCGATCC TTGGGTGACG AGTATCTATA 7800
TGTTTATATG TACTATATGT TATGTTGTAC AGAAGAAGTC GTGTCCACAA ATATCAATTG 7860
ATGTCAGATG TCTAGTAAGT GATCATGTGT AGCATACAAA CTGGAGTAAT TTAAAAAGTG 7920
172
CA 02280196 2000-06-02
AATAAACAAA AATAATTACT AAACGTTATT CCAAGTAGCT TTCCAAGACA GTCACTTGCC 7980
CTTTTCCAAT TTCCCTTGCA ATTAACTAAA TTGCTCTTCA CGATATGATA TTATACCAAA 8040
ATGGTGATAC CTTGGGAATT GTTAATTTGA CTCATTTGAA CAAATCTCAT CTATAAAATC 8100
ATCCCACCTC TCCACCACAT TTGTTCTCAC TACCAATCAA AAAATAATCT AGTCTTAAAC 8160
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3195 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
ATGAATTCAA ATGGCCATGA GGAGGAGAAG AAGTTGGGGA ATGGAGTTGT GGGGATTCTA 60
TCTGAAACAG TTAACAAATG GGAGAGACGA ACACCATTGA CGCCATCGCA TTGCGCTCGC 120
CTTTTACACG GTGGGAAAGA CAGAACCGGC ATTTCCCGCA TTGTGGTTCA GCCATCTGCT 180
AAGCGTATCC ATCATGATGC CTTGTATGAA CATGTTGGGT GTGAAATTTC TGATGATTTG 240
TCTGATTGTG GGCTTATACT TGGAATCAAA CAACCTGAGC TAGAAATGAT TCTTCCAGAG 300
AGAGCATACG CTTTCTTTTC ACATACTCAT AAGGCACAGA AAGAGAACAT GCCTTTGTTG 360
GATAAAATTC TTTCTGAGAG AGTGACTTTG TGTGATTATG AGCTCATTGT TGGGGATCAT 420
GGGAAACGAT TATTGGCGTT TGGTAAATAT GCAGGCAGAG CTGGTCTTGT TGACTTCTTA 480
CACGGACTTG GACAGCGATA TCTAAGTCTA GGATACTCAA CACCTTTCCT CTCGCTCGGT 540
GCATCGTATA TGTATTCCTC ATTGGCTGCT GCAAAAGCCG CTGTAATTTC TGTTGGTGAA 600
GAAATTGCAA GCCAGGGACT GCCATTAGGA ATCTGCCCTC TTGTATTTGT CTTCACCGGA 660
ACAGGAAATG TTTCTCTGGG GGCGCAAGAA ATTTTCAAGC TTCTTCCTCA CACTTTTGTT 720
GAACCAAGCA AACTTCCTGA ACTATTTGTA AAAGACAAAG GAATTAGTCA AAATGGGATT 780
TCAACAAAGC GAGTCTATCA AGTATATGGT TGTATTATTA CCAGCCAAGA CATGGTTGAA 840
CACAAAGATC CATCAAAGTC ATTCGACAAA GCCGACTATT ATGCACACCC GGAACATTAC 900
AATCCAGTTT TCCACGAAAA GATATCGCCA TATACGTCTG TTCTTGTAAA CTGTATGTAC 960
TGGGAGAAGA GGTTTCCCTG TCTTCTGAGC ACAAAACAGC TTCAAGATTT AACAAAAAAA 1020
GGACTCCCAC TAGTAGGCAT ATGTGATATA ACTTGTGACA TCGGTGGCTC CATTGAATTT 1080
GTTAACCGAG CTACTTTAAT CGATTCCCCT TTCTTCAGGT TTAATCCCTC GAACAATTCA 1140
173
CA 02280196 2000-06-02
TACTACGATG ACATGGATGG GGATGGCGTA CTATGCATGG CTGTTGACAT TTTACCCACA 1200
GAATTTGCAA AAGAGGCATC CCAGCATTTT GGAGATATTC TTTCCGGATT TGTCGGTAGT 1260
TTGGCTTCAA TGACTGAAAT TTCAGATCTA CCAGCACATC TGAAGAGGGC TTGCATAAGC 1320
TATAGGGGAG AATTGACATC TTTGTATGAG TATATTCCAC GTATGAGGAA GTCAAATCCA 1380
GAAGAGGCAC AAGATAATAT TATCGCCAAC GGGGTTTCCA GCCAGAGAAC ATTCAACATA 1440
TTGGTATCTC TGAGCGGACA CCTATTTGAT AAGTTTCTGA TAAACGAAGC TCTTGATATG 1500
ATCGAAGCGG CTGGTGGCTC ATTTCATTTG GCTAAATGTG AACTGGGGCA GAGCGCTGAT 1560
GCTGAATCGT ACTCAGAACT TGAAGTTGGT GCGGATGATA AGAGAGTATT GGATCAAATC 1620
ATTGATTCAT TAACTCGGTT AGCTAATCCA AATGAAGATT ATATATCCCC ACATAGAGAA 1680
GCAAATAAGA TCTCACTGAA GATTGGTAAA GTCCAGCAAG AAAATGAGAT AAAAGAGAAG 1740
CCTGAAATGA CGAAAAAATC AGGTGTTTTG ATTCTTGGTG CTGGACGTGT GTGTCGCCCA 1800
GCTGCTGATT TCCTAGCTTC AGTTAGAACC ATTTCGTCAC AGCAATGGTA CAAAACATAT 1860
TTCGGAGCAG ACTCTGAAGA GAAAACAGAT GTTCATGTGA TTGTCGCGTC TCTGTATCTT 1920
AAGGATGCCA AAGAGACGGT TGAAGGTATT TCAGATGTAG AAGCAGTTCG GCTAGATGTA 1980
TCTGATAGTG AAAGTCTCCT TAAGTATGTT TCTCAGGTTG ATGTTGTCCT AAGTTTATTA 2040
CCTGCAAGTT GTCATGCTGT TGTAGCAAAG ACATGCATTG AGCTGAAGAA GCATCTCGTC 2100
ACTGCTAGCT ATGTTGATGA TGAAACGTCC ATGTTACATG AGAAGGCTAA GAGTGCTGGG 2160
ATAACGATTC TAGGCGAAAT GGGACTGGAC CCTGGAATCG ATCACATGAT GGCGATGAAA 2220
ATGATCAACG ATGCTCATAT CAAAAAAGGG AAAGTGAAGT CTTTTACCTC TTATTGTGGA 2280
GGGCTTCCCT CTCCTGCTGC AGCAAATAAT CCATTAGCAT ATAAATTTAG CTGGAACCCT 2340
GCTGGAGCAA TTCGAGCTGG TCAAAACCCC GCCAAATACA AAAGCAACGG CGACATAATA 2400
CATGTTGATG GGAAGAATCT CTATGATTCC GCGGCAAGAT TCCGAGTACC TAATCTTCCA 2460
GCTTTTGCAT TGGAGTGTTT TCCAAATCGT GACTCCTTGG TTTACGGGGA ACATTATGGC 2520
ATCGAGAGCG AAGCAACAAC GATATTTCGT GGAACACTCA GATATGAAGG GTTTAGTATG 2580
ATAATGGCAA CACTTTCGAA ACTTGGATTC TTTGACAGTG AAGCAAATCA AGTACTCTCC 2640
ACTGGAAAGA GGATTACGTT TGGTGCTCTT TTAAGTAACA TTCTAAATAA GGATGCAGAC 2700
AATGAATCAG AGCCCCTAGC GGGAGAAGAA GAGATAAGCA AGAGAATTAT CAAGCTTGGA 2760
CATTCCAAGG AGACTGCAGC CAAAGCTGCC AAAACAATTG TATTCTTGGG GTTCAACGAA 2820
GAGAGGGAGG TTCCATCACT GTGTAAAAGC GTATTTGATG CAACTTGTTA CCTAATGGAA 2880
174
CA 02280196 2000-06-02
GAGAAACTAG CTTATTCCGG AAATGAACAG GACATGGTGC TTTTGCATCA CGAAGTAGAA 2940
GTGGAATTCC TTGAAAGCAA ACGTATAGAG AAGCACACTG CGACTCTTTT GGAATTCGGG 3000
GACATCAAGA ATGGACAAAC AACAACCGCT ATGGCCAAGA CTGTTGGGAT CCCTGCAGCC 3060
ATTGGAGCTC TGGTGTTAAT TGAAGACAAG ATCAAGACAA GAGGAGTCTT AAGGCCTCTC 3120
GAAGCAGAGG TGTATTTGCC AGCTTTGGAT ATATTGCAAG CATATGGTAT AAAGCTGATG 3180
GAGAAGGCAG AATGA 3195
(2) INFORMATION FOR SEQ ID NO:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1064 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:
Met Asn Ser Asn Gly His Glu Glu Glu Lys Lys Leu Gly Asn Gly Val
1 5 10 15
Val Gly Ile Leu Ser Glu Thr Val Asn Lys Trp Glu Arg Arg Thr Pro
20 25 30
Leu Thr Pro Ser His Cys Ala Arg Leu Leu His Gly Gly Lys Asp Arg
35 40 45
Thr Gly Ile Ser Arg Ile Val Val Gln Pro Ser Ala Lys Arg Ile His
50 55 60
His Asp Ala Leu Tyr Glu His Val Gly Cys Glu Ile Ser Asp Asp Leu
65 70 75 80
Ser Asp Cys Gly Leu Ile Leu Gly Ile Lys Gln Pro Glu Leu Glu Met
85 90 95
Ile Leu Pro Glu Arg Ala Tyr Ala Phe Phe Ser His Thr His Lys Ala
100 105 110
Gln Lys Glu Asn Met Pro Leu Leu Asp Lys Ile Leu Ser Glu Arg Val
115 120 125
Thr Leu Cys Asp Tyr Glu Leu Ile Val Gly Asp His Gly Lys Arg Leu
130 135 140
Leu Ala Phe Giy Lys Tyr Ala Gly Arg Ala Gly Leu Val Asp Phe Leu
145 150 155 160
His Gly Leu Gly Gln Arg Tyr Leu Ser Leu Gly Tyr Ser Thr Pro Phe
165 170 175
175
CA 02280196 2000-06-02
Leu Ser Leu Gly Ala Ser Tyr Met Tyr Ser Ser Leu Ala Ala Ala Lys
180 185 190
Ala Ala Val Ile Ser Val Gly Glu Glu Ile Ala Ser Gln Gly Leu Pro
195 200 205
Leu Gly Ile Cys Pro Leu Val Phe Val Phe Thr Gly Thr Gly Asn Val
210 215 220
Ser Leu Gly Ala Gln Glu Ile Phe Lys Leu Leu Pro His Thr Phe Val
225 230 235 240
Glu Pro Ser Lys Leu Pro Glu Leu Phe Val Lys Asp Lys Gly Ile Ser
245 250 255
Gln Asn Gly Ile Ser Thr Lys Arg Val Tyr Gln Val Tyr Gly Cys Ile
260 265 270
Ile Thr Ser Gln Asp Met Val Glu His Lys Asp Pro Ser Lys Ser Phe
275 280 285
Asp Lys Ala Asp Tyr Tyr Ala His Pro Glu His Tyr Asn Pro Val Phe
290 295 300
His Glu Lys Ile Ser Pro Tyr Thr Ser Val Leu Val Asn Cys Met Tyr
305 310 315 320
Trp Glu Lys Arg Phe Pro Cys Leu Leu Ser Thr Lys Gln Leu Gln Asp
325 330 335
Leu Thr Lys Lys Gly Leu Pro Leu Val Gly Ile Cys Asp Ile Thr Cys
340 345 350
Asp Ile Gly Gly Ser Ile Glu Phe Val Asn Arg Ala Thr Leu Ile Asp
355 360 365
Ser Pro Phe Phe Arg Phe Asn Pro Ser Asn Asn Ser Tyr Tyr Asp Asp
370 375 380
Met Asp Gly Asp Gly Val Leu Cys Met Ala Val Asp Ile Leu Pro Thr
385 390 395 400
Glu Phe Ala Lys Glu Ala Ser Gln His Phe Gly Asp Ile Leu Ser Gly
405 410 415
Phe Val Gly Ser Leu Ala Ser Met Thr Glu Ile Ser Asp Leu Pro Ala
420 425 430
His Leu Lys Arg Ala Cys Ile Ser Tyr Arg Gly Glu Leu Thr Ser Leu
435 440 445
Tyr Glu Tyr Ile Pro Arg Met Arg Lys Ser Asn Pro Glu Glu Ala Gln
450 455 460
Asp Asn Ile Ile Ala Asn Gly Val Ser Ser Gln Arg Thr Phe Asn Ile
465 470 475 480
176
CA 02280196 2000-06-02
Leu Val Ser Leu Ser Gly His Leu Phe Asp Lys Phe Leu Ile Asn Glu
485 490 495
Ala Leu Asp Met Ile Glu Ala Ala Gly Gly Ser Phe His Leu Ala Lys
500 505 510
Cys Glu Leu Gly Gln Ser Ala Asp Ala Glu Ser Tyr Ser Glu Leu Glu
515 520 525
Val Gly Ala Asp Asp Lys Arg Val Leu Asp Gln Ile Ile Asp Ser Leu
530 535 540
Thr Arg Leu Ala Asn Pro Asn Glu Asp Tyr Ile Ser Pro His Arg Glu
545 550 555 560
Ala Asn Lys Ile Ser Leu Lys Ile Gly Lys Val Gln Gln Glu Asn Glu
565 570 575
Ile Lys Glu Lys Pro Glu Met Thr Lys Lys Ser Gly Val Leu Ile Leu
580 585 590
Gly Ala Gly Arg Val Cys Arg Pro Ala Ala Asp Phe Leu Ala Ser Val
595 600 605
Arg Thr Ile Ser Ser Gln Gln Trp Tyr Lys Thr Tyr Phe Gly Ala Asp
610 615 620
Ser Glu Glu Lys Thr Asp Val His Val Ile Val Ala Ser Leu Tyr Leu
625 630 635 640
Lys Asp Ala Lys Glu Thr Val Glu Gly Ile Ser Asp Val Glu Ala Val
645 650 655
Arg Leu Asp Val Ser Asp Ser Glu Ser Leu Leu Lys Tyr Val Ser Gln
660 665 670
Val Asp Val Val Leu Ser Leu Leu Pro Ala Ser Cys His Ala Val Val
675 680 685
Ala Lys Thr Cys Ile Glu Leu Lys Lys His Leu Val Thr Ala Ser Tyr
690 695 700
Val Asp Asp Glu Thr Ser Met Leu His Glu Lys Ala Lys Ser Ala Gly
705 710 715 720
Ile Thr Ile Leu Gly Glu Met Gly Leu Asp Pro Gly Ile Asp His Met
725 730 735
Met Ala Met Lys Met Ile Asn Asp Ala His Ile Lys Lys Gly Lys Val
740 745 750
Lys Ser Phe Thr Ser Tyr Cys Gly Gly Leu Pro Ser Pro Ala Ala Ala
755 760 765
Asn Asn Pro Leu Ala Tyr Lys Phe Ser Trp Asn Pro Ala Gly Ala Ile
770 775 780
177
CA 02280196 2000-06-02
Arg Ala Gly Gln Asn Pro Ala Lys Tyr Lys Ser Asn Gly Asp Ile Ile
785 790 795 800
His Val Asp Gly Lys Asn Leu Tyr Asp Ser Ala Ala Arg Phe Arg Val
805 810 815
Pro Asn Leu Pro Ala Phe Ala Leu Glu Cys Phe Pro Asn Arg Asp Ser
820 825 830
Leu Val Tyr Gly Glu His Tyr Gly Ile Glu Ser Glu Ala Thr Thr Ile
835 840 845
Phe Arg Gly Thr Leu Arg Tyr Glu Gly Phe Ser Met Ile Met Ala Thr
850 855 860
Leu Ser Lys Leu Gly Phe Phe Asp Ser Glu Ala Asn Gln Val Leu Ser
865 870 875 880
Thr Gly Lys Arg Ile Thr Phe Gly Ala Leu Leu Ser Asn Ile Leu Asn
885 890 895
Lys Asp Ala Asp Asn Glu Ser Glu Pro Leu Ala Gly Glu Glu Glu Ile
900 905 910
Ser Lys Arg Ile Ile Lys Leu Gly His Ser Lys Glu Thr Ala Ala Lys
915 920 925
Ala Ala Lys Thr Ile Val Phe Leu Gly Phe Asn Glu Glu Arg Glu Val
930 935 940
Pro Ser Leu Cys Lys Ser Val Phe Asp Ala Thr Cys Tyr Leu Met Glu
945 950 955 960
Glu Lys Leu Ala Tyr Ser Gly Asn Glu Gln Asp Met Val Leu Leu His
965 970 975
His Glu Val Glu Val Glu Phe Leu Glu Ser Lys Arg Ile Glu Lys His
980 985 990
Thr Ala Thr Leu Leu Glu Phe Gly Asp Ile Lys Asn Gly Gln Thr Thr
995 1000 1005
Thr Ala Met Ala Lys Thr Val Gly Ile Pro Ala Ala Ile Gly Ala Leu
1010 1015 1020
Val Leu Ile Glu Asp Lys Ile Lys Thr Arg Gly Val Leu Arg Pro Leu
1025 1030 1035 1040
Glu Ala Glu Val Tyr Leu Pro Ala Leu Asp Ile Leu Gln Ala Tyr Gly
1045 1050 1055
Ile Lys Leu Met Glu Lys Ala Glu
1060
178
CA 02280196 2000-06-02
(2) INFORMATION FOR SEQ ID NO:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 6
(D) OTHER INFORMATION: /mod base=i
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 12
(D) OTHER INFORMATION: /mod base=i
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 21
(D) OTHER INFORMATION: /mod base=i
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:
TTYTCNCAYA CNCAYAARGC NCA 23
(2) INFORMATION FOR SEQ ID NO:114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
TTYTCCCART ACATRCARTT 20
(2) INFORMATION FOR SEQ ID NO:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 619 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:
GAAAACATGC CTTTGCTGGA TAAGATTCTA GCTGAGAGGG CATCGTTATA TGACTATGAA 60
TTAATTGTTG GGGACACTGG GAAAAGGTTA CTTGCATTTG GAAAATTCGC TGGTAGGGCT 120
179
CA 02280196 2000-06-02
GGAATGATCG ACTTTTTGCG CGGATTAGGA CAGCGGTTTT TAAGTCTTGG ATATTCAACA 180
CCTTTCTTGT CACTTGGATC ATCTTACATG TACCCTTCCC TGGCTGCTGC TAAGGCTGCT 240
GTGATTTCTG TTGGTGAAAA ATTGCGACGC AGGGATTGCC ATTGGGGATT TGTCCCCTGG 300
TTTGTTTATT TACTGGTTCA GGAAATGTTT GTTCTGGTGC ACAGGAGATA TTTAAGCTTC 360
TTCCTCATAC CTTTGTTGAT CCATCTAAAC TACGCGACCT ACATAGAACG GACCCAGATC 420
AACCAAGGCA TGCTTCAAAA AGAGTTTTCC AAGTTTATGG TTGTGTTGTG ACTGCCCAAG 480
ACATGGTTGA ACCCAAAGAT CACGTGATAG TGTTTGACAA AGCAGACTAC TATGCACATC 540
CTGAGCATTA CAATCCCACT TTCCATGAAA AAATAGCACC ATATGCATCT GTTATTGTCA 600
ATTGCATGTA TTGGGAAAA 619
(2) INFORMATION FOR SEQ ID NO:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 620 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
GAGAATATGC CACTGTTAGA CAAGATCCTT GAAGAAAGGG TGTCCTTGTT TGATTATGAG 60
CTAATTGTTG GAGATGATGG GAAAAGATCA CTAGCATTTG GGAAATTTGC TGGTAGAGCT 120
GGACTGATAG ATTTCTTACA TGGTCTCGGA CAGCGATATT TGAGCCTTGG ATACTCCACT 180
CCATTTCTCT CTCTGGGACA TCTCATATGT TCCTTCGCTC GCTGCAGCCA AGGCTGCAGT 240
CATTGTCGTT GCAGAAGAGA TAGCAACATT TGGACTTCCA TCCGGAATTT GTCCGATAGT 300
GTTTGTGTTC ACTGGAGTTG GAAACGTCTC TCAGGGTGCG CAGGAGATAT TCAAGTTATT 360
GCCCCATACC TTTGTTGATG CTGAGAAGCT TCCCGAAATT TTTCAGGCCA GGAATCTGTC 420
TAAGCAATCT CAGTCGACCA AGAGAGTATT TCAACTTTAT GGTTGTGTTG TGACCTCTAG 480
AGACATAGTT TCTCACAAGG ATCCCACCAG ACAATTTGAC AAAGGTGACT ATTATGCTCA 540
TCCAGAACAC TACACCCCTG TTTTTCATGA AAGAATTGCT CCATATGCAT CTGTCATCGT 600
AAACTGCATG TATTGGGAAA 620
(2) INFORMATION FOR SEQ ID NO:117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 amino acids
(B) TYPE: amino acid
180
CA 02280196 2000-06-02
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
Glu Asn Met Pro Leu Leu Asp Lys Ile Leu Ala Glu Arg Ala Ser Leu
1 5 10 15
Tyr Asp Tyr Glu Leu Ile Val Gly Asp Thr Gly Lys Arg Leu Leu Ala
20 25 30
Phe Gly Lys Phe Ala Gly Arg Ala Gly Met Ile Asp Phe Leu Arg Gly
35 40 45
Leu Gly Gln Arg Phe Leu Ser Leu Gly Tyr Ser Thr Pro Phe Leu Ser
50 55 60
Leu Gly Ser Ser Tyr Met Tyr Pro Ser Leu Ala Ala Ala Lys Ala Ala
65 70 75 80
Val Ile Ser Val Gly Glu Xaa Ile Ala Thr Gln Gly Leu Pro Leu Gly
85 90 95
Ile Cys Pro Leu Val Cys Leu Phe Thr Gly Ser Gly Asn Val Cys Ser
100 105 110
Gly Ala Gln Glu Ile Phe Lys Leu Leu Pro His Thr Phe Val Asp Pro
115 120 125
Ser Lys Leu Arg Asp Leu His Arg Thr Asp Pro Asp Gln Pro Arg His
130 135 140
Ala Ser Lys Arg Val Phe Gln Val Tyr Gly Cys Val Val Thr Ala Gln
145 150 155 160
Asp Met Val Glu Pro Lys Asp His Val Ile Val Phe Asp Lys Ala Asp
165 170 175
Tyr Tyr Ala His Pro Glu His Tyr Asn Pro Thr Phe His Glu Lys Ile
180 185 190
Ala Pro Tyr Ala Ser Val Ile Val Asn Cys Met Tyr Trp Glu
195 200 205
(2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 207 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
181
CA 02280196 2000-06-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:
Glu Asn Met Pro Leu Leu Asp Lys Ile Leu Glu Glu Arg Val Ser Leu
1 5 10 15
Phe Asp Tyr Glu Leu Ile Val Gly Asp Asp Gly Lys Arg Ser Leu Ala
20 25 30
Phe Gly Lys Phe Ala Gly Arg Ala Gly Leu Ile Asp Phe Leu His Gly
35 40 45
Leu Gly Gin Arg Tyr Leu Ser Leu Gly Tyr Ser Thr Pro Phe Leu Ser
50 55 60
Leu Gly Xaa Ser His Met Xaa Pro Ser Leu Ala Ala Ala Lys Ala Ala
65 70 75 80
Val Ile Val Val Ala Glu Glu Ile Ala Thr Phe Gly Leu Pro Ser Gly
85 90 95
Ile Cys Pro Ile Val Phe Val Phe Thr Gly Val Gly Asn Val Ser Gln
100 105 110
Gly Ala Gln Glu Ile Phe Lys Leu Leu Pro His Thr Phe Val Asp Ala
115 120 125
Glu Lys Leu Pro Glu Ile Phe Gln Ala Arg Asn Leu Ser Lys Gin Ser
130 135 140
Gln Ser Thr Lys Arg Val Phe Gln Leu Tyr Gly Cys Val Val Thr Ser
145 150 155 160
Arg Asp Ile Val Ser His Lys Asp Pro Thr Arg Gln Phe Asp Lys Gly
165 170 175
Asp Tyr Tyr Ala His Pro Glu His Tyr Thr Pro Val Phe His Glu Arg
180 185 190
Ile Ala Pro Tyr Ala Ser Val Ile Val Asn Cys Met Tyr Trp Glu
195 200 205
(2) INFORMATION FOR SEQ ID NO:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2582 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max
182
CA 02280196 2000-06-02
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..2357
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:
TTGAACCCAA AGATCACGTG ATAGTGTTTG ACAAAGCAGA CTACTATTCA CACCCTGAGC 60
ATTACAATCC CACTTTCCAT GATAAAATAG CACCATATGC ATCTGTTATT GTCAATTGCA 120
TGTATTGGGA GAAAAGATTT CCTCAATTGC CGAGCTATAA GCAGATGCAA GACTTAATGG 180
GCCGGGGGAG CCCCCTTGTT GGAATAGCTG ACATAACGTG TGATATAGGG GGTTCAATTG 240
AGTTTGTTAA CCGCGGTACT TCAATTGATT CACCCTTCTT CAGATATGAT CCCTTAACAA 300
ATTCCTACCA TGATGATATG GAGGGGAATG GAGTGATATG CTTAGCTGTT GACATTCTTC 360
CAACAGAATT TGCAAAGGAG GCTTCCCAAC ATTTTGGAAA CATACTTTCC CAATTTGTTG 420
TAAATTTGGC TTCTGCTACA GACATTACAA AGTTGCCTGC TCACTTAAGG AGAGCTTGCA 480
TAGCCCATAA AGGAGTGCTA ACCTCCTTAT ATGATTATAT CCCACGCATG CGGAGTTCTG 540
ATTCAGAGGA AGTATCAGAA AACGCAGAAA ATTCTCTATC CAACAAAAGG AAGTACAATA 600
TATCGGTGTC TCTGAGTGGT CACTTATTTG ATCAGTTTCT GATAAATGAG GCCTTAGATA 660
TTATTGAAGC TGCAGGAGGC TCCTTCCACT TAGTCAACTG CCATGTGGGT CAGAGCATTG 720
AAGCCGTATC ATTCTCTGAA CTTGAAGTTG GTGCAGATAA CAGGGCTGTT CTGGATCAAA 780
TCATTGATTC TTTAACTGCT ATTGCTAGTC CAACTGAACA TGATAGATTT TCAAATCAAG 840
ATTCAAGTAA AATTTCACTT AAGCTTGGTA AAGTTGAAGA GAATGGCATA GAGAAGGAAT 900
CTGACCCCAG AAAGAAGGCT GCGGTTTTAA TTCTTGGAGC TGGTCGGGTC TGTCAACCAG 960
CTGCTGAAAT GTTATCATCA TTTGGAAGGC CATCATCGAG CCAATGGTAT AAAACATTGT 1020
TGGAAGATGA TTTTGAATGT CAAACTGATG TAGAAGTCAT TGTGGGATCT CTGTACCTGA 1080
AGGATGCAGA GCAGACTGTT GAGGGCATTC CAAATGTAAC CGGAATTCAG CTTGATGTGA 1140
TGGATCGTGC CAATTTGTGT AAGTACATTT CACAGGTTGA CGTTGTTATA AGTTTGCTGC 1200
CCCCAAGTTG TCATATTATT GTAGCAAATG CTTGCATTGA GCTGAAAAAA CATCTTGTCA 1260
CTGCTAGCTA TGTTGATAGC TCCATGTCAA TGCTAAATGA TAAGGCTAAA GATGCTGGCA 1320
TAACAATTCT TGGAGAGATG GGCTTGGACC CAGGAATTGG TCATATGATG GCAATGAAGA 1380
TGATCAACCA AGCACATGTG AGGAAGGGGA AAATAAAGTC TTTCACTTCT TATTGTGGTG 1440
GACTTCCATC TCCTGAAGCT GCTAACAATC CATTAGCATA TAAATTCAGT TGGAATCCTG 1500
CAGGAGCCAT CCGAGCTGGG CGCAATCCTG CCACCTACAA ATGGGGTGGT GAAACTGTAC 1560
ATATTGATGG GGACGATCTT TATGATTCGG CTACAAGACT AAGGCTACCG GACCTTCCTG 1620
CTTTTGCTTT GGAATGTCTC CCAAATCGCA ATTCATTACT TTATGGGGAT TTGTATGGAA 1680
TAACTGAAGC ATCAACCATT TTCCGTGGAA CCCTCCGCTA TGAAGGATTT AGTGAGATCA 1740
TGGGGACACT GTCTAGGATT AGCTTATTTA ACAATGAAGC CCATTCGTTG CTAATGAATG 1800
183
CA 02280196 2000-06-02
GACAAAGACC AACTTTCAAA AAATTCTTAT TTGAACTTCT CAAAGTTGTT GGTGATAATC 1860
CAGATGAACT ATTGATAGGA GAGAATGACA TCATGGAGCA AATATTAATA CAAGGGCACT 1920
GCAAAGATCA AAGAACGGCA ATGGAGACAG CAAAAACAAT CATTTTCTTG GGACTTCTTG 1980
ACCAAACTGA AATCCCTGCT TCCTGCAAAA GTGCTTTTGA TGTTGCTTGT TTCCGCATGG 2040
AGGAGAGGTT ATCATACACC AGCACAGAAA AGGATATGGT GCTTTTGCAT CATGAAGTGG 2100
AAATAGAATA CCCAGATAGC CAAATTACAG AGAAGCATAG AGCTACTTTA CTTGAATTTG 2160
GGAAGACTCT TGATGAAAAA ACCACAACTG CCATGGCCCT TACTGTTGGT ATTCCAGCTG 2220
CTGTTGGAGC TTTGCTTTTA TTGACAAACA AAATTCAGAC AAGAGGAGTC TTAAGGCCTA 2280
TCGAACCTGA AGTATACAAT CCAGCACTGG ATATTATAGA AGCTTATGGG ATCAAGTTGA 2340
TAGAGAAGAC CGAGTAATTT GCATYTATGA ATTGATGTAT AGGTGTACAT TAATGTACAC 2400
CATGCAATGT TTGATTTGAA TAAGATAAAA TATAATAATT ACTGCAGTCA TGGAATTGCA 2460
ACTGCCATTC TATGCAACTG TCAGAAATGG ACCACACGGT ACCAGCATAG TTAAAACACT 2520
TAGGCAGATA CCAATTTCAA TTGCAGCAGT ACAATCCAAC CAGTTATGAA GTATGGTTCT 2580
AG 2582
(2) INFORMATION FOR SEQ ID NO:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3265 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..3071
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:
ATTGTGCCCG CCTTCTGCTA GGAGGAGGCA AGAACGGACC TCGAGTAAAC CGGATTATTG 60
TGCAGCCAAG CACAAGGAGG ATCCATCATG ACGCTCAGTA TGAGGATGCA GGATGCGAGA 120
TTTCAGAAGA CCTGTCAGAA TGCGGCCTTA TCATAGGCAT CAAACAACCC AAGCTGCAGA 180
TGATTCTTTC AGATAGAGCG TACGCTTTCT TTTCACACAC ACACAAAGCC CAAAAAGAGA 240
ATATGCCACT GTTAGACAAG ATCCTTGAAG AAAGGGTGTC CTTGTTTGAT TATGAGCTAA 300
TTGTTGGAGA TGATGGGAAA AGATCACTAG CATTTGGGAA ATTTGCTGGT AGAGCTGGAC 360
184
CA 02280196 2000-06-02
TGATAGATTT CTTACATGGT CTCGGACAGC GATATTTGAG CCTTGGATAC TCGACTCCAT 420
TTCTCTCTCT GGGACAATCT CATATGTATC CTTCGCTCGC TGCAGCCAAG GCTGCAGTCA 480
TTGTCGTTGC AGAAGAGATA GCAACATTTG GACTTCCATC CGGAATTTGT CCGATAGTGT 540
TTGTGTTCAC TGGAGTTGGA AACGTCTCTC AGGGTGCGCA GGAGATATTC AAGTTATTGC 600
CCCATACCTT TGTTGATGCT GAGAAGCTTC CCGAAATTTT TCAGGCCAGG AATCTGTCTA 660
AGCAATCTCA GTCGACCAAG AGAGTATTTC AACTTTATGG TTGTGTTGTG ACCTCTAGAG 720
ACATAGTTTC TCACAAGGAT CCCACCAGAC AATTTGACAA AGGTGACTAT TATGCTCATC 780
CAGAACACTA CACCCCTGTT TTTCATGAAA GAATTGCTCC ATATGCATCT GTCATCGTAA 840
ACTGTATGTA TTGGGAGAAG AGGTTTCCAC CATTACTAAA TATGGATCAG TTACAGCAAT 900
TGATGGAGAC TGGTTGTCCT TTAGTCGGCG TTTGTGACAT AACTTGTGAT ATTGGAGGTT 960
CCATTGAATT TATCAACAAG AGTACATCAA TAGAGAGGCC TTTCTTTCGG TATGATCCTT 1020
CTAAGAATTC ATACCATGAT GATATGGAAG GTGCCGGAGT GGTCTGCTTG GCTGTTGACA 1080
TTCTCCCTAC AGAATTCTCT AAAGAGGCCT CCCAACATTT TGGAAACATA CTATCTAGAC 1140
TTGTTGCTAG TTTGGCCTCA GTGAAGCAAC CGGCAGAACT TCCTTCCTAC TTGAGAAGAG 1200
CTTGCATTGC ACATGCTGGC AGATTAACTC CTTTGTATGA ATATATCCCT AGGATGAGAA 1260
ATACTATGAT AGATTTGGCA CCCGCAAAAA CAAATCCATT GCCTGACAAG AAGTATAGCA 1320
CCCTGGTATC TCTCAGTGGG CACCTATTTG ATAAGTTCCT TATAAATGAA GCTTTGGACA 1380
TCATTGAGAC AGCTGGAGGT TCATTTCACT TGGTTAGATG TGAAGTTGGA CAAAGCACGG 1440
ATGATATGTC ATACTCAGAG CTTGAAGTAG GAGCAGATGA TACTGCCACA TTGGATAAAA 1500
TTATTGATTC CTTGACTTCT TTAGCTAATG AACATGGTGG AGATCACGAT GCCGGGCAAG 1560
AAATTGAATT AGCTCTGAAG ATAGGAAAAG TCAATGAGTA TGAAACTGAC GTCACAATTG 1620
ATAAAGGAGG GCCAAAGATT TTAATTCTTG GAGCTGGAAG AGTCTGTCGG CCAGCTGCTG 1680
AGTTTCTGGC ATCTTACCCA GACATATGTA CCTATGGTGT TGATGACCAT GATGCAGATC 1740
AAATTCATGT TATCGTGGCA TCTTTGTATC AAAAAGATGC AGAAGAGACA GTTGATGGTA 1800
TTGAAAATAC AACTGCTACC CAGCTTGATG TTGCTGATAT TGGAAGCCTT TCAGATCTTG 1860
TTTCTCAGGT TGAGGTTGTA ATTAGCTTGC TGCCTGCTAG TTTTCATGCT GCCATTGCAG 1920
GAGTATGCAT AGAGTTGAAG AAGCACATGG TAACGGCAAG CTATGTTGAT GAATCCATGT 1980
CAAACTTGAG CCAAGCTGCC AAAGATGCAG GTGTAACTAT ACTTTGTGAA ATGGGCCTAG 2040
ATCCTGGCAT AGATCACTTG ATGTCAATGA AGATGATTGA TGAAGCTCAT GCACGAAAGG 2100
GAAAAATAAA GGCATTTACA TCTTACTGTG GTGGATTGCC ATCTCCAGCT GCAGCAAACA 2160
ATCCGCTTGC CTATAAATTC AGTTGGAACC CAGCTGGTGC ACTCCGGTCA GGGAAAAATC 2220
CTGCAGTCTA CAAATTTCTT GGTGAGACGA TCCATGTAGA TGGTCATAAC TTGTATGAAT 2280
CAGCAAAGAG GCTCAGACTA CGAGAGCTTC CAGCTTTTGC TCTGGAACAC TTGCCAAATC 2340
185
CA 02280196 2000-06-02
GGAATTCCTT GATATATGGT GACCTTTATG GTATCTCCAA AGAAGCATCC ACCATATATA 2400
GGGCTACTYT TCGTTACGAA GGTTTTAGTG AGATTATGGT AACCCTTTCC AAAACTGGGT 2460
TCTTTGATGC TGCAAATCAT CCACTGCTGC AAGATACTAG TCGTCCAACA TATAAGGGTT 2520
TCCTTGATGA ACTACTGAAT AATATCTCCA CAATTAACAC GGACTTAGAT ATTGAAGCTT 2580
CTGGTGGATA CGATGATGAC CTGATTGCCA GACTGTTGAA GCTCGGGTGT TGCAAAAATA 2640
AGGAAATAGC TGTTAAGACA GTCAAAACCA TCAAGTTCTT GGGACTACAT GAAGAGACTC 2700
AAATACCTAA GGGTTGTTCG AGCCCATTTG ATGTGATTTG CCAGCGAATG GAACAGAGGA 2760
TGGCCTATGG CCACAATGAG CAAGACATGG TACTGCTCCA CCACGAAGTC GAGGTGGAAT 2820
ACCCGGACGG GCAACCCGCC GAAAAGCACC AAGCGACGCT ACTGGAGTTC GGGAAGGTTG 2880
AAAATGGCAG GTCCACCACT GCCATGGCGC TGACCGTCGG CATTCCAGCA GCAATAGGGG 2940
CCCTGCTATT GCTAAAGAAT AAGGTCCAGA CGAAAGGAGT GATCAGGCCT CTGCAACCGG 3000
AAATCTACGT TCCAGCATTG GAGATCTTGG AGTCGTCGGG CATCAAGCTG GTTGAGAAAG 3060
TGGAGACTTG AAAGTTCCCT GATACACAGA TAAAGATAGT ATGATATAGC AGGGCACATG 3120
TATCTTTTGT ATTAACTCCG TTCTGGAATA TATATTTGTG AACTAAAATG TGACAAATAA 3180
AAAGAACGGG TGGAGTATAT TGTAAGAGAC GGCAAAGAAA CCTCTGTATA TATGACCTGT 3240
CGATATCAAA TAATGCCGAT CAGTT 3265
(2) INFORMATION FOR SEQ ID NO:121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 784 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:
Glu Pro Lys Asp His Val Ile Val Phe Asp Lys Ala Asp Tyr Tyr Ser
1 5 10 15
His Pro Glu His Tyr Asn Pro Thr Phe His Glu Lys Ile Ala Pro Tyr
20 25 30
Ala Ser Val Ile Val Asn Cys Met Tyr Trp Glu Lys Arg Phe Pro Gin
35 40 45
Leu Pro Ser Tyr Lys Gln Met Gln Asp Leu Met Gly Arg Gly Ser Pro
50 55 60
186
CA 02280196 2000-06-02
Leu Val Gly Ile Ala Asp Ile Thr Cys Asp Ile Gly Gly Ser Ile Glu
65 70 75 80
Phe Val Asn Arg Gly Thr Ser Ile Asp Ser Pro Phe Phe Arg Tyr Asp
85 90 95
Pro Leu Thr Asn Ser Tyr His Asp Asp Met Glu Gly Asn Gly Val Ile
100 105 110
Cys Leu Ala Val Asp Ile Leu Pro Thr Glu Phe Ala Lys Glu Ala Ser
115 120 125
Gln His Phe Gly Asn Ile Leu Ser Gln Phe Val Val Asn Leu Ala Ser
130 135 140
Ala Thr Asp Ile Thr Lys Leu Pro Ala His Leu Arg Arg Ala Cys Ile
145 150 155 160
Ala His Lys Gly Val Leu Thr Ser Leu Tyr Asp Tyr Ile Pro Arg Met
165 170 175
Arg Ser Ser Asp Ser Glu Glu Val Ser Glu Asn Ala Glu Asn Ser Leu
180 185 190
Ser Asn Lys Arg Lys Tyr Asn Ile Ser Val Ser Leu Ser Gly His Leu
195 200 205
Phe Asp Gln Phe Leu Ile Asn Glu Ala Leu Asp Ile Ile Glu Ala Ala
210 215 220
Gly Gly Ser Phe His Leu Val Asn Cys His Val Gly Gln Ser Ile Glu
225 230 235 240
Ala Val Ser Phe Ser Glu Leu Glu Val Gly Ala Asp Asn Arg Ala Val
245 250 255
Leu Asp Gln Ile Ile Asp Ser Leu Thr Ala Ile Ala Ser Pro Thr Glu
260 265 270
His Asp Arg Phe Ser Asn Gln Asp Ser Ser Lys Ile Ser Leu Lys Leu
275 280 285
Gly Lys Val Glu Glu Asn Gly Ile Glu Lys Glu Ser Asp Pro Arg Lys
290 295 300
Lys Ala Ala Val Leu Ile Leu Gly Ala Gly Arg Val Cys Gln Pro Ala
305 310 315 320
Ala Glu Met Leu Ser Ser Phe Gly Arg Pro Ser Ser Ser Gln Trp Tyr
325 330 335
Lys Thr Leu Leu Glu Asp Asp Phe Glu Cys Gln Thr Asp Val Glu Val
340 345 350
Ile Val Gly Ser Leu Tyr Leu Lys Asp Ala Glu Gln Thr Val Glu Gly
355 360 365
Ile Pro Asn Val Thr Gly Ile Gln Leu Asp Val Met Asp Arg Ala Asn
370 375 380
Leu Cys Lys Tyr Ile Ser Gln Val Asp Val Val Ile Ser Leu Leu Pro
385 390 395 400
Pro Ser Cys His Ile Ile Val Ala Asn Ala Cys Ile Glu Leu Lys Lys
405 410 415
187
CA 02280196 2000-06-02
His Leu Val Thr Ala Ser Tyr Val Asp Ser Ser Met Ser Met Leu Asn
420 425 430
Asp Lys Ala Lys Asp Ala Gly Ile Thr Ile Leu Gly Glu Met Gly Leu
435 440 445
Asp Pro Gly Ile Gly His Met Met Ala Met Lys Met Ile Asn Gln Ala
450 455 460
His Val Arg Lys Gly Lys Ile Lys Ser Phe Thr Ser Tyr Cys Gly Gly
465 470 475 480
Leu Pro Ser Pro Glu Ala Ala Asn Asn Pro Leu Ala Tyr Lys Phe Ser
485 490 495
Trp Asn Pro Ala Gly Ala Ile Arg Ala Gly Arg Asn Pro Ala Thr Tyr
500 505 510
Lys Trp Gly Gly Glu Thr Val His Ile Asp Gly Asp Asp Leu Tyr Asp
515 520 525
Ser Ala Thr Arg Leu Arg Leu Pro Asp Leu Pro Ala Phe Ala Leu Glu
530 535 540
Cys Leu Pro Asn Arg Asn Ser Leu Leu Tyr Gly Asp Leu Tyr Gly Ile
545 550 555 560
Thr Glu Ala Ser Thr Ile Phe Arg Gly Thr Leu Arg Tyr Glu Gly Phe
565 570 575
Ser Glu Ile Met Gly Thr Leu Ser Arg Ile Ser Leu Phe Asn Asn Glu
580 585 590
Ala His Ser Leu Leu Met Asn Gly Gln Arg Pro Thr Phe Lys Lys Phe
595 600 605
Leu Phe Glu Leu Leu Lys Val Val Gly Asp Asn Pro Asp Glu Leu Leu
610 615 620
Ile Gly Glu Asn Asp Ile Met Glu Gln Ile Leu Ile Gln Gly His Cys
625 630 635 640
Lys Asp Gln Arg Thr Ala Met Glu Thr Ala Lys Thr Ile Ile Phe Leu
645 650 655
Gly Leu Leu Asp Gln Thr Glu Ile Pro Ala Ser Cys Lys Ser Ala Phe
660 665 670
Asp Val Ala Cys Phe Arg Met Glu Glu Arg Leu Ser Tyr Thr Ser Thr
675 680 685
Glu Lys Asp Met Val Leu Leu His His Glu Val Glu Ile Glu Tyr Pro
690 695 700
Asp Ser Gln Ile Thr Glu Lys His Arg Ala Thr Leu Leu Glu Phe Gly
705 710 715 720
Lys Thr Leu Asp Glu Lys Thr Thr Thr Ala Met Ala Leu Thr Val Gly
725 730 735
Ile Pro Ala Ala Val Gly Ala Leu Leu Leu Leu Thr Asn Lys Ile Gln
740 745 750
188
CA 02280196 2000-06-02
Thr Arg Gly Val Leu Arg Pro Ile Glu Pro Glu Val Tyr Asn Pro Ala
755 760 765
Leu Asp Ile Ile Glu Ala Tyr Gly Ile Lys Leu Ile Glu Lys Thr Glu
770 775 780
(2) INFORMATION FOR SEQ ID NO:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1022 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:
Cys Ala Arg Leu Leu Leu Gly Gly Gly Lys Asn Gly Pro Arg Val Asn
1 5 10 15
Arg Ile Ile Val Gln Pro Ser Thr Arg Arg Ile His His Asp Ala Gln
20 25 30
Tyr Glu Asp Ala Gly Cys Glu Ile Ser Glu Asp Leu Ser Glu Cys Gly
35 40 45
Leu Ile Ile Gly Ile Lys Gln Pro Lys Leu Gln Met Ile Leu Ser Asp
50 55 60
Arg Ala Tyr Ala Phe Phe Ser His Thr His Lys Ala Gln Lys Glu Asn
65 70 75 80
Met Pro Leu Leu Asp Lys Ile Leu Glu Glu Arg Val Ser Leu Phe Asp
85 90 95
Tyr Glu Leu Ile Val Gly Asp Asp Gly Lys Arg Ser Leu Ala Phe Gly
100 105 110
Lys Phe Ala Gly Arg Ala Gly Leu Ile Asp Phe Leu His Gly Leu Gly
115 120 125
Gln Arg Tyr Leu Ser Leu Gly Tyr Ser Thr Pro Phe Leu Ser Leu Gly
130 135 140
Gln Ser His Met Tyr Pro Ser Leu Ala Ala Ala Lys Ala Ala Val Ile
145 150 155 160
Val Val Ala Glu Glu Ile Ala Thr Phe Gly Leu Pro Ser Gly Ile Cys
165 170 175
Pro Ile Val Phe Val Phe Thr Gly Val Gly Asn Val Ser Gln Gly Ala
180 185 190
Gln Glu Ile Phe Lys Leu Leu Pro His Thr Phe Val Asp Ala Glu Lys
195 200 205
Leu Pro Glu Ile Phe Gin Ala Arg Asn Leu Ser Lys Gln Ser Gln Ser
210 215 220
189
CA 02280196 2000-06-02
Thr Lys Arg Val Phe Gln Leu Tyr Gly Cys Val Val Thr Ser Arg Asp
225 230 235 240
Ile Val Ser His Lys Asp Pro Thr Arg Gln Phe Asp Lys Gly Asp Tyr
245 250 255
Tyr Ala His Pro Glu His Tyr Thr Pro Val Phe His Giu Arg Ile Ala
260 265 270
Pro Tyr Ala Ser Val Ile Val Asn Cys Met Tyr Trp Glu Lys Arg Phe
275 280 285
Pro Pro Leu Leu Asn Met Asp Gln Leu Gln Gln Leu Met Glu Thr Gly
290 295 300
Cys Pro Leu Val Gly Val Cys Asp Ile Thr Cys Asp Ile Gly Gly Ser
305 310 315 320
Ile Glu Phe Ile Asn Lys Ser Thr Ser Ile Glu Arg Pro Phe Phe Arg
325 330 335
Tyr Asp Pro Ser Lys Asn Ser Tyr His Asp Asp Met Glu Gly Ala Gly
340 345 350
Val Val Cys Leu Ala Val Asp Ile Leu Pro Thr Glu Phe Ser Lys Glu
355 360 365
Ala Ser Gln His Phe Gly Asn Ile Leu Her Arg Leu Val Ala Ser Leu
370 375 380
Ala Ser Val Lys Gln Pro Ala Glu Leu Pro Ser Tyr Leu Arg Arg Ala
385 390 395 400
Cys Ile Ala His Ala Gly Arg Leu Thr Pro Leu Tyr Glu Tyr Ile Pro
405 410 415
Arg Met Arg Asn Thr Met Ile Asp Leu Ala Pro Ala Lys Thr Asn Pro
420 425 430
Leu Pro Asp Lys Lys Tyr Ser Thr Leu Val Ser Leu Ser Gly His Leu
435 440 445
Phe Asp Lys Phe Leu Ile Asn Glu Ala Leu Asp Ile Ile Glu Thr Ala
450 455 460
Gly Gly Ser Phe His Leu Val Arg Cys Glu Val Gly Gln Ser Thr Asp
465 470 475 480
Asp Met Ser Tyr Ser Glu Leu Glu Val Gly Ala Asp Asp Thr Ala Thr
485 490 495
Leu Asp Lys Ile Ile Asp Ser Leu Thr Ser Leu Ala Asn Glu His Gly
500 505 510
Gly Asp His Asp Ala Gly Gln Glu Ile Glu Leu Ala Leu Lys Ile Gly
515 520 525
Lys Val Asn Glu Tyr Glu Thr Asp Val Thr Ile Asp Lys Gly Gly Pro
530 535 540
Lys Ile Leu Ile Leu Gly Ala Gly Arg Val Cys Arg Pro Ala Ala Glu
545 550 555 560
Phe Leu Ala Ser Tyr Pro Asp Ile Cys Thr Tyr Gly Val Asp Asp His
565 570 575
190
CA 02280196 2000-06-02
Asp Ala Asp Gln Ile His Val Ile Val Ala Ser Leu Tyr Gln Lys Asp
580 585 590
Ala Glu Glu Thr Val Asp Gly Ile Glu Asn Thr Thr Ala Thr Gln Leu
595 600 605
Asp Val Ala Asp Ile Gly Ser Leu Ser Asp Leu Val Ser Gln Val Glu
610 615 620
Val Val Ile Ser Leu Leu Pro Ala Ser Phe His Ala Ala Ile Ala Gly
625 630 635 640
Val Cys Ile Glu Leu Lys Lys His Met Val Thr Ala Ser Tyr Val Asp
645 650 655
Glu Ser Met Ser Asn Leu Ser Gln Ala Ala Lys Asp Ala Gly Val Thr
660 665 670
Ile Leu Cys Glu Met Gly Leu Asp Pro Gly Ile Asp His Leu Met Ser
675 680 685
Met Lys Met Ile Asp Glu Ala His Ala Arg Lys Gly Lys Ile Lys Ala
690 695 700
Phe Thr Ser Tyr Cys Gly Gly Leu Pro Ser Pro Ala Ala Ala Asn Asn
705 710 715 720
Pro Leu Ala Tyr Lys Phe Ser Trp Asn Pro Ala Gly Ala Leu Arg Ser
725 730 735
Gly Lys Asn Pro Ala Val Tyr Lys Phe Leu Gly Glu Thr Ile His Val
740 745 750
Asp Gly His Asn Leu Tyr Glu Ser Ala Lys Arg Leu Arg Leu Arg Glu
755 760 765
Leu Pro Ala Phe Ala Leu Glu His Leu Pro Asn Arg Asn Ser Leu Ile
770 775 780
Tyr Gly Asp Leu Tyr Gly Ile Ser Lys Glu Ala Ser Thr Ile Tyr Arg
785 790 795 800
Ala Thr Xaa Arg Tyr Glu Gly Phe Ser Glu Ile Met Val Thr Leu Ser
805 810 815
Lys Thr Gly Phe Phe Asp Ala Ala Asn His Pro Leu Leu Gln Asp Thr
820 825 830
Ser Arg Pro Thr Tyr Lys Gly Phe Leu Asp Glu Leu Leu Asn Asn Ile
835 840 845
Ser Thr Ile Asn Thr Asp Leu Asp Ile Glu Ala Ser Gly Gly Tyr Asp
850 855 860
Asp Asp Leu Ile Ala Arg Leu Leu Lys Leu Gly Cys Cys Lys Asn Lys
865 870 875 880
Glu Ile Ala Val Lys Thr Val Lys Thr Ile Lys Phe Leu Gly Leu His
885 890 895
Glu Glu Thr Gln Ile Pro Lys Gly Cys Ser Ser Pro Phe Asp Val Ile
900 905 910
191
CA 02280196 2000-06-02
Cys Gln Arg Met Glu Gln Arg Met Ala Tyr Gly His Asn Glu Gln Asp
915 920 925
Met Val Leu Leu His His Glu Val Glu Val Glu Tyr Pro Asp Gly Gln
930 935 940
Pro Ala Glu Lys His Gln Ala Thr Leu Leu Glu Phe Gly Lys Val Glu
945 950 955 960
Asn Gly Arg Ser Thr Thr Ala Met Ala Leu Thr Val Gly Ile Pro Ala
965 970 975
Ala Ile Gly Ala Leu Leu Leu Leu Lys Asn Lys Val Gln Thr Lys Gly
980 985 990
Val Ile Arg Pro Leu Gln Pro Glu Ile Tyr Val Pro Ala Leu Glu Ile
995 1000 1005
Leu Glu Ser Ser Gly Ile Lys Leu Val Glu Lys Val Glu Thr
1010 1015 1020
(2) INFORMATION FOR SEQ ID NO:123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1908 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..1908
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:
ATTGTGCCCG CCTTCTGCTA GGAGGAGGCA AGAACGGACC TCGAGTAAAC CGGATTATTG 60
TGCAGCCAAG CACAAGGAGG ATCCATCATG ACGCTCAGTA TGAGGATGCA GGATGCGAGA 120
TTTCAGAAGA CCTGTCAGAA TGCGGCCTTA TCATAGGCAT CAAACAACCC AAGCTGCAGA 180
TGATTCTTTC AGATAGAGCG TACGCTTTCT TTTCACACAC ACACAAAGCC CAAAAAGAGA 240
ATATGCCACT GTTAGACAAG ATCCTTGAAG AAAGGGTGTC CTTGTTTGAT TATGAGCTAA 300
TTGTTGGAGA TGATGGGAAA AGATCACTAG CATTTGGGAA ATTTGCTGGT AGAGCTGGAC 360
TGATAGATTT CTTACATGGT CTCGGACAGC GATATTTGAG CCTTGGATAC TCGACTCCAT 420
TTCTCTCTCT GGGACAATCT CATATGTATC CTTCGCTCGC TGCAGCCAAG GCTGCAGTCA 480
TTGTCGTTGC AGAAGAGATA GCAACATTTG GACTTCCATC CGGAATTTGT CCGATAGTGT 540
TTGTGTTCAC TGGAGTTGGA AACGTCTCTC AGGGTGCGCA GGAGATATTC AAGTTATTGC 600
192
CA 02280196 2000-06-02
CCCATACCTT TGTTGATGCT GAGAAGCTTC CCGAAATTTT TCAGGCCAGG AATCTGTCTA 660
AGCAATCTCA GTCGACCAAG AGAGTATTTC AACTTTATGG TTGTGTTGTG ACCTCTAGAG 720
ACATAGTTTC TCACAAGGAT CCCACCAGAC AATTTGACAA AGGTGACTAT TATGCTCATC 780
CAGAACACTA CACCCCTGTT TTTCATGAAA GAATTGCTCC ATATGCATCT GTCATCGTAA 840
ACTGTATGTA TTGGGAGAAG AGGTTTCCAC CATTACTAAA TATGGATCAG TTACAGCAAT 900
TGATGGAGAC TGGTTGTCCT TTAGTCGGCG TTTGTGACAT AACTTGTGAT ATTGGAGGTT 960
CCATTGAATT TATCAACAAG AGTACATCAA TAGAGAGGCC TTTCTTTCGG TATGATCCTT 1020
CTAAGAATTC ATACCATGAT GATATGGAAG GTGCCGGAGT GGTCTGCTTG GCTGTTGACA 1080
TTCTCCCTAC AGAATTCTCT AAAGAGGCCT CCCAACATTT TGGAAACATA CTATCTAGAC 1140
TTGTTGCTAG TTTGGCCTCA GTGAAGCAAC CGGCAGAACT TCCTTCCTAC TTGAGAAGAG 1200
CTTGCATTGC ACATGCTGGC AGATTAACTC CTTTGTATGA ATATATCCCT AGGATGAGAA 1260
ATACTATGAT AGATTTGGCA CCCGCAAAAA CAAATCCATT GCCTGACAAG AAGTATAGCA 1320
CCCTGGTATC TCTCAGTGGG CACCTATTTG ATAAGTTCCT TATAAATGAA GCTTTGGACA 1380
TCATTGAGAC AGCTGGAGGT TCATTTCACT TGGTTAGATG TGAAGTTGGA CAAAGCACGG 1440
ATGATATGTC ATACTCAGAG CTTGAAGTAG GAGCAGATGA TACTGCCACA TTGGATAAAA 1500
TTATTGATTC CTTGACTTCT TTAGCTAATG AACATGGTGG AGATCACGAT GCCGGGCAAG 1560
AAATTGAATT AGCTCTGAAG ATAGGAAAAG TCAATGAGTA TGAAACTGAC GTCACAATTG 1620
ATAAAGGAGG GCCAAAGATT TTAATTCTTG GAGCTGGAAG AGTCTGTCGG CCAGCTGCTG 1680
AGTTTCTGGC ATCTTACCCA GACATATGTA CCTATGGTGT TGATGACCAT GATGCAGATC 1740
AAATTCATGT TATCGTGGCA TCTTTGTATC AAAAAGATGC AGAAGAGACA GTTGATGGTA 1800
TTGAAAATAC AACTGCTACC CAGCTTGATG TTGCTGATAT TGGAAGCCTT TCAGATCTTG 1860
TTTCTCAGGT TGAGGTTGTA ATTAGCTTGC TGCCTGCTAG TTTTCATG 1908
(2) INFORMATION FOR SEQ ID NO:124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 640 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays
193
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:
Cys Ala Arg Leu Leu Leu Gly Gly Gly Lys Asn Gly Pro Arg Val Asn
1 5 10 15
Arg Ile Ile Val Gln Pro Ser Thr Arg Arg Ile His His Asp Ala Gln
20 25 30
Tyr Glu Asp Ala Gly Cys Glu Ile Ser Glu Asp Leu Ser Glu Cys Gly
35 40 45
Leu Ile Ile Gly Ile Lys Gln Pro Lys Leu Gln Met Ile Leu Ser Asp
50 55 60
Arg Ala Tyr Ala Phe Phe Ser His Thr His Lys Ala Gln Lys Glu Asn
65 70 75 80
Met Pro Leu Leu Asp Lys Ile Leu Glu Glu Arg Val Ser Leu Phe Asp
85 90 95
Tyr Glu Leu Ile Val Gly Asp Asp Gly Lys Arg Ser Leu Ala Phe Gly
100 105 110
Lys Phe Ala Gly Arg Ala Gly Leu Ile Asp Phe Leu His Gly Leu Gly
115 120 125
Gln Arg Tyr Leu Ser Leu Gly Tyr Ser Thr Pro Phe Leu Ser Leu Gly
130 135 140
Gln Ser His Met Tyr Pro Ser Leu Ala Ala Ala Lys Ala Ala Val Ile
145 150 155 160
Val Val Ala Glu Glu Ile Ala Thr Phe Gly Leu Pro Ser Gly Ile Cys
165 170 175
Pro Ile Val Phe Val Phe Thr Gly Val Gly Asn Val Ser Gln Giy Ala
180 185 190
Gln Glu Ile Phe Lys Leu Leu Pro His Thr Phe Val Asp Ala Glu Lys
195 200 205
Leu Pro Glu Ile Phe Gin Ala Arg Asn Leu Ser Lys Gln Ser Gln Ser
210 215 220
Thr Lys Arg Val Phe Gln Leu Tyr Gly Cys Val Val Thr Ser Arg Asp
225 230 235 240
Ile Val Ser His Lys Asp Pro Thr Arg Gln Phe Asp Lys Gly Asp Tyr
245 250 255
Tyr Ala His Pro Glu His Tyr Thr Pro Val Phe His Glu Arg Ile Ala
260 265 270
Pro Tyr Ala Ser Val Ile Val Asn Cys Met Tyr Trp Glu Lys Arg Phe
275 280 285
Pro Pro Leu Leu Asn Met Asp Gln Leu Gln Gln Leu Met Glu Thr Gly
290 295 300
Cys Pro Leu Val Gly Val Cys Asp Ile Thr Cys Asp Ile Gly Gly Ser
305 310 315 320
Ile Glu Phe Ile Asn Lys Ser Thr Ser Ile Giu Arg Pro Phe Phe Arg
325 330 335
194
CA 02280196 2000-06-02
Tyr Asp Pro Ser Lys Asn Ser Tyr His Asp Asp Met Glu Gly Ala Gly
340 345 350
Val Val Cys Leu Ala Val Asp Ile Leu Pro Thr Glu Phe Ser Lys Glu
355 360 365
Ala Ser Gln His Phe Gly Asn Ile Leu Ser Arg Leu Val Ala Ser Leu
370 375 380
Ala Ser Val Lys Gln Pro Ala Glu Leu Pro Ser Tyr Leu Arg Arg Ala
385 390 395 400
Cys Ile Ala His Ala Gly Arg Leu Thr Pro Leu Tyr Glu Tyr Ile Pro
405 410 415
Arg Met Arg Asn Thr Met Ile Asp Leu Ala Pro Ala Lys Thr Asn Pro
420 425 430
Leu Pro Asp Lys Lys Tyr Ser Thr Leu Val Ser Leu Ser Gly His Leu
435 440 445
Phe Asp Lys Phe Leu Ile Asn Glu Ala Leu Asp Ile Ile Glu Thr Ala
450 455 460
Gly Gly Ser Phe His Leu Val Arg Cys Glu Val Gly Gln Ser Thr Asp
465 470 475 480
Asp Met Ser Tyr Ser Glu Leu Glu Val Gly Ala Asp Asp Thr Ala Thr
485 490 495
Leu Asp Lys Ile Ile Asp Ser Leu Thr Ser Leu Ala Asn Glu His Gly
500 505 510
Gly Asp His Asp Ala Gly Gln Glu Ile Glu Leu Ala Leu Lys Ile Gly
515 520 525
Lys Val Asn Glu Tyr Glu Thr Asp Val Thr Ile Asp Lys Gly Gly Pro
530 535 540
Lys Ile Leu Ile Leu Gly Ala Gly Arg Val Cys Arg Pro Ala Ala Glu
545 550 555 560
Phe Leu Ala Ser Tyr Pro Asp Ile Cys Thr Tyr Gly Val Asp Asp His
565 570 575
Asp Ala Asp Gln Ile His Val Ile Val Ala Ser Leu Tyr Gln Lys Asp
580 585 590
Ala Glu Glu Thr Val Asp Gly Ile Glu Asn Thr Thr Ala Thr Gln Leu
595 600 605
Asp Val Ala Asp Ile Gly Ser Leu Ser Asp Leu Val Ser Gln Val Glu
610 615 620
Val Val Ile Ser Leu Leu Pro Ala Ser Phe His Ala Ala Ile Ala Gly
625 630 635 640
(2) INFORMATION FOR SEQ ID NO:125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 720 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
195
CA 02280196 2000-06-02
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..720
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 215
(D) OTHER INFORMATION: /label= unknown
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 678
(D) OTHER INFORMATION: /label= unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:
GTTTAAACAT CTTTCCAATC TTGTTTCTCA GGTTGAAGTA GTAGTTAGCT TGCTGCCTGC 60
CAGTTTTCAT GCTGCCATAG CAAGAGTATG CATAGAGATG AAGAAGCACT TGGTCACTGC 120
AAGCTATGTT GATGAGTCCA TGTCAAAGTT GGAACAATCT GCAGAAGGTG CTGGTGTAAC 180
TATTCTCTGT GAAATGGGCC TGGATCCTGG CATANATCAT ATGATGTCAA TGAAGATGAT 240
TGACGAAGCA CATTCACGGA AGGGGAAAAT AAAGTCATTT ACATCCTTTT GTGGAGGACT 300
TCCATCTCCA GCTTCTGCAA ACAATCCACT TGCTTATAAG TTCAGTTGGA GTCCAGCTGG 360
TGCCATCCGT GCAGGGAGAA ACCCTGCTGT CTACAAATTT CATGGAGAAA TCATCCATGT 420
AGATGGTGAT AAATTGTATG AATCCGCAAA GAGGCTCAGA TTACMAGAAC TTCCAGCTTT 480
TGCACTGGAA CACTTGCCAA ACCGGAATTC CTTGATGTAT GGAGACCTGT ATGGGATCTC 540
CAAAGAAGCA TCTACTGTGT ACAGGGCTAC TCTTCGTTAT GAAGGATTTA ATGAGATAAT 600
GGCAACCTTC GCGAAAATTG GGTTTTTTGA TGCTGCAAGT CATCCACTGT TGCAACAAAC 660
TACTCGCCCT ACATACANGG ATTTCCTGTT GAACCCTCAA TGCTTGTACA TCTCCAAAAC 720
(2) INFORMATION FOR SEQ ID NO:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 239 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
196
CA 02280196 2000-06-02
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:
Phe Lys His Leu Ser Asn Leu Val Ser Gln Val Glu Val Val Val Ser
1 5 10 15
Leu Leu Pro Ala Ser Phe His Ala Ala Ile Ala Arg Val Cys Ile Glu
20 25 30
Met Lys Lys His Leu Val Thr Ala Ser Tyr Val Asp Glu Ser Met Ser
35 40 45
Lys Leu Glu Gln Ser Ala Glu Gly Ala Gly Val Thr Ile Leu Cys Glu
50 55 60
Met Gly Leu Asp Pro Gly Ile Xaa His Met Met Ser Met Lys Met Ile
65 70 75 80
Asp Glu Ala His Ser Arg Lys Gly Lys Ile Lys Ser Phe Thr Ser Phe
85 90 95
Cys Gly Gly Leu Pro Ser Pro Ala Ser Ala Asn Asn Pro Leu Ala Tyr
100 105 110
Lys Phe Ser Trp Ser Pro Ala Gly Ala Ile Arg Ala Gly Arg Asn Pro
115 120 125
Ala Val Tyr Lys Phe His Gly Glu Ile Ile His Val Asp Gly Asp Lys
130 135 140
Leu Tyr Glu Ser Ala Lys Arg Leu Arg Leu Xaa Glu Leu Pro Ala Phe
145 150 155 160
Ala Leu Glu His Leu Pro Asn Arg Asn Ser Leu Met Tyr Gly Asp Leu
165 170 175
Tyr Gly Ile Ser Lys Glu Ala Ser Thr Val Tyr Arg Ala Thr Leu Arg
180 185 190
Tyr Glu Gly Phe Asn Glu Ile Met Ala Thr Phe Ala Lys Ile Gly Phe
195 200 205
Phe Asp Ala Ala Ser His Pro Leu Leu Gln Gln Thr Thr Arg Pro Thr
210 215 220
Tyr Xaa Asp Phe Leu Leu Asn Pro Gln Cys Leu Tyr Ile Ser Lys
225 230 235
(2) INFORMATION FOR SEQ ID NO:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 308 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
197
CA 02280196 2000-06-02
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..129
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:
CTGCTGTTGC TCCAGAACAA GATCCAAAAG AAAGGAGTGA TCAGGCCTCT GGAACCTGAA 60
ATTTACATTC CAGCGTTGGA GATCTTGGAG TCATCGGGTA TCAAGCTGGC GGAGAGAGTG 120
GAGACCTGAG AATCGGACCC AATATGTATA ATGTAGCATG GTGGTAGCTT CTCTATATAT 180
ATGCTTCAGT GAATAATTGA TTTGCCGTTG TGTGGTAATT AAGCAATGCC CGCTAATAAA 240
TTGTACCGTA GAAGTCCTTC TATGTACATC CGTATCAAAA AATAAAAAAA GCATCGATTA 300
GCTTGAAT 308
(2) INFORMATION FOR SEQ ID NO:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:
Leu Leu Leu Leu Gln Asn Lys Ile Gln Lys Lys Gly Val Ile Arg Pro
1 5 10 15
Leu Glu Pro Glu Ile Tyr Ile Pro Ala Leu Glu Ile Leu Glu Ser Ser
20 25 30
Gly Ile Lys Leu Ala Glu Arg Val Glu Thr
35 40
(2) INFORMATION FOR SEQ ID NO:129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 429 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
198
CA 02280196 2000-06-02
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..252
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 172
(D) OTHER INFORMATION: /label= unknown
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 186
(D) OTHER INFORMATION: /label= unknown
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 331
(D) OTHER INFORMATION: /label= unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:
TACCCCGACG GGGACCCCAC CGAGAAGCAC CAAGCGACGC TGCTGGAGTT CGGAAAGACC 60
GAGAACGGCA GGCCCACCAC CGCCATGGCC CTCACCGTTG GGGTACCGGC AGCGATAGGA 120
GCCCTGCTCT TGCTCCAGAA CAAGGTCCAG AGGAAAGGGG TGATCCGGCC TNTGGAACCG 180
GAGATNTACA TCCCTGCGCT GGAGATCTTG GAAGCGTCGG GCATCAAGCT GATCGAGAGA 240
GTGGAGACCT GAGGATGTCA GGATGGGATG AGAATCTATC GAGTATATAT GCTGCAGCAA 300
CAGAGGCAGT GAGTAAATAA AATGATGATT NTCGCCGTTG TAAGTAAAAT GAGTGGACTG 360
TATGTATGTA TGTGACTATC TATTGTACTA CATATATACC AAATCTGTCG CCGGTTGATT 420
CTGTTGGTG 429
(2) INFORMATION FOR SEQ ID NO:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:
Tyr Pro Asp Gly Asp Pro Thr Glu Lys His Gln Ala Thr Leu Leu Glu
1 5 10 15
Phe Gly Lys Thr Glu Asn Gly Arg Pro Thr Thr Ala Met Ala Leu Thr
20 25 30
199
CA 02280196 2000-06-02
Val Gly Val Pro Ala Ala Ile Gly Ala Leu Leu Leu Leu Gln Asn Lys
35 40 45
Val Gln Arg Lys Gly Val Ile Arg Pro Xaa Glu Pro Glu Xaa Tyr Ile
50 55 60
Pro Ala Leu Glu Ile Leu Glu Ala Ser Gly Ile Lys Leu Ile Glu Arg
65 70 75 80
Val Glu Thr
(2) INFORMATION FOR SEQ ID NO:131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:
ATGACGAAAA AATCAGGTGT TTTGATTCTT GGTGCTGGAC GTGTGTGTCG CCCAGCTGCT 60
GATTTCCTAG CTTCAGTTAG AACCATTTCG TCACAGCAAT GGTACAAAAC ATATTTCGGA 120
GCAGACTCTG AAGAGAAAAC AGATGTTCAT GTGATTGTCG CGTCTCTGTA TCTTAAGGAT 180
GCCAAAGAGA CGGTTGAAGG TATTTCAGAT GTAGAAGCAG TTCGGCTAGA TGTATCTGAT 240
AGTGAAAGTC TCCTTAAGTA TGTTTCTCAG GTTGATGTTG TCCTAAGTTT ATTACCTGCA 300
AGTTGTCATG CTGTTGTAGC AAAGACATGC ATTGAGCTGA AGAAGCATCT CGTCACTGCT 360
AGCTATGTTG ATGATGAAAC GTCCATGTTA CATGAGAAGG CTAAGAGTGC TGGGATAACG 420
ATTCTAGGCG AAATGGGACT GGACCCTGGA ATCGATCACA TGATGGCGAT GAAAATGATC 480
AACGATGCTC ATATCAAAAA AGGGAAAGTG AAGTCTTTTA CCTCTTATTG TGGAGGGCTT 540
CCCTCTCCTG CTGCAGCAAA TAATCCATTA GCATATAAAT TTAGCTGGAA CCCTGCTGGA 600
GCAATTCGAG CTGGTCAAAA CCCCGCCAAA TACAAAAGCA ACGGCGACAT AATACATGTT 660
GATGGGAAGA ATCTCTATGA TTCCGCGGCA AGATTCCGAG TACCTAATCT TCCAGCTTTT 720
GCATTGGAGT GTTTTCCAAA TCGTGACTCC TTGGTTTACG GGGAACATTA TGGCATCGAG 780
AGCGAAGCAA CAACGATATT TCGTGGAACA CTCAGATATG AAGGGTTTAG TATGATAATG 840
GCAACACTTT CGAAACTTGG ATTCTTTGAC AGTGAAGCAA ATCAAGTACT CTCCACTGGA 900
AAGAGGATTA CGTTTGGTGC TCTTTTAAGT AACATTCTAA ATAAGGATGC AGACAATGAA 960
TCAGAGCCCC TAGCGGGAGA AGAAGAGATA AGCAAGAGAA TTATCAAGCT TGGACATTCC 1020
AAGGAGACTG CAGCCAAAGC TGCCAAAACA ATTGTATTCT TGGGGTTCAA CGAAGAGAGG 1080
200
CA 02280196 2000-06-02
GAGGTTCCAT CACTGTGTAA AAGCGTATTT GATGCAACTT GTTACCTAAT GGAAGAGAAA 1140
CTAGCTTATT CCGGAAATGA ACAGGACATG GTGCTTTTGC ATCACGAAGT AGAAGTGGAA 1200
TTCCTTGAAA GCAAACGTAT AGAGAAGCAC ACTGCGACTC TTTTGGAATT CGGGGACATC 1260
AAGAATGGAC AAACAACAAC CGCTATGGCC AAGACTGTTG GGATCCCTGC AGCCATTGGA 1320
GCTCTGGTGT TAATTGAAGA CAAGATCAAG ACAAGAGGAG TCTTAAGGCC TCTCGAAGCA 1380
GAGGTGTATT TGCCAGCTTT GGATATATTG CAAGCATATG GTATAAAGCT GATGGAGAAG 1440
GCAGAATGA 1449
(2) INFORMATION FOR SEQ ID NO:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 482 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:
Met Thr Lys Lys Ser Gly Val Leu Ile Leu Gly Ala Gly Arg Val Cys
1 5 10 15
Arg Pro Ala Ala Asp Phe Leu Ala Ser Val Arg Thr Ile Ser Ser Gln
20 25 30
Gln Trp Tyr Lys Thr Tyr Phe Gly Ala Asp Ser Glu Glu Lys Thr Asp
35 40 45
Val His Val Ile Val Ala Ser Leu Tyr Leu Lys Asp Ala Lys Glu Thr
50 55 60
Val Glu Gly Ile Ser Asp Val Glu Ala Val Arg Leu Asp Val Ser Asp
65 70 75 80
Ser Glu Ser Leu Leu Lys Tyr Val Ser Gln Val Asp Val Val Leu Ser
85 90 95
Leu Leu Pro Ala Ser Cys His Ala Val Val Ala Lys Thr Cys Ile Glu
100 105 110
Leu Lys Lys His Leu Val Thr Ala Ser Tyr Val Asp Asp Glu Thr Ser
115 120 125
Met Leu His Glu Lys Ala Lys Ser Ala Gly Ile Thr Ile Leu Gly Glu
130 135 140
Met Gly Leu Asp Pro Gly Ile Asp His Met Met Ala Met Lys Met Ile
145 150 155 160
201
CA 02280196 2000-06-02
Asn Asp Ala His Ile Lys Lys Gly Lys Val Lys Ser Phe Thr Ser Tyr
165 170 175
Cys Gly Gly Leu Pro Ser Pro Ala Ala Ala Asn Asn Pro Leu Ala Tyr
180 185 190
Lys Phe Ser Trp Asn Pro Ala Gly Ala Ile Arg Ala Gly Gln Asn Pro
195 200 205
Ala Lys Tyr Lys Ser Asn Gly Asp Ile Ile His Val Asp Gly Lys Asn
210 215 220
Leu Tyr Asp Ser Ala Ala Arg Phe Arg Val Pro Asn Leu Pro Ala Phe
225 230 235 240
Ala Leu Glu Cys Phe Pro Asn Arg Asp Ser Leu Val Tyr Gly Glu His
245 250 255
Tyr Gly Ile Glu Ser Glu Ala Thr Thr Ile Phe Arg Gly Thr Leu Arg
260 265 270
Tyr Glu Gly Phe Ser Met Ile Met Ala Thr Leu Ser Lys Leu Gly Phe
275 280 285
Phe Asp Ser Glu Ala Asn Gln Val Leu Ser Thr Gly Lys Arg Ile Thr
290 295 300
Phe Gly Ala Leu Leu Ser Asn Ile Leu Asn Lys Asp Ala Asp Asn Glu
305 310 315 320
Ser Glu Pro Leu Ala Gly Glu Glu Glu Ile Ser Lys Arg Ile Ile Lys
325 330 335
Leu Gly His Ser Lys Glu Thr Ala Ala Lys Ala Ala Lys Thr Ile Val
340 345 350
Phe Leu Gly Phe Asn Glu Glu Arg Glu Val Pro Ser Leu Cys Lys Ser
355 360 365
Val Phe Asp Ala Thr Cys Tyr Leu Met Glu Glu Lys Leu Ala Tyr Ser
370 375 380
Gly Asn Glu Gln Asp Met Val Leu Leu His His Glu Val Glu Val Glu
385 390 395 400
Phe Leu Glu Ser Lys Arg Ile Glu Lys His Thr Ala Thr Leu Leu Glu
405 410 415
Phe Gly Asp Ile Lys Asn Gly Gln Thr Thr Thr Ala Met Ala Lys Thr
420 425 430
Val Gly Ile Pro Ala Ala Ile Gly Ala Leu Val Leu Ile Glu Asp Lys
435 440 445
Ile Lys Thr Arg Gly Val Leu Arg Pro Leu Glu Ala Glu Val Tyr Leu
450 455 460
202
CA 02280196 2000-06-02
Pro Ala Leu Asp Ile Leu Gln Ala Tyr Gly Ile Lys Leu Met Glu Lys
465 470 475 480
Ala Glu
203
