Note: Descriptions are shown in the official language in which they were submitted.
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NUCLEIC ACID SEQUENCES ENCODING POLYENOIC
FATTY ACID ISOMERASE AND USES THEREOF
Cross-Reference to Related Applications
This application claims priority from U.S. provisional patent application Ser.
No.
60/146,458 filed July 30, 1999, which is hereby incorporated by reference in
its entirety.
Technical Field
The present invention is directed to nucleic acid and amino acid sequences and
constructs, and methods related thereto.
Background
Novel vegetable oils compositions and/or improved means to obtain or
manipulate
fatty acid compositions, from biosynthetic or natural plant sources, are
needed.
Depending upon the intended oil use, various different oil compositions are
desired. For
example, edible oil sources containing the minimum possible amounts of
saturated fatty
acids are desired for dietary reasons and alternatives to current sources of
highly saturated
1 S oil products, such as tropical oils, are also needed. Furthermore, oils
compositions
containing rare or exotic fatty acid species having nutritional benefits are
also needed in
the art.
Conjugated fatty acids, such as conjugated linoleic acid (CLA), are gaining
recognition for their health benefits in animal feed and in human nutrition.
Conjugated
fatty acid is a general term for fatty acids containing double bonds
alternating with single
bonds. For example, conjugated linoleic acid refers to a series of positional
and geometric
isomers of linoleic acid (an 18 carbon molecule that contains double bonds in
the cis-9 and
cis-12 positions).
Of the various isomers of CLA, the cis-9, trans-11 and trans-10, cis-12
isomers
have received the most attention. Recent data suggests (Parks, et al. (1999),
Lipids, 34:
235-243) that the trans-10, cis-12 is the biologically active form. However,
it is
recognized that other CLA isomers, and/or other conjugated fatty acids, may
also be
shown to have biological activities.
CLA is now recognized as a nutritional supplement and an effective inhibitor
of
epidermal carcinogenesis and forestomach neoplasia in mice, and of carcinogen-
induced
rat mammary and colon tumors. Furthermore, CLA has been shown to reduce LDL
and
atherosclerosis in hamsters and rabbits, reduce body fat and increase lean
body mass in
chickens, swine, rats and mice, increase feed efficiency in chickens and
swine, reduce
serum PGE2 in rats, increase bone mass in mice and chickens, as well as
reducing weight
loss during immune challenge in mice, chickens and rats.
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Thus, the identification of enzyme targets and sources for nucleic acid
sequences of
such enzyme targets capable of producing conjugated fatty acids in host cells
is needed in
the art. Ultimately, useful nucleic acid constructs having the necessary
elements to provide
a phenotypic modification and host cells containing such constructs are
needed.
Summary of the Invention
The present invention is directed to polyenoic fatty acid isomerases (PFI),
and in
particular to PFI polypeptides and polynucleotides. The polypeptides and
polynucleotides
of the present invention include those derived from plant and fungal sources.
In another aspect of the invention, polynucleotides encoding novel
polypeptides,
particularly, polynucleotides that encode PFI, are provided.
In a further aspect, the invention relates to oligonucleotides derived from
the PFI
proteins and oligonucleotides which include partial or complete PFI encoding
sequences.
It is also an aspect of the present invention to provide recombinant DNA
constructs
which can be used for either transcription and/or expression of PFI. In
particular,
constructs are provided which are capable of both transcription or and/or in
host cells.
Particularly preferred constructs are those capable of both transcription
and/or expression
in plant cells.
In yet another aspect of the present invention, methods are provided for
production
of PFI in a host cell or progeny thereof. In particular, host cells are
transformed or
transfected with a DNA construct which can be used for transcription and/or
expression of
PFI. The recombinant cells which contain PFI are also part of the present
invention.
In a further aspect, the present invention relates to methods of using
polynucleotide
and polypeptide sequences to modify the fatty acid composition in a host cell,
particularly
in seed oil of oilseed crops. In particular, the modified fatty acid
composition comprises
an altered amount of conjugated fatty acids. Plant cells having such a
modified fatty acids
are also contemplated herein.
The modified plants, seeds and oils obtained by the expression of the plant
PFI
proteins are also considered part of the invention.
Detailed description of the Invention
In accordance with the subject invention, nucleotide sequences are provided
that
code for a protein, polypeptide or peptide, which are active in the formation
of conjugated
fatty acids from polyenoic fatty acid substrates. Such sequences are referred
to herein as
polyenoic fatty acid isomerases (also referred to as PFI). The novel nucleic
acid sequences
find use in the preparation of constructs to direct their expression in a host
cell.
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Furthermore, the novel nucleic acid sequences find use in the preparation of
plant
expression constructs to modify the fatty acid composition of a plant cell.
A polyenoic fatty acid isomerase nucleic acid sequence of this invention
includes
any nucleic acid sequence that codes for a protein, polypeptide, or peptide
fragment,
obtainable from a source which is active in the formation of conjugated fatty
acids from a
polyunsaturated fatty acid substrate in a plant host cell, i.e., in vivo, or
in a plant cell-like
environment, i.e. in vitro. As used herein, "conjugated" refers to the
interaction of the pi
electron systems when the carbon chain contains alternating double and single
bonds
(C=C-C=C) such that the electrons of the double bonds are close enough to
interact with
each other. This is in contrast to "isolated" double bonds where the pi
electron systems are
separated by a saturated carbon (C=C-C-C=C) or "cumulated" where the double
bonds
share a central carbon (C=C=C). "A plant cell-like environment" means that any
necessary conditions are available in said environment (i.e., such factors as
temperatures,
pH, lack of inhibiting substances) which will permit the enzyme to function.
The fatty acids used as substrates by the protein encoded by the
polynucleotide
sequence of the present invention include any polyunsaturated fatty acid
substrate. Such
fatty acid substrates include, but are not limited to dimes, trienes,
tetraenes, pentaenes and
hexaenes. Fatty acid substrates of particular interest in the present
invention include, but
are not limited to, linoleic acid, linolenic acid, stearidonic,
eicosapentaenoic acid, dihomo-
y-linolenic acid, adrenic acid, eicosatrienonic acid, a-linolenic acid,
docosahexaenoic acid
and arachidonic acid.
Isolated proteins, Polypeptides and Polynucleotides
A first aspect of the present invention relates to isolated PFI polypeptides.
Such
polypeptides include isolated polypeptides set forth in the Sequence Listing,
as well as
polypeptides and fragments thereof, particularly those polypeptides which
exhibit PFI
activity and also those polypeptides which have approximately at least 50-79%
identity,
more preferably approximately at least 80% identity, even more preferably
approximately
at least 90% identity, and most preferably approximately at least 95% identity
to a
polypeptide sequence selected from the group of sequences set forth in the
Sequence
Listing, and also includes portions of such polypeptides, wherein such portion
of the
polypeptide preferably include at least 30 amino acids and more preferably
include at least
50 amino acids.
"Identity", as is well understood in the art, is a relationship between two or
more
polypeptide sequences or two or more polynucleotide sequences, as determined
by
comparing the sequences. In the art, "identity" also means the degree of
sequence
relatedness between polypeptide or polynucleotide sequences, as determined by
the match
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between strings of such sequences. "Identity" can be readily calculated by
known
methods including, but not limited to, those described in Computational
Molecular
Biology, Lesk, A.M., ed., Oxford University Press, New York (1988);
Biocomputing:
Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York,
1993;
S Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G.,
eds.,
Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von
Heinje,
G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and
Devereux, J.,
eds., Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM
JApplied
Math, 48:1073 (1988). Methods to determine identity are designed to give the
largest
match between the sequences tested. Moreover, methods to determine identity
are
codified in publicly available programs. Computer programs which can be used
to
determine identity between two sequences include, but are not limited to, GCG
(Devereux,
J., et al., Nucleic Acids Research 12(1):387 (1984); suite of five BLAST
programs, three
designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and
two
designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in
Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, l: 543-559
(1997)).
The BLAST X program is publicly available from NCBI and other sources (BLAST
Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, MD 20894; Altschul, S.,
et al., J.
Mol. Biol., 215:403-410 (1990)). The well known Smith Waterman algorithm can
also be
used to determine identity.
Parameters for polypeptide sequence comparison typically include the
following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.
Acad. Sci USA 89:10915-10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4
A program which can be used with these parameters is publicly available as the
"gap" program from Genetics Computer Group, Madison Wisconsin. The above
parameters along with no penalty for end gap are the default parameters for
peptide
comparisons.
Parameters for polynucleotide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)
Comparison matrix: matches = +10; mismatches = 0
Gap Penalty: SO
Gap Length Penalty: 3
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A program which can be used with these parameters is publicly available as the
"gap" program from Genetics Computer Group, Madison Wisconsin. The above
parameters are the default parameters for nucleic acid comparisons.
The invention also includes polypeptides of the formula:
X-(R~)n-(Rz)-(R3)n1'
wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y
is
hydrogen or a metal, R, and R3 are any amino acid residue, n is an integer
between 1 and
1000, and Rz is an amino acid sequence of the invention, particularly an amino
acid
sequence selected from the group set forth in the Sequence Listing and
preferably SEQ ID
NOs: 2 and 4. In the formula, Rz is oriented so that its amino terminal
residue is at the
left, bound to Rl, and its carboxy terminal residue is at the right, bound to
R3 Any stretch
of amino acid residues denoted by either R group, where R is greater than 1,
may be either
a heteropolymer or a homopolymer, preferably a heteropolymer.
Polypeptides of the present invention include isolated polypeptides encoded by
a
polynucleotide comprising a sequence selected from the group of a sequence
contained in
SEQ ID NOs: 1 and 3 .
Polypeptides of the present invention have been shown to have PFI activity and
are
of interest because PFI is involved in the production of conjugated fatty
acids from
polyenoic fatty acyl substrate molecules.
The polypeptides of the present invention can be a mature protein or can be
part of
a fusion protein.
Fragments and variants of the polypeptides are also considered to be a part of
the
invention. A fragment is a variant polypeptide which has an amino acid
sequence that is
entirely the same as part but not all of the amino acid sequence of the
previously described
polypeptides. The fragments can be "free-standing" or comprised within a
larger
polypeptide of which the fragment forms a part or a region, most preferably as
a single
continuous region. Preferred fragments are biologically active fragments which
are those
fragments that mediate activities of the polypeptides of the invention,
including those with
similar activity or improved activity or with a decreased activity. Also
included are those
fragments that are antigenic or immunogenic in an animal, particularly a
human.
Variants of the polypeptide also include polypeptides that vary from the
sequences
set forth in the Sequence Listing by conservative amino acid substitutions,
which are
substitution of a residue by another residue with like characteristics and/or
properties. In
general, such substitutions are between Ala, Val, Leu and Ile; between Ser and
Thr;
between Asp and Glu; between Asn and Gln; between Lys and Arg; or between Phe
and
Tyr. Particularly preferred are variants in which 5 to 10; 1 to 5; 1 to 3 or
one amino
acids) are substituted, deleted, or added, in any combination.
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Variants that are fragments of the polypeptides of the invention can be used
to
produce the corresponding full length polypeptide by peptide synthesis.
Therefore, these
variants can be used as intermediates for producing the full-length
polypeptides of the
invention.
Another aspect of the present invention relates to isolated PFI
polynucleotides.
The polynucleotide sequences of the present invention include isolated
polynucleotides
that encode the polypeptides of the invention having a deduced amino acid
sequence
selected from the group of sequences set forth in the Sequence Listing and to
other
polynucleotide sequences closely related to such sequences and variants
thereof.
The invention also provides a polynucleotide sequence identical over its
entire
length to each coding sequence as set forth in the Sequence Listing. The
invention also
provides the coding sequence for the mature polypeptide or a fragment thereof,
as well as
the coding sequence for the mature polypeptide or a fragment thereof in a
reading frame
with other coding sequences, such as those encoding a leader or secretory
sequence, a pre-,
pro-, or prepro- protein sequence. The polynucleotide can also include non-
coding
sequences, including for example, but not limited to, non-coding 5' and 3'
sequences, such
as the transcribed, untranslated sequences, termination signals, ribosome
binding sites,
sequences that stabilize mRNA, introns, polyadenylation signals, and
additional coding
sequence that encodes additional amino acids. For example, a marker sequence
can be
included to facilitate the purification of the fused polypeptide.
Polynucleotides of the
present invention also include polynucleotides comprising a structural gene
and the
naturally associated sequences that control gene expression.
The invention also includes polynucleotides of the formula:
X'R~)n'Rz'R3)n Y
wherein, at the 5' end, X is hydrogen, and at the 3' end, Y is hydrogen or a
metal, R, and
R3 are any nucleic acid residue, n is an integer between 1 and 3000,
preferably between 1
and 1000 and RZ is a nucleic acid sequence of the invention, particularly a
nucleic acid
sequence selected from the group set forth in the Sequence Listing and
preferably SEQ ID
NOs: 1 and 3. In the formula, Rz is oriented so that its 5' end residue is at
the left, bound
to RI, and its 3' end residue is at the right, bound to R3. Any stretch of
nucleic acid
residues denoted by either R group, where R is greater than l, may be either a
heteropolymer or a homopolymer, preferably a heteropolymer.
The invention also relates to variants of the polynucleotides described herein
that
encode for variants of the polypeptides of the invention. Variants that are
fragments of the
polynucleotides of the invention can be used to synthesize full-length
polynucleotides of
the invention. Preferred embodiments are polynucleotides encoding polypeptide
variants
wherein 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues of a
polypeptide sequence of
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the invention are substituted, added or deleted, in any combination.
Particularly preferred
are substitutions, additions, and deletions that are silent such that they do
not alter the
properties or activities of the polynucleotide or polypeptide.
Nucleotide sequences encoding polyenoic fatty acid isomerases may be obtained
from natural sources or be partially or wholly artificially synthesized. They
may directly
correspond to a polyenoic fatty acid isomerase endogenous to a natural source
or contain
modified amino acid sequences, such as sequences which have been mutated,
truncated,
increased or the like. Polyenoic fatty acid isomerases may be obtained by a
variety of
methods, including but not limited to, partial or homogenous purification of
protein
extracts, protein modeling, nucleic acid probes, antibody preparations and
sequence
comparisons. Typically a polyenoic fatty acid isomerase will be derived in
whole or in
part from a natural source. A natural source includes, but is not limited to,
prokaryotic and
eukaryotic sources, including, bacteria, yeasts, plants, including algae, and
the like.
Of special interest are polyenoic fatty acid isomerases which are obtainable
from
algae sources, including those which are obtained, from Ptilota, Bossiella,
Lithotham, for
example P. filicina, or from polyenoic fatty acid isomerases which are
obtainable through
the use of these sequences. "Obtainable" refers to those polyenoic fatty acid
isomerases
which have sufficiently similar sequences to that of the sequences provided
herein to
provide a biologically active polyenoic fatty acid isomerase.
Further preferred embodiments of the invention that are approximately at least
50-79% identical over their entire length to a polynucleotide encoding a
polypeptide of the
invention, and polynucleotides that are complementary to such polynucleotides.
More
preferable are polynucleotides that comprise a region that is approximately at
least 80%
identical over its entire length to a polynucleotide encoding a polypeptide of
the invention
and polynucleotides that are complementary thereto. Polynucleotides
approximately at
least 90% identical over their entire length are particularly preferred, those
approximately
at least 95% identical are especially preferred. Further, those with
approximately at least
97% identity are highly preferred and those with approximately at least 98%
and 99%
identity are particularly highly preferred, with those approximately at least
99% being the
most highly preferred.
Preferred embodiments are polynucleotides that encode polypeptides that retain
substantially the same biological function or activity as the mature
polypeptides encoded
by the polynucleotides set forth in the Sequence Listing.
The invention further relates to polynucleotides that hybridize to the above-
3~ described sequences. In particular, the invention relates to
polynucleotides that hybridize
under stringent conditions to the above-described polynucleotides. As used
herein, the
terms "stringent conditions" and "stringent hybridization conditions" mean
that
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hybridization will generally occur if there is approximately at least 95% and
preferably
approximately at least 97% identity between the sequences. An example of
stringent
hybridization conditions is overnight incubation at 42°C in a solution
comprising 50%
formamide, Sx SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium
phosphate
(pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20
micrograms/milliliter
denatured, sheared salmon sperm DNA, followed by washing the hybridization
support in
O.lx SSC at approximately 65°C. Other hybridization and wash conditions
are well
known and are exemplified in Sambrook, et al., Molecular Cloning: A Laboratory
Manual,
Second Edition, cold Spring Harbor, NY (1989), particularly Chapter 11.
The invention also provides a polynucleotide consisting essentially of a
polynucleotide sequence obtainable by screening an appropriate library
containing the
complete gene for a polynucleotide sequence set for in the Sequence Listing
under
stringent hybridization conditions with a probe having the sequence of said
polynucleotide
sequence or a fragment thereof; and isolating said polynucleotide sequence.
Fragments
useful for obtaining such a polynucleotide include, for example, probes and
primers as
described herein.
As discussed herein regarding polynucleotide assays of the invention, for
example,
polynucleotides of the invention can be used as a hybridization probe for RNA,
cDNA, or
genomic DNA to isolate full length cDNAs or genomic clones encoding a
polypeptide and
to isolate cDNA or genomic clones of other genes that have a high sequence
similarity to a
polynucleotide set forth in the Sequence Listing. Such probes will generally
comprise at
least 15 bases. Preferably such probes will have at least 30 bases and can
have at least 50
bases. Particularly preferred probes will have between 30 bases and 50 bases,
inclusive.
The coding region of each gene that comprises or is comprised by a
polynucleotide
sequence set forth in the Sequence Listing may be isolated by screening using
a DNA
sequence provided in the Sequence Listing to synthesize an oligonucleotide
probe. A
labeled oligonucleotide having a sequence complementary to that of a gene of
the
invention is then used to screen a library of cDNA, genomic DNA or mRNA to
identify
members of the library which hybridize to the probe. For example, synthetic
oligonucleotides are prepared which correspond to the N-terminal sequence of
the PFI
peptide. The partial sequences so prepared are then used as probes to obtain
PFI clones
from a gene library prepared from Ptilota filicina, a red marine algae.
Alternatively,
where oligonucleotides of low degeneracy can be prepared from particular PFI
peptides,
such probes may be used directly to screen gene libraries for PFI gene
sequences. In
particular, screening of cDNA libraries in phage vectors is useful in such
methods due to
lower levels of background hybridization.
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Typically, a PFI sequence obtainable from the use of nucleic acid probes will
show
approximately 60-70% sequence identity between the target PFI sequence and the
encoding sequence used as a probe. However, lengthy sequences with as little
as
approximately 50-60% sequence identity may also be obtained. The nucleic acid
probes
may be a lengthy fragment of the nucleic acid sequence, or may also be a
shorter,
oligonucleotide probe. When longer nucleic acid fragments are employed as
probes
(greater than about 100 bp), one may screen at lower stringencies in order to
obtain
sequences from the target sample which have 20-50% deviation (i.e., 50-80%
sequence
homology) from the sequences used as probe. Oligonucleotide probes can be
considerably
shorter than the entire nucleic acid sequence encoding an PFI enzyme, but
should be at
least about 10, preferably at least about 15, and more preferably at least
about 20
nucleotides. A higher degree of sequence identity is desired when shorter
regions are used
as opposed to longer regions. It may thus be desirable to identify regions of
highly
conserved amino acid sequence to design oligonucleotide probes for detecting
and
recovering other related PFI genes. Shorter probes are often particularly
useful for
polymerase chain reactions (PCR), especially when highly conserved sequences
can be
identified. (See, Gould, et al., PNAS USA (1989) 86:1934-1938).
The skilled artisan will appreciate that, in many cases, an isolated cDNA
sequence
will be incomplete, in that the region coding for the polypeptide is truncated
with respect
to the 5' terminus of the cDNA. This is a consequence of the reverse
transcriptase, an
enzyme with low 'processivity' (a measure of the ability of the enzyme to
remain attached
to the template during the polymerization reaction) employed during the first
strand cDNA
synthesis.
There are several methods available and are well know to the skilled artisan
to
obtain full-length cDNAs, or extend short cDNAs, for example those based on
the method
of Rapid Amplification of cDNA Ends (RACE) (see, for example, Frohman et al.
(1988)
Proc. Natl. Acad. Sci. USA 85:8998-9002). Recent modifications of the
technique,
exemplified by the Marathona technology (Clonetech Laboratories, Inc.) for
example,
have significantly simplified obtaining full-length cDNA sequences.
The polynucleotides and polypeptides of the invention can be used, for
example, in
the transformation of various host cells, as further discussed herein.
The invention also provides polynucleotides that encode a polypeptide that is
a
mature protein plus additional amino or carboxyl-terminal amino acids, or
amino acids
within the mature polypeptide (for example, when the mature form of the
protein has more
than one polypeptide chain). Such sequences can, for example, play a role in
the
processing of a protein from a precursor to a mature form, allow protein
transport, shorten
or lengthen protein half life, or facilitate manipulation of the protein in
assays or
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production. It is contemplated that cellular enzymes can be used to remove any
additional
amino acids from the mature protein.
A precursor protein, having the mature form of the polypeptide fused to one or
more prosequences may be an inactive form of the polypeptide. The inactive
precursors
generally are activated when the prosequences are removed. Some or all of the
prosequences may be removed prior to activation. Such precursor protein are
generally
called proproteins.
The polynucleotide and polypeptide sequences can also be used to identify
additional sequences which are homologous to the sequences of the present
invention.
10 The most preferable and convenient method is to store the sequence in a
computer
readable medium, for example, floppy disk, CD ROM, hard disk drives, external
disk
drives and DVD, and then to use the stored sequence to search a sequence
database with
well known searching tools. Examples of public databases include the DNA
Database of
Japan (DDBJ)(http://www.ddbj.nig.ac.jp~; Genebank
(http://www.ncbi.nlm.nih.~ov/web/Genbank/Index.htlm); and the European
Molecular
Biology Laboratory Nucleic Acid Sequence Database (EMBL)
(http://www.ebi.ac.uk/ebi_docs/embl db.html). A number of different search
algorithms
are available to the skilled artisan, one example of which are the suite of
programs referred
to as BLAST programs. There are five implementations of BLAST, three designed
for
nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed
for
protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in
Biotechnology,
12: 76-80 (1994); Birren, et al., Genome Analysis, l: 543-559 (1997)).
Additional
programs are available in the art for the analysis of identified sequences,
such as sequence
alignment programs, programs for the identification of more distantly related
sequences,
and the like, and are well known to the skilled artisan.
Plant Constructs and Methods of Use
Of particular interest is the use of the nucleotide sequences, or
polynucleotides, in
recombinant DNA constructs to direct the transcription and/or expression of
the PFI
sequences of the present invention in a host plant cell. The expression
constructs
generally comprise a promoter functional in a plant cell operably linked to a
nucleic acid
sequence encoding a polyenoic fatty acid isomerase of the present invention
and a
transcriptional termination region functional in a plant cell.
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Those skilled in the art will recognize that there are a number of promoters
which
are functional in plant cells that have been described in the literature. In
addition, organelle
and plastid specific promoters such as chloroplast or plastid functional
promoters, and
chloroplast or plastid operable promoters are also envisioned.
One set of promoters are constitutive promoters such as the CaMV35S or FMV35S
promoters that yield high levels of expression in most plant organs. Enhanced
or
duplicated versions of the CaMV35S and FMV35S promoters are useful in the
practice of
this invention (Odell, et al. (1985) Nature 313:810-812; Rogers, U.S. Patent
Number
5,378, 619). In addition, it may also be preferred to bring about expression
of the PFI gene
in specific tissues of the plant, such as leaf, stem, root, tuber, seed,
fruit, etc., and the
promoter chosen should have the desired tissue and developmental specificity.
Of particular interest is the expression of the nucleic acid sequences of the
present
invention from transcription initiation regions which are preferentially
expressed in a plant
seed tissue. Examples of such seed preferential transcription initiation
sequences include
those sequences derived from sequences encoding plant storage protein genes or
from
genes involved in fatty acid biosynthesis in oilseeds. Examples of such
promoters include
the 5' regulatory regions from such genes as napin (Kridl et al., Seed Sci.
Res. 1:209:219
(1991)), phaseolin, zero, soybean trypsin inhibitor, ACP, stearoyl-ACP
desaturase,
soybean a subunit of b-conglycinin (soy 7s, (Chen et al., Proc. Natl. Acad.
Sci., 83:8560-
8564 (1986))) and oleosin.
It may be advantageous to direct the localization of proteins confernng PFI to
a
particular subcellular compartment, for example, to the mitochondrion,
endoplasmic
reticulum, vacuoles, chloroplast or other plastidic compartment. For example,
where the
genes of interest of the present invention will be targeted to plastids, such
as chloroplasts
for expression, the constructs will also employ the use of sequences to direct
the gene to
the plastid. Such sequences are referred to herein as chloroplast transit
peptides (CTP) or
plastid transit peptides (PTP). In this manner, where the gene of interest is
not directly
inserted into the plastid, the expression construct will additionally contain
a gene encoding
a transit peptide to direct the gene of interest to the plastid. The
chloroplast transit
peptides may be derived from the gene of interest, or may be derived from a
heterologous
sequence having a CTP. Such transit peptides are known in the art. See, for
example,
Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989)
J. Biol.
Chem. 264:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84:965-968;
Romer et
al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and, Shah et al.
(1986)
Science 233:478-481. Additional transit peptides for the translocation of the
PFI protein
to the endoplasmic reticulum (ER), or vacuole may also find use in the
constructs of the
present invention.
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12
Depending upon the intended use, the constructs may contain the nucleic acid
sequence which encodes the entire PFI protein, or a portion thereof. For
example, where
antisense inhibition of a given PFI protein is desired, the entire PFI
sequence is not
required. Furthermore, where PFI sequences used in constructs are intended for
use as
probes, it may be advantageous to prepare constructs containing only a
particular portion
of a PFI encoding sequence, for example a sequence which is discovered to
encode a
highly conserved PFI region.
The skilled artisan will recognize that there are various methods for the
inhibition
of expression of endogenous sequences in a host cell. Such methods include,
but are not
limited to antisense suppression (Smith, et al. (1988) Nature 334:724-726) ,
co
suppression (Napoli, et al. (1989) Plant Cell 2:279-289), ribozymes (PCT
Publication
WO 97/10328), and combinations of sense and antisense Waterhouse, et al.
(1998) Proc.
Natl. Acad. Sci. USA 95:13959-13964. Methods for the suppression of endogenous
sequences in a host cell typically employ the transcription or transcription
and translation
1 S of at least a portion of the sequence to be suppressed. Such sequences may
be homologous
to coding as well as non-coding regions of the endogenous sequence.
Regulatory transcript termination regions may be provided in plant expression
constructs of this invention as well. Transcript termination regions may be
provided by
the DNA sequence encoding the polyenoic fatty acid isomerase or a convenient
transcription termination region derived from a different gene source, for
example, the
transcript termination region which is naturally associated with the
transcript initiation
region. The skilled artisan will recognize that any convenient transcript
termination region
which is capable of terminating transcription in a plant cell may be employed
in the
constructs of the present invention.
Alternatively, constructs may be prepared to direct the expression of the PFI
sequences directly from the host plant cell plastid. Such constructs and
methods are
known in the art and are generally described, for example, in Svab, et al.
(1990) Proc.
Natl. Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad.
Sci.
USA 90:913-917 and in U.S. Patent Number 5,693,507.
A plant cell, tissue, organ, or plant into which the recombinant DNA
constructs
containing the expression constructs have been introduced is considered
transformed,
transfected, or transgenic. A transgenic or transformed cell or plant also
includes progeny
of the cell or plant and progeny produced from a breeding program employing
such a
transgenic plant as a parent in a cross and exhibiting an altered phenotype
resulting from
the presence of a PFI nucleic acid sequence.
Plant expression or transcription constructs having a plant PFI as the DNA
sequence of interest for increased or decreased expression thereof may be
employed with a
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13
wide variety of plant life, particularly, plant life involved in the
production of vegetable
oils for edible and industrial uses. Most especially preferred are temperate
oilseed crops.
Plants of interest include, but are not limited to, rapeseed (Canola and High
Erucic Acid
varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil
palms, and corn.
S Depending on the method for introducing the recombinant constructs into the
host cell,
other DNA sequences may be required. Importantly, this invention is applicable
to
dicotyledyons and monocotyledons species alike and will be readily applicable
to new
and/or improved transformation and regulation techniques.
Of particular interest, is the use of plant PFI constructs in plants which
have been
genetically engineered to produce a particular fatty acid in the plant seed
oil, where TAG
in the seeds of nonengineered plants of the engineered species, do not
naturally contain
that particular fatty acid. Thus, the expression of novel PFI in plants may be
desirable for
the incorporation of unique fatty acyl groups into the sn-3 position.
Further plant genetic engineering applications for PFI proteins of this
invention
include their use in preparation of structured plant lipids which contain TAG
molecules
having desirable fatty acyl groups incorporated into particular positions on
the TAG
molecules.
It is contemplated that the gene sequences may be synthesized, either
completely
or in part, especially where it is desirable to provide plant-preferred
sequences. Thus, all
or a portion of the desired structural gene (that portion of the gene which
encodes the PFI
protein) may be synthesized using codons preferred by a selected host. Host-
preferred
codons may be determined, for example, from the codons used most frequently in
the
proteins expressed in a desired host species.
One skilled in the art will readily recognize that antibody preparations,
nucleic acid
probes (DNA and RNA) and the like may be prepared and used to screen and
recover
"homologous" or "related" PFIs from a variety of plant sources. Homologous
sequences
are found when there is an identity of sequence, which may be determined upon
comparison of sequence information, nucleic acid or amino acid, or through
hybridization
reactions between a known PFI and a candidate source. Conservative changes,
such as
Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also be considered in
determining
sequence homology. Amino acid sequences are considered homologous by as little
as
25% sequence identity between the two complete mature proteins. (See
generally,
Doolittle, R.F., OF URFS and ORES (University Science Books, CA, 1986.)
Thus, other PFIs may be obtained from the specific exemplified Ptilota PFI
sequences provided herein. Furthermore, it will be apparent that one can
obtain natural
and synthetic PFIs, including modified amino acid sequences and starting
materials for
synthetic-protein modeling from the exemplified PFIs and from PFIs which are
obtained
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14
through the use of such exemplified sequences. Modified amino acid sequences
include
sequences which have been mutated, truncated, increased and the like, whether
such
sequences were partially or wholly synthesized. Sequences which are actually
purified
from plant preparations or are identical or encode identical proteins thereto,
regardless of
the method used to obtain the protein or sequence, are equally considered
naturally
derived.
For immunological screening, antibodies to the PFI protein can be prepared by
injecting rabbits or mice with the purified protein or portion thereof, such
methods of
preparing antibodies being well known to those in the art. Either monoclonal
or
polyclonal antibodies can be produced, although typically polyclonal
antibodies are more
useful for gene isolation. Western analysis may be conducted to determine that
a related
protein is present in a crude extract of the desired plant species, as
determined by cross-
reaction with the antibodies to the P. filicina PFI protein. When cross-
reactivity is
observed, genes encoding the related proteins are isolated by screening
expression libraries
1 S representing the desired plant species. Expression libraries can be
constructed in a variety
of commercially available vectors, including lambda gtl l, as described in
Sambrook, et al.
(Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring
Harbor
Laboratory, Cold Spring Harbor, New York).
The nucleic acid sequences associated with plant PFI proteins may be utilized
for a
variety of uses. For example, recombinant constructs can be prepared which may
be
employed as probes, or which will direct expression of the PFI protein in host
cells to
produce a ready source of the enzyme and/or to modify the composition of
triglycerides
found therein. Other useful applications may be found when the host cell is a
plant host
cell, either in vitro or in vivo. For example, by expressing a PFI protein in
a host plant
cell, various conjugate fatty acids may be produced in a given plant tissue.
In a like
manner, for some applications it may be desired to decrease the amount of PFI
endogenously expressed in a plant cell by various gene suppression
technologies discussed
supra.
It is appreciated that the expression constructs containing the polynucleotide
sequences of the present invention find use with additional expression
constructs having
sequences responsible for the alteration of fatty acids in a host cell.
Examples of such
sequences include, but are not limited to thioesterases, desaturases,
elongases, KASes, and
the like.
The modification of fatty acid compositions may also affect the fluidity of
plant
membranes. Different lipid concentrations have been observed in cold-hardened
plants,
for example. By this invention, one may be capable of introducing traits which
will lend
CA 02379813 2002-O1-17
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to chill tolerance. Constitutive or temperature inducible transcription
initiation regulatory
control regions may have special applications for such uses.
As discussed above, nucleic acid sequence encoding a plant PFI of this
invention
may include genomic, cDNA or mRNA sequence. By "encoding" is meant that the
5 sequence corresponds to a particular amino acid sequence either in a sense
or anti-sense
orientation. By "extrachromosomal" is meant that the sequence is outside of
the plant
genome of which it is naturally associated. By "recombinant" is meant that the
sequence
contains a genetically engineered modification through manipulation via
mutagenesis,
restriction enzymes, and the like.
10 Once the desired plant PFI nucleic acid sequence is obtained, it may be
manipulated in a variety of ways. Where the sequence involves non-coding
flanking
regions, the flanking regions may be subjected to resection, mutagenesis, etc.
Thus,
transitions, transversions, deletions, and insertions may be performed on the
naturally
occurnng sequence. In addition, all or part of the sequence may be
synthesized. In the
15 structural gene, one or more codons may be modified to provide for a
modified amino acid
sequence, or one or more codon mutations may be introduced to provide for a
convenient
restriction site or other purpose involved with construction or expression.
The structural
gene may be further modified by employing synthetic adapters, linkers to
introduce one or
more convenient restriction sites, or the like.
The nucleic acid or amino acid sequences encoding a plant PFI of this
invention
may be combined with other non-native or heterologous sequences in a variety
of ways.
By heterologous sequences is meant any sequence which is not naturally found
joined to
the plant PFI, including, for example, combinations of nucleic acid sequences
from the
same plant which are not naturally found joined together.
The DNA sequence encoding a plant PFI of this invention may be employed in
conjunction with all or part of the gene sequences normally associated with
the PFI. In its
component parts, a DNA sequence encoding PFI is combined in a DNA construct
having,
in the 5' to 3' direction of transcription, a transcription initiation control
region capable of
promoting transcription and translation in a host cell, the DNA sequence
encoding plant
PFI and a transcription and translation termination region.
Potential host cells include both prokaryotic and eukaryotic cells. A host
cell may
be unicellular or found in a multicellular differentiated or undifferentiated
organism
depending upon the intended use. Cells of this invention may be distinguished
by having
a plant PFI foreign to the wild-type cell present therein, for example, by
having a
recombinant nucleic acid construct encoding a plant PFI therein.
The methods used for the transformation of the host plant cell are not
critical to the
present invention. The transformation of the plant is preferably permanent,
i.e. by
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16
integration of the introduced expression constructs into the host plant
genome, so that the
introduced constructs are passed onto successive plant generations. The
skilled artisan
will recognize that a wide variety of transformation techniques exist in the
art, and new
techniques are continually becoming available. Any technique that is suitable
for the
S target host plant can be employed within the scope of the present invention.
For example,
the constructs can be introduced in a variety of forms including, but not
limited to, as a
strand of DNA, in a plasmid, or in an artificial chromosome. The introduction
of the
constructs into the target plant cells can be accomplished by a variety of
techniques,
including, but not limited to calcium-phosphate-DNA co-precipitation,
electroporation,
microinjection, Agrobacterium infection, liposomes or microprojectile
transformation.
The skilled artisan can refer to the literature for details and select
suitable techniques for
use in the methods of the present invention.
Normally, included with the DNA construct will be a structural gene having the
necessary regulatory regions for expression in a host and providing for
selection of
transformant cells. The gene may provide for resistance to a cytotoxic agent,
e.g.
antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to
an
auxotrophic host, viral immunity or the like. Depending upon the number of
different host
species, the expression construct or components thereof are introduced, one or
more
markers may be employed, where different conditions for selection are used for
the
different hosts.
Where Agrobacterium is used for plant cell transformation, a vector may be
used
which may be introduced into the Agrobacterium host for homologous
recombination with
T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host. The Ti- or
Ri-plasmid
containing the T-DNA for recombination may be armed (capable of causing gall
formation) or disarmed (incapable of causing gall formation), the latter being
permissible,
so long as the vir genes are present in the transformed Agrobacterium host.
The armed
plasmid can give a mixture of normal plant cells and gall.
In some instances where Agrobacterium is used as the vehicle for transforming
host plant cells, the expression or transcription construct bordered by the T-
DNA border
regions) will be inserted into a broad host range vector capable of
replication in E. coli
and Agrobacterium, there being broad host range vectors described in the
literature.
Commonly used is pRK2 or derivatives thereof. See, for example, Ditta, et al.,
(roc. Nat.
Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0 120 515, which are
incorporated
herein by reference. Alternatively, one may insert the sequences to be
expressed in plant
cells into a vector containing separate replication sequences, one of which
stabilizes the
vector in E. coli, and the other in Agrobacterium. See, for example, McBride
and
Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein the pRiHRI (Jouanin,
et al.,
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17
Mol. Gen. Genet. (1985) 201:370-374) origin of replication is utilized and
provides for
added stability of the plant expression vectors in host Agrobacterium cells.
Included with the expression construct and the T-DNA will be one or more
markers, which allow for selection of transformed Agrobacterium and
transformed plant
cells. A number of markers have been developed for use with plant cells, such
as
resistance to chloramphenicol, kanamycin, the aminoglycoside 6418, hygromycin,
or the
like. The particular marker employed is not essential to this invention. The
preferred
marker will depend upon the particular host and specific construct employed
for
transformation of such host.
For transformation of plant cells using Agrobacterium, plants may be combined
and incubated with the modified Agrobacterium for sufficient time to enable
said plant cell
to be transformed by the Agrobacterium. The bacteria are then killed, and the
plant cells
cultured in an appropriate selective medium. Once a callus forms, shoot
formation can be
encouraged by employing the appropriate plant hormones in accordance with
known
methods and the shoots transferred to rooting medium for regeneration of
plants. The
plants may then be grown to seed and the seed used to establish repetitive
generations and
for isolation of vegetable oils.
There are several possible ways to obtain the plant cells of this invention
which
contain multiple expression constructs. Any means for producing a plant
comprising a
construct having a DNA sequence encoding the polyenoic fatty acid isomerase of
the
present invention, and at least one other construct having another DNA
sequence encoding
an enzyme are encompassed by the present invention. For example, the
expression
construct can be used to transform a plant at the same time as the second
construct either
by inclusion of both expression constructs in a single transformation vector
or by using
separate vectors, each of which express desired genes. The second construct
can be
introduced into a plant which has already been transformed with the PFI
expression
construct, or alternatively, transformed plants, one expressing the PFI
construct and one
expressing the second construct, can be crossed to bring the constructs
together in the
same plant.
Other Constructs and Methods of Use
The invention also relates to vectors that include a polynucleotide or
polynucleotides of the invention, host cells that are genetically engineered
with vectors of
the invention and the production of polypeptides of the invention by
recombinant
techniques. Cell free translation systems can be employed to produce such
protein using
RNAs derived from the DNA constructs of the invention.
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18
For recombinant production, host cells can be genetically engineered to
incorporate
expression systems or portions thereof or polynucleotides of the present
invention.
Introduction of a polynucleotide into a host cell can be effected by methods
described in
many standard laboratory manuals, such as Davis et al., Basic Methods in
Molecular
Biology, (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual,
2°a
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989).
Such
methods include, but are not limited to, calcium phosphate transfection, DEAF
dextran
mediated transfection, transvection, microinjection, cationic lipid-mediated
transfection,
electroporation, transduction, scrape loading ballistic introduction and
infection.
Representative examples of appropriate hosts include bacterial cells, such as
streptococci, staphylococci, enterococci, E. coli, streptomyces, and Bacillus
subtilis cells;
fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as
Drosophila S2
and Spodoptera Sf~3 cells; animal cells such as CHO, COS, HeLa, C127, 3T3,
BHK, 293
and Bowes melanoma cells; and plant cells as described above.
A variety of expression systems can be used to produce the polypeptides of the
invention. Such vectors include, but are not limited to, chromosomal,
episomal, and virus
derived vectors, for example vectors from bacterial plasmids, bacteriophage,
transposons,
yeast episomes, insertion elements, yeast chromosomal elements, viruses such
as
baculoviruses, papova viruses, such as SB40, vaccinia viruses, adenoviruses,
fowl pox
viruses, pseudorabies viruses and retroviruses, and vectors derived from
combinations of
such viruses, such as those derived from plasmid and bacteriophage genetic
elements, such
as cosmids and phagemids. The expression system constructs may contain control
regions
that regulate as well as engender expression. Generally, any system or vector
which is
suitable to maintain, propagate or express polynucleotides and/or to express a
polypeptide
in a host can be used for expression. The appropriate DNA sequence can be
inserted into
the chosen expression by any of a variety of well-known and routine
techniques, such as,
for example, those set forth in Sambrook et al, Molecular Cloning, A
Laboratory Manual,
(supra).
Appropriate secretion signals, either homologous or heterologous, can be
incorporated into the expressed polypeptide to allow the secretion of the
protein into the
lumen of the endoplasmic reticulum, the periplasmic space or the extracellular
environment.
The polypeptides of the present invention can be recovered and purified from
recombinant cell cultures by any of a number of well known methods, including,
but not
limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation
exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction
chromatography, affinity chromatography, hydroxylapatite chromatography, and
lectin
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19
chromatography. It is most preferable to use high performance liquid
chromatography
(HPLC) for purification. Any of the well known techniques for protein
refolding can be
used to regenerate an active confirmation if the polypeptide is denatured
during isolation
and/or purification.
The oil compositions obtained from host cells expressing the PFI sequences of
the
present invention find use in a variety of industrial, animal feed and human
nutritional
applications.
The oil produced by the methods of the present invention containing the
conjugated fatty acids find a number of uses. For example methods for the use
of various
conjugated fatty acids, for example, conjugated linolenic acid (CLA) are known
in the art.
A number of methods for the use of CLA are described in US Patents 5,428,072,
5,430,066, 5,504,114, 5,554,646, 5,585,400, 5,674,901, 5,760,082, 5,760,083,
5,770,247,
5,804,210, 5,814,663, 5,827,885, 5,851,572, 5,855,917.
Thus, the oil compositions of the present invention having an altered
conjugated
fatty acid content find use in the preparation of foods, food products,
processed foods,
food ingredients, food additive compositions, or dietary supplements that
contain oils
and/or fats. Examples of such uses include but are not limited to margarines,
butters,
shortenings, cooking oils, frying oils, dressings, spreads, mayonnaises, and
vitamin/mineral supplements. Additional examples include, but are not limited
to
toppings, dairy products such as cheese and processed cheese, processed meat
and meat
mimetics, pastas, cereals, sauces, desserts including frozen and shelf stable
desserts, dips,
chips, baked goods, pastries, cookies, snack bars, confections, chocolates,
beverages,
unextracted seed, and unextracted seed that has been ground, cracked, milled,
rolled,
extruded, pelleted, defatted, dehydrated, or otherwise processed, but which
still contains
the oils, etc., disclosed herein.
The oil compositions of the present invention having an altered conjugated
fatty
acid also find use in pharmaceutical compositions comprising an effective
amount of the
conjugated fatty acid composition, along with a pharmaceutically acceptable
Garner,
excipient, or diluent. These pharmaceutical compositions can be in the form of
a solid or
liquid. Solids can be in the form of a powder, a granule, a pill, a tablet, a
gel, or an
extrudate; liquids can be solutions or suspensions.
The invention now being generally described, it will be more readily
understood by
reference to the following examples which are included for purposes of
illustration only
and are not intended to limit the present invention.
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EXAMPLES
Example 1 Identification of a Polyenoic Fatty Acid Isomerase Nucleic Acid
Sequence
A. Complementary DNA Library Preparation
Total RNA from the red marine algae Ptilota filicina was isolated for use in
5 construction of complementary (cDNA) libraries. Fresh material was ground in
liquid
nitrogen with a mortar/pestle. Approximately 5 g P. filicina powder was mixed
with 5 ml
extraction buffer (1 % SDS, 10 mM EDTA, 0.2 M NaAC, pH 4.8) and 3 ml acid
phenol
(pH 4.3). The mixture was incubated at 60°C for 30 min with shaking
every 5 min. The
mixture was cooled to room temperature and 3 ml chlorophorm was added. After
shaking
10 at room temperature for 10 min, the mixture was centrifuged at 5000 X g for
30 min. The
aqueous phase was extracted once with 6 ml phenol/ chlorophorm (1:1 v/v) for
10 min and
then centrifuged at 5000 X g for 5 min. The upper aqueous layer was recovered
, extracted
once with 6 ml chloroform. After the last extraction, RNA was precipitated
from the
aqueous layer with equal volume of 4M LiCI on ice overnight, and spun down at
10000 X
15 g for 30 min. Precipitated RNA was washed with 70% ethanol, vacuum-dried
and
dissolved in water.
The resulting total RNA was used to prepare cDNA libraries using the
Superscript
plasmid system for cDNA synthesis and plasmid cloning kit (BRL Life-
Technologies,
Gaithersburg, MD).
20 In order to identify candidate nucleic acid sequences, a pair of synthetic
oligonucleotides (5'-GAYYYNGAYGAYACNATHGC-3', 5'-
TGYTGNBWRTADATYTCNAC-3' (Y=CT, N=ATGC, H=ACT, B=GCT, W=AT,
R=AG, D=AGT)(SEQ ID NO:S and 6)) were prepared corresponding to the 38 N-
terminal
amino acids (DDFDDTIAVVGAGYSGLSAAFTLVKKGYTNVEIYSQQY, SEQ ID
N0:7)(Wise (1995) Biosynthesis and enzymology of conjugated polyenoic fatty
acid
production in macrophytic marine algae, Ph.D. Thesis, Oregon State University,
Corvalis,
OR, UMI Dissertation Services) for use in PCR reactions to amplify probes for
use in
hybridization screening of the P. filicina cDNA library. PCR amplification
included an
initial denaturation step of 95 °C for 5 min followed by a 5 cycles of
94°C (30 sec) - 45°C
(30 sec) - 72°C (30 sec) and a 30 cycles of 94°C (30 sec) -
52°C (30 sec) - 72°C (30 sec).
PCR products between 70 and 150 by were gel purified and used as templates for
a second
PCR reaction. Following amplification, PCR products were cloned into pCR2.1
using TA
cloning system from Invitrogen Co. Library screenings were carned out using
standard
colony blot protocols (Sambrook, et al. Molecular Cloning, A Laboratory
Manual,
(supra)).
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21
Two cDNA sequences were identified as having hybridized with the
oligonucleotide probe. These two DNA sequences, referred to as PFI-B2 (SEQ ID
NO:1)
and PFI-F3 (SEQ ID N0:3), contained in the pSPORTl cloning vector (BRL Life-
Technologies, Gaithersburg, MD), pCGN10100 and pCGN10101, respectively. The
deduced amino acid sequence for both PFI-B2 (SEQ ID N0:2) and PFI-F3 (SEQ ID
N0:4) are also determined.
Example 2 Construct Preparation
2A. Bacterial Expression Constructs
A series of constructs were prepared to express the PFI sequences in host
cells.
For expression in E. coli, constructs were prepared that either contained tags
or lacked
such tag sequences.
The vector pCGN10102 was designed to express the protein encoded by the PFI-
B2 sequence with the native leader peptide with a C-terminal 6 residue His-tag
sequence
in the pQE-60 vector (Qiagen). The vector pCGN10103 is similar to pCGN10102,
except
it contains the sequence encoding the PFI-F3 protein with its native leader
peptide.
A set of constructs were also prepared to express the PFI sequences in E. coli
without the leader sequences. The vector pCGN10104 contains the PFI-B2
sequence from
pCGN10100 without the leader peptide cloned into the pQE-60 vector with a 6
residue His
tag on the PFI C-terminus. The vector pCGN10105 is similar to pCGN10104,
except it
contains the PFI-F3 encoding sequence from pCGN10101.
Finally, for expression in E. coli a set of constructs lacking the C-terminal
6
residue His tag were prepared. The construct pCGN10106 contains the PFI-B2
encoding
sequence from pCGN10100 including the native leader peptide in the pQE-60
vector. The
vector pCGN10107 is similar to pCGN10106 except containing the PFI-F3 encoding
sequence, including the native leader peptide.
For expression of PFI in the cytoplasm of E. coli, the pQE60 system from
Qiagen
Inc. was used. The PFI coding region with and without the putative leader
peptide was
PCR amplified using forward primers: 5'-CGCCATGGCTTTGAATAGAGTTCTTCAC-
3' or 5'-CGCCATGGACGATTTTGATGACACGATTGC-3'(SEQ ID N0:8 and 9),
reverse primer: 5'- CGAGATCTGAAGAAATCCTTGATCAAATTATCCG-3'(SEQ ID
NO:10). The NcoI sites (underlined) were introduced in the forward primers
while the
BgIII site (underlined) was introduced in the reverse primer. The PCR products
were
subcloned in pCR2.1 using TA cloning system (Invitrogen Co.). The resulting
product
was then sent for sequencing. After sequence confirmation, the inserts were
excised using
complete digestion of BgIII and partial digestion of NcoI followed by gel
purification
using the gel purification system from Qiagen Inc. Subcloning of the inserts
into pQE60
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22
vector and expression of the recombinant proteins were done as recommended by
the
manufacturer. The E. coli transformants were grown at different temperatures
to an
OD600 of 0.7- 0.8 before being induced by 1 mM isopropyl-b-D-
thiogalactopyranoside
for 1-5 h. The induced cells were then harvested and lysed by sonication
followed by
enzyme assays.
Periplasmic expression of PFI was carried out by fusing E. coli alkaline
phosphotase (PhoA) leader peptide and PFI without its native leader peptide.
The fusion
protein coding region was designed to be driven by E. coli alkaline
phosphotase gene
(phoA) native promoter. Three primers were designed: PPF, S'-AAGCTTTGGAGATTA
TCGTC-3'(SEQ ID NO:11), was derived from the sequence upstream ofphoA
promoter;
PPM, 5'-TCGTGTCATC AAAATCATGGGCTTTTGTCACAGGGGTAA-3'(SEQ ID
N0:12), contained partial coding sequence of PhoA leader peptide (underlined)
and partial
coding region of PFI without leader peptide; and EPR, 5'-
GCAGGATCCGTATCGAGCTC T GATT CG-3' (SEQ ID N0:13) was derived from
down stream sequence of PFI coding region of the cDNA clone. To make the
fusion
construct, two PCR reactions were conducted. The first PCR reaction was done
using
primers PPF paired with PPM and E. coli K-12 genomic DNA as template. The
second
PCR reaction was carned out using primers PPF and EPR. The templates for the
second
PCR reaction were generated by mixing 1 ml of the first PCR product and 1 ml
of 1:50
diluted plasmid DNA of the PFI cDNA clone. The final PCR product was cloned
into
pCR2.l using the Topo-TA cloning system (Invitrogen Co. ) and the insert was
verified by
sequencing of both strands. The E. coli transformants were grown in ECLB media
at 37°C
to early stationary phase and subsequently harvested for protein analysis by
centrifugation.
2B. Plant Expression Constructs
A series of constructs are prepared for the expression of the PFI encoding
sequences in host plant cells. Constructs are prepared to direct the
expression of the PFI
encoding sequences constitutively as well as preferentially in particular
plant tissues.
A plasmid containing the napin cassette derived from pCGN3223 (described in
USPN 5,639,790, the entirety of which is incorporated herein by reference) was
modified
to make it more useful for cloning large DNA fragments containing multiple
restriction
sites, and to allow the cloning of multiple napin fusion genes into plant
binary
transformation vectors. An adapter comprised of the self annealed
oligonucleotide of
sequence CGCGATTTAAATGGCGCGCCCTGCAGGCGGCCGCCTG
CAGGGCGCGCCATTTAAAT (SEQ ID N0:14) was ligated into the cloning vector pBC
SK+ (Stratagene) after digestion with the restriction endonuclease BssHII to
construct
vector pCGN7765. Plasmids pCGN3223 and pCGN7765 were digested with NotI and
CA 02379813 2002-O1-17
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23
ligated together. The resultant vector, pCGN7770, contains the pCGN7765
backbone with
the napin seed specific expression cassette from pCGN3223.
The cloning cassette, pCGN7787, has essentially the same regulatory elements
as
pCGN7770, with the exception of the napin regulatory regions of pCGN7770 have
been
replaced with the double CAMV 35S promoter and the tml polyadenylation and
transcriptional termination region.
A binary vector for plant transformation, pCGN5139, was constructed from
pCGN1558 (McBride and Summerfelt, (1990) Plant Molecular Biology, 14:269-276).
The
polylinker of pCGN1558 was replaced as a HindIII/Asp718 fragment with a
polylinker
containing unique restriction endonuclease sites, AscI, PacI, XbaI, SwaI,
BamHI, and
NotI. The Asp718 and HindIII restriction endonuclease sites are retained in
pCGN5139.
A series of turbo binary vectors were constructed to allow for the rapid
cloning of
DNA sequences into binary vectors containing transcriptional initiation
regions
(promoters) and transcriptional termination regions.
The plasmid pCGN8618 was constructed by ligating oligonucleotides 5'-
TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO:15) and 5'-
TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID N0:16) into SaII/XhoI
double-digested pCGN7770. A fragment containing the napin promoter, polylinker
and
napin 3' region was excised from pCGN8618 by digestion with Asp718I; the
fragment
was blunt-ended by filling in the 5' overhangs with Klenow fragment then
ligated into
pCGN5139 that had been digested with Asp718I and HindIII and blunt-ended by
filling in
the S' overhangs with Klenow fragment. A plasmid containing the insert
oriented so that
the napin promoter was closest to the blunted Asp718I site of pCGN5139 and the
napin 3'
was closest to the blunted HindIII site. Subsequently, these regions were
subjected to
sequence analysis to confirm both the insert orientation and the integrity of
cloning
junctions. The resulting plasmid was designated pCGN8622.
The plasmid pCGN8619 was constructed by ligating oligonucleotides 5'-
TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC -3' (SEQ ID N0:17) and 5'-
TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:18) into SaII/XhoI
double-digested pCGN7770. A fragment containing the napin promoter, polylinker
and
napin 3' region was removed from pCGN8619 by digestion with Asp718I; the
fragment
was blunt-ended by filling in the 5' overhangs with Klenow fragment then
ligated into
pCGN5139 that had been digested with Asp718I and HindIII and blunt-ended by
filling in
the 5' overhangs with Klenow fragment. A plasmid containing the insert
oriented so that
the napin promoter was closest to the blunted Asp718I site of pCGN5139 and the
napin 3'
was closest to the blunted HindIII site was subjected to sequence analysis to
confirm both
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24
the insert orientation and the integrity of cloning junctions. The resulting
plasmid was
designated pCGN8623.
The plasmid pCGN8620 was constructed by ligating oligonucleotides 5'-
TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGGAGCT -3' (SEQ ID N0:19) and 5'-
CCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID N0:20) into SaII/SacI
double-digested pCGN7787. A fragment containing the d35S promoter, polylinker
and
tml 3' region was removed from pCGN8620 by complete digestion with Asp718I and
partial digestion with NotI. The fragment was blunt-ended by filling in the 5'
overhangs
with Klenow fragment then ligated into pCGN5139 that had been digested with
Asp718I
and HindIII and blunt-ended by filling in the 5' overhangs with Klenow
fragment. A
plasmid containing the insert oriented so that the d35S promoter was closest
to the blunted
Asp718I site of pCGN5139 and the tml 3' was closest to the blunted HindIII
site was
subjected to sequence analysis to confirm both the insert orientation and the
integrity of
cloning junctions. The resulting plasmid was designated pCGN8624.
The plasmid pCGN8621 was constructed by ligating oligonucleotides 5'-
TCGACCTGCAGGAAGCTTGCGGCCGCGGATCCAGCT -3' (SEQ ID N0:21) and 5'-
GGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:22) into SaII/SacI
double-digested pCGN7787. A fragment containing the d35S promoter, polylinker
and
tml 3' region was removed from pCGN8621 by complete digestion with Asp718I and
partial digestion with NotI. The fragment was blunt-ended by filling in the 5'
overhangs
with Klenow fragment then ligated into pCGN5139 that had been digested with
Asp718I
and HindIII and blunt-ended by filling in the 5' overhangs with Klenow
fragment. A
plasmid containing the insert oriented so that the d35S promoter was closest
to the blunted
Asp718I site of pCGN5139 and the tml 3' was closest to the blunted HindIII
site was
subjected to sequence analysis to confirm both the insert orientation and the
integrity of
cloning junctions. The resulting plasmid was designated pCGN8625.
For cloning in plant expression constructs, the PFI coding regions with or
without
the putative leader peptide were PCR-amplified using forward primers, PLF1 5'-
GGATCCGCGGCCGCATGTCTTTGAATAGAGTTCTTC-3'(with the leader peptide)
(SEQ ID N0:23) and PLF2 5'-GGATCCGCGGCCGCATGGATTTT
GATGACACGATTGC-3' (without the leader peptide) (SEQ ID N0:24), and the reverse
primer PLR 5'-CCTGCAGGAAGCTTCTAGAAGA AATCCT TGATC-3'(SEQ ID
N0:25). The NotI sites (underlined) were placed upstream of the start codons
(in boldface)
in primers PLF1 and PLF2 while the PstI site (underlined) was placed
downstream of the
stop codon (in boldface) in PLR. The PCR products were first cloned into
pCR2.l
(Invitrogen) and the presence of inserts possessing the correct sequence was
verified by
sequencing of both strands.
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Two constructs employing the PFI-B2 encoding sequence were prepared in the
vector pCGN8622 for expression from the napin promoter. The vector pCGN10108
contains the PFI-B2 encoding sequence containing the native leader sequence.
The vector
pCGN10109 contains the PFI-B2 encoding sequence lacking the native leader
sequence.
Two constructs employing the PFI-B2 encoding sequence were prepared in the
vector pCGN8624 for expression from the 35S promoter. The vector pCGN10110
contains the PFI-B2 encoding sequence containing the native leader sequence.
The vector
pCGN10111 contains the PFI-B2 encoding sequence lacking the native leader
sequence.
Two constructs employing the PFI-F3 encoding sequence were prepared in the
10 vector pCGN8622 for expression from the napin promoter. The vector
pCGN10112
contains the PFI-F3 encoding sequence containing the native leader sequence.
The vector
pCGN10113 contains the PFI-F3 encoding sequence lacking the native leader
sequence.
A single construct employing the PFI-F3 encoding sequence was prepared in the
vector pCGN8624 for expression from the 35S promoter. The vector pCGN10114
15 contains the PFI-B2 encoding sequence containing the native leader
sequence.
Example 3 Host Cell Transformation and Analysis
To express the PFI protein in E.coli, constructs were made using the
QIAexpressionist system (Qiagen). Transformation and induction of the M15
cells were
performed according to the manufacturers protocol.
20 Yeast competent cell preparation and transformation were performed using
the
Frozen-EZ yeast transformation kit (Zymo Research). The expression vector
employed
was pYES2 (Invitrogen) and the yeast strain selected was INVcI (Invitrogen).
A variety of methods have been developed to insert a DNA sequence of interest
into the genome of a plant host to obtain the transcription or transcription
and translation
25 of the sequence to effect phenotypic changes.
Transgenic Brassica plants were obtained by Agrobacterium-mediated
transformation as described by Radke et al. (Theor. Appl. Genet. (1988) 75:685-
694; Plant
Cell Reports (1992) 11:499-505). Transgenic Arabidopsis thaliana plants may be
obtained
by Agrobacterium-mediated transformation as described by Valverkens et al.,
(Proc. Nat.
Acad. Sci. (1988) 85:5536-5540), or as described by Bent et al. ((1994),
Science
265:1856-1860), or Bechtold et al. ((1993), C.R.Acad.Sci, Life Sciences
316:1194-1199).
Other plant species may be similarly transformed using related techniques.
Alternatively, microprojectile bombardment methods, such as described by Klein
et al. (Bio/Technology 10:286-291 ) may also be used to obtain nuclear
transformed plants.
The expressed PFI protein, as well as the PFI protein from the wild-type P.
filicina,
was assayed as described herein. The enzyme activity was assayed as described
by Wise
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26
((1995) PhD Thesis, supra) with some modifications. Preparation of protein
crude extract
from Ptilota filicina: frozen tissue was ground in a mortar/pestle with liquid
nitrogen.
Approximately 200 mg of the tissue powder was mixed with 1 ml of the
extraction buffer
(100 mM NaH2P04, 5 mM EGTA, 5 mM DTT, and 5 mM MgCl2, pH6.5) and
homogenized with a glass homogenizer. The homogenate was microfuged at 14 K
rpm for
min and the supernatant was collected for the enzyme assays. Similar methods
were
used for preparation of protein crude extract from transgenic material (E.
coli, yeast,
Schizochitrium, and Arabidopsis): The sample materials were broken and
homogenized in
the extraction buffer and microcentrifuged. The supernatant was used for the
enzyme
assay.
The collected supernatant was analyzed for PFI activity as follows. About 20
ml
of the protein crude extract was mixed with 300 ml of the reaction buffer (100
mM
NaH2P04, pH7.2 with 0.02% Tween 20) and the spectrophotometer (DU650, Beckman)
was blanked. The reaction was initiated by adding 2 ml of the stock substrate
solution and
the conjugated fatty acid formation was measured by wave length scanning
between
200nm and 300nm. Specifically, the conjugated triene and dime formation was
measured
by monitoring absorbance at 278nm and 234run, respectively. The stock
substrate
solution was prepared by adding free polyunsaturated fatty acids in 95%
ethanol to a final
concentration of 25 mg/ml.
All publications and patent applications mentioned in this specification are
indicative of the level of skill of those skilled in the art to which this
invention pertains.
All publications and patent applications are herein incorporated by reference
to the same
extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
obvious that
certain changes and modifications may be practiced within the scope of the
appended
claim.
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1
SEQUENCE LISTING
<110> Zheng, Wei
Yuan, Ling
Metz, James G.
<120> NUCLEIC ACID SEQUENCES ENCODING POLYENOIC FATTY ACID
ISOMERASE AND USES THEREOF
<130> MTC6711
<140>
<141>
<150> 60/146,458
<151> 1999-07-30
<160> 27
<170> PatentIn Ver. 2.1
<210> 1
<211> 1507
<212> DNA
<213> Ptilota filicina
<400> 1
cgcaaaatgt ctttgaatag agttcttcac attttcctta tcgcatatct cgcatgcact 60
gccctaaccc atgattttga tgacacgatt gccgttgtgg gagctggcta ctctggactg 120
agcgctgctt ttactctcgt caagaaaggg tacaccaacg ttgagattta cgaatcccag 180
ggcgaagttg gggggtatgt ctactctgtt gactataaca acgtcgcgca tgacctggcc 240
acgtacgctc tgactcctgc atactggaaa ttccaggagg ccatgaaaag tatcggcgtt 300
gggttttgtg agctcgatgt tgcaattgtg caaacgaatt ctacgcctgt ctcagtcccg 360
ttcgagaaat ggatggccgc ctactgggct gcgaaagtcc caaacccact caacctcgtg 420
aggaaggtct cgactcaagt ttcgacgtac gttgaagttt ggaagaagct cttcaatatg 480
gacttcattg acacgagcac gaagcgcact aatcgcctct ttccgttgaa gaccaacgac 540
gtcgacgtcc ttgcccaatt ttcaatgccc atgaaagatt ttgttgcatt gcataagctg 600
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2
gacttgctcg agcctctttt tatccaggca accgactccc aggcgtacgg tccgtatgac 660
acgacaccgg cactctacta catggtgtgg ttccctccga accttttcaa cggtgaggaa 720
aataccgttc catgtggtac gtataactcg atgcagtcca tggccgagca catggccgaa 780
tggttgaaga gcaaaggagt cacgttccac atgaatacga aggtgacgaa aatctctcgc 840
gccaccgatg gatctagtcc atccctcttg gaagaaggtg tagctacgcc gaagctcttc 900
gacaccataa tcagtacgaa caagctgccg tctgcgaacc gtgccgaagt tgtgacacct 960
ctgcttccga aggagcggga ggccgccgat acgtacgagg agctacaaat gttctctgct 1020
cttctcgaga cgaatcgcag cgatgccatt ccgacgacag gcttcttgat ggtggatgcg 1080
gacgcaatta tagctcacga ccctaacacc gggttttggg gttgtttgaa tgctgagcgt 1140
cgcggaggct attcggatga gaatgctatt ctaagctcgg atactgtgac gcgcgtcagc 1200
gccatctact actatacaga gcgtgcaaac aacgaacgca tcgacttttc tctcgacgag 1260
aagattcagc aggtgaagac caatcttgcg acgtgggact cggctacctg gaccaatcta 1320
acctcccgta cgttcggtgg atatttccag aggtggagga cgccggatgt tatgggtcaa 1380
aagccgtgga atctggctga cattcaagga gaaggagatg tgtactacgt caactcggct 1440
gcatgcgggt tcgagtccgt cggccacgtt ttcgattgcg cggataattt gatcaaggat 1500
tttttct 1507
<210> 2
<211> 500
<212> PRT
<213> Ptilota filicina
<400> 2
Met Ser Leu Asn Arg Val Leu His Ile Phe Leu Ile Ala Tyr Leu Ala
1 5 10 15
Cys Thr Ala Leu Thr His Asp Phe Asp Asp Thr Ile Ala Val Val Gly
20 25 30
Ala Gly Tyr Ser Gly Leu Ser Ala Ala Phe Thr Leu Val Lys Lys Gly
40 45
Tyr Thr Asn Val Glu Ile Tyr Glu Ser Gln Gly Glu Val Gly Gly Tyr
50 55 60
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3
Val Tyr Ser Val Asp Tyr Asn Asn Val Ala His Asp Leu Ala Thr Tyr
65 70 75 80
Ala Leu Thr Pro Ala Tyr Trp Lys Phe Gln Glu Ala Met Lys Ser Ile
85 90 95
Gly Val Gly Phe Cys Glu Leu Asp Val Ala Ile Val Gln Thr Asn Ser
100 105 110
Thr Pro Val Ser Val Pro Phe Glu Lys Trp Met Ala Ala Tyr Trp Ala
115 120 125
Ala Lys Val Pro Asn Pro Leu Asn Leu Val Arg Lys Val Ser Thr Gln
130 135 140
Val Ser Thr Tyr Val Glu Val Trp Lys Lys Leu Phe Asn Met Asp Phe
145 150 155 160
Ile Asp Thr Ser Thr Lys Arg Thr Asn Arg Leu Phe Pro Leu Lys Thr
165 170 175
Asn Asp Val Asp Val Leu Ala Gln Phe Ser Met Pro Met Lys Asp Phe
180 185 190
Val Ala Leu His Lys Leu Asp Leu Leu Glu Pro Leu Phe Ile Gln Ala
195 200 205
Thr Asp Ser Gln Ala Tyr Gly Pro Tyr Asp Thr Thr Pro Ala Leu Tyr
210 215 220
Tyr Met Val Trp Phe Pro Pro Asn Leu Phe Asn Gly Glu Glu Asn Thr
225 230 235 240
Val Pro Cys Gly Thr Tyr Asn Ser Met Gln Ser Met Ala Glu His Met
245 250 255
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Ala Glu Trp Leu Lys Ser Lys Gly Val Thr Phe His Met Asn Thr Lys
260 265 270
Val Thr Lys Ile Ser Arg Ala Thr Asp Gly Ser Ser Pro Ser Leu Leu
275 280 285
Glu Glu Gly Val Ala Thr Pro Lys Leu Phe Asp Thr Ile Ile Ser Thr
290 295 300
Asn Lys Leu Pro Ser Ala Asn Arg Ala Glu Val Val Thr Pro Leu Leu
305 310 315 320
Pro Lys Glu Arg Glu Ala Ala Asp Thr Tyr Glu Glu Leu Gln Met Phe
325 330 335
Ser Ala Leu Leu Glu Thr Asn Arg Ser Asp Ala Ile Pro Thr Thr Gly
340 345 350
Phe Leu Met Val Asp Ala Asp Ala Ile Ile Ala His Asp Pro Asn Thr
355 360 365
Gly Phe Trp Gly Cys Leu Asn Ala Glu Arg Arg Gly Gly Tyr Ser Asp
370 375 380
Glu Asn Ala Ile Leu Ser Ser Asp Thr Val Thr Arg Val Ser Ala Ile
385 390 395 400
Tyr Tyr Tyr Thr Glu Arg Ala Asn Asn Glu Arg Ile Asp Phe Ser Leu
405 410 415
Asp Glu Lys Ile Gln Gln Val Lys Thr Asn Leu Ala Thr Trp Asp Ser
420 425 430
Ala Thr Trp Thr Asn Leu Thr Ser Arg Thr Phe Gly Gly Tyr Phe Gln
435 440 445
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Arg Trp Arg Thr Pro Asp Val Met Gly Gln Lys Pro Trp Asn Leu Ala
450 455 460
Asp Ile Gln Gly Glu Gly Asp Val Tyr Tyr Val Asn Ser Ala Ala Cys
465 470 475 480
5 Gly Phe Glu Ser Val Gly His Val Phe Asp Cys Ala Asp Asn Leu Ile
485 490 495
Lys Asp Phe Phe
500
<210> 3
<211> 1539
<212> DNA
<213> Ptilota filicina
<400> 3
atgtctttga atagagttct tcacattttc cttatcgcat atctcgcatg cactgcccta 60
1 S acccatgatt ttgatgacac gattgccgtt gtgggagctg gctactctgg actgagcgct 120
gcttttactc tcgtcaagaa agggtacacc aacgttgaga tttacgaatc ccagggcgaa 180
gttggggggt atgtctactc tgttgactat aacaacgtcg cgcatgacct ggccacgtac 240
gctctgactc ctgcatactg gaaattccag gaggccatga aaagtatcgg cgttgggttt 300
tgtgagctcg atgttgcaat tgtgcaaacg aattctacgc ctgtctcagt cccgttcgag 360
aaatggatgg ccgcctactg ggctgcgaaa gtcccaaacc cactcaacct cgtgaggaag 420
gtctcgactc aagtttcgac gtacgttgaa gtttggaaga agctcttcaa tatggacttc 480
attgacacga gcacgaagcg cactaatcgc ctctttccgt tgaagaccaa cgacgtcgac 540
gtccttgccc aattttcaat gcccatgaaa gattttgttg cattgcataa gctggacttg 600
ctcgagcctc tttttatcca ggcaaccgac tcccaggcgt acggtccgta tgacacgaca 660
ccggcactct actacatggt gtggttccct ccgaaccttt tcaacggtga ggaaaatacc 720
gttccatgtg gtacgtataa ctcgatgcag tccatggccg agcacatggc cgaatggttg 780
aagagcaaag gagtcacgtt ccacatgaat acgaaggtga cgaaaatctc tcgcgccacc 840
gatggatcta gtccatccct cttggaagaa ggtgtagcta cgccgaagct cttcgacacc 900
ataatcagta cgaacaagct gccgtctgcg aaccgtgccg aagttgtgac acctctgctt 960
ccgaaggagc gggaggccac caatacgtac gaggagctac aaatgttctc tgctcttctc 1020
gagacgaatc gcagcgatgc cattccgacg acaggcttct tgatggtgga tgcggacgca 1080
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6
attatagctc acgaccctga caccgggttt tggggttgtt tgaatgctga gcgtcgcgga 1140
ggctattcgg atgagaatgc tattctaagc tcggatactg tgacgcgcgt cagcgccatc 1200
tactactata cagagcgtgc aaacaacgaa cgcatcgact tttctctcga cgagaagatt 1260
cagcaggtga agaccaatct tgcgacgtgg gactcggcta cctggaccaa tctaacttcc 1320
S cgtacgttcg gtggatattt ccagaggtgg aggacgccgg atgttatggg tcaaaagccg 1380
tggaatctgg ctgacattca aggagaagga gatgtgtact acgtcaacgc ggctgcatgc 1440
gggttcgagt ccgtcggcca cgttttcgat tgcgcggata atttgatcaa ggatttcttc 1500
tagataaaca caacagaagt agactgccgc caaagtctg 1539
<210> 4
<211> 500
<212> PRT
<213> Ptilota filicina
<400> 4
Met Ser Leu Asn Arg Val Leu His Ile Phe Leu Ile Ala Tyr Leu Ala
1 5 10 15
Cys Thr Ala Leu Thr His Asp Phe Asp Asp Thr Ile Ala Val Val Gly
25 30
Ala Gly Tyr Ser Gly Leu Ser Ala Ala Phe Thr Leu Val Lys Lys Gly
35 40 45
20 Tyr Thr Asn Val Glu Ile Tyr Glu Ser Gln Gly Glu Val Gly Gly Tyr
50 55 60
Val Tyr Ser Val Asp Tyr Asn Asn Val Ala His Asp Leu Ala Thr Tyr
65 70 75 80
Ala Leu Thr Pro Ala Tyr Trp Lys Phe Gln Glu Ala Met Lys Ser Ile
85 90 95
Gly Val Gly Phe Cys Glu Leu Asp Val Ala Ile Val Gln Thr Asn Ser
100 105 110
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Thr Pro Val Ser Val Pro Phe Glu Lys Trp Met Ala Ala Tyr Trp Ala
115 120 125
Ala Lys Val Pro Asn Pro Leu Asn Leu Val Arg Lys Val Ser Thr Gln
130 135 140
S Val Ser Thr Tyr Val Glu Val Trp Lys Lys Leu Phe Asn Met Asp Phe
145 150 155 160
Ile Asp Thr Ser Thr Lys Arg Thr Asn Arg Leu Phe Pro Leu Lys Thr
165 170 175
Asn Asp Val Asp Val Leu Ala Gln Phe Ser Met Pro Met Lys Asp Phe
180 185 190
Val Ala Leu His Lys Leu Asp Leu Leu Glu Pro Leu Phe Ile Gln Ala
195 200 205
Thr Asp Ser Gln Ala Tyr Gly Pro Tyr Asp Thr Thr Pro Ala Leu Tyr
210 215 220
Tyr Met Val Trp Phe Pro Pro Asn Leu Phe Asn Gly Glu Glu Asn Thr
225 230 235 240
Val Pro Cys Gly Thr Tyr Asn Ser Met Gln Ser Met Ala Glu His Met
245 250 255
Ala Glu Trp Leu Lys Ser Lys Gly Val Thr Phe His Met Asn Thr Lys
260 265 270
Val Thr Lys Ile Ser Arg Ala Thr Asp Gly Ser Ser Pro Ser Leu Leu
275 280 285
Glu Glu Gly Val Ala Thr Pro Lys Leu Phe Asp Thr Ile Ile Ser Thr
290 295 300
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Asn Lys Leu Pro Ser Ala Asn Arg Ala Glu Val Val Thr Pro Leu Leu
305 310 315 320
Pro Lys Glu Arg Glu Ala Thr Asn Thr Tyr Glu Glu Leu Gln Met Phe
325 330 335
S Ser Ala Leu Leu Glu Thr Asn Arg Ser Asp Ala Ile Pro Thr Thr Gly
340 345 350
Phe Leu Met Val Asp Ala Asp Ala Ile Ile Ala His Asp Pro Asp Thr
355 360 365
Gly Phe Trp Gly Cys Leu Asn Ala Glu Arg Arg Gly Gly Tyr Ser Asp
370 375 380
Glu Asn Ala Ile Leu Ser Ser Asp Thr Val Thr Arg Val Ser Ala Ile
385 390 395 400
Tyr Tyr Tyr Thr Glu Arg Ala Asn Asn Glu Arg Ile Asp Phe Ser Leu
405 410 415
Asp Glu Lys Ile Gln Gln Val Lys Thr Asn Leu Ala Thr Trp Asp Ser
420 425 430
Ala Thr Trp Thr Asn Leu Thr Ser Arg Thr Phe Gly Gly Tyr Phe Gln
435 440 445
Arg Trp Arg Thr Pro Asp Val Met Gly Gln Lys Pro Trp Asn Leu Ala
450 455 460
Asp Ile Gln Gly Glu Gly Asp Val Tyr Tyr Val Asn Ala Ala Ala Cys
465 470 475 480
Gly Phe Glu Ser Val Gly His Val Phe Asp Cys Ala Asp Asn Leu Ile
485 490 495
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9
Lys Asp Phe Phe
500
<210> S
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide
<220>
<221> misc difference
<222> (3)..(6)
<223>y=cort,n=aortorgorc
<220>
<221> misc difference
<222> (9)
<223> y=c or t
<220>
<221> misc difference
<222> (12)
<223> y=c or t
<220>
<221> misc difference
<222> (15)
<223> n=a or t or g or c
CA 02379813 2002-O1-17
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<220>
<221> misc difference
<222> (18)
<223> h=a or c or t
5 <400> 5
gayyyngayg ayacnathgc 20
<210> 6
<211> 20
<212> DNA
10 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide
<220>
<221> misc difference
<222> (3)
<223> y=c or t
<220>
<221> misc difference
<222> (6)..(9)
<223> n=a or t or g or c, b=g or c or t, w=a or t, r=a
or g
<220>
<221> misc difference
<222> (12)
<223> d=a or g or t
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<220>
<221> misc difference
<222> (15)
<223> y=c or t
<220>
<221 > misc difference
<222> (18)
<223> n=a or t or g or c
<400> 6
tgytgnbwrt adatytcnac 20
<210> 7
<211> 38
<212> PRT
<213> Ptilota filicina
<400> 7
Asp Asp Phe Asp Asp Thr Ile Ala Val Val Gly Ala Gly Tyr Ser Gly
1 5 10 15
Leu Ser Ala Ala Phe Thr Leu Val Lys Lys Gly Tyr Thr Asn Val Glu
25 30
20 Ile Tyr Ser Gln Gln Tyr
<210> 8
<211> 28
<212> DNA
25 <213> Artificial Sequence
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<220>
<223> Description of Artificial Sequence: Forward Primer
<400> 8
cgccatggct ttgaatagag ttcttcac 28
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Forward Primer
<400> 9
cgccatggac gattttgatg acacgattgc 30
<210> 10
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Reverse Primer
<400> 10
cgagatctga agaaatcctt gatcaaatta tccg 34
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
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<220>
<223> Description of Artificial Sequence: PPF Primer
<400> 11
aagctttgga gattatcgtc 20
<210> 12
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PPM Primer
<400> 12
tcgtgtcatc aaaatcatgg gcttttgtca caggggtaa 39
<210> 13
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: EPR Primer
<400> 13
gcaggatccg tatcgagctc tgattcg 27
<210> 14
<211> 56
<212> DNA
<213> Artificial Sequence
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<220>
<223> Description of Artificial Sequence: Adapter
<400> 14
cgcgatttaa atggcgcgcc ctgcaggcgg ccgcctgcag ggcgcgccat ttaaat 56
<210> 15
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 15
tcgaggatcc gcggccgcaa gcttcctgca gg 32
<210> 16
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 16
tcgacctgca ggaagcttgc ggccgcggat cc 32
<210> 17
<211> 32
<212> DNA
<213> Artificial Sequence
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<220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 17
5 tcgacctgca ggaagcttgc ggccgcggat cc 32
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence
10 <220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 18
tcgaggatcc gcggccgcaa gcttcctgca gg 32
15 <210> 19
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 19
tcgaggatcc gcggccgcaa gcttcctgca ggagct 36
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<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 20
cctgcaggaa gcttgcggcc gcggatcc 28
<210> 21
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 21
tcgacctgca ggaagcttgc ggccgcggat ccagct 36
<210> 22
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ligating
Oligonucleotide
<400> 22
ggatccgcgg ccgcaagctt cctgcagg 28
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<210> 23
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Forward Primer
PLF1
<400> 23
ggatccgcgg ccgcatgtct ttgaatagag ttcttc 36
<210> 24
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Forward Primer
PLF2
<400> 24
ggatccgcgg ccgcatggat tttgatgaca cgattgc 37
<210> 25
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Reverse Primer
PLR
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<400> 25
cctgcaggaa gcttctagaa gaaatccttg atc 33
<210> 26
<211> 1920
<212> DNA
<213> Ptyas mucosus
<400> 26
gggcaatcag ctgttgccgt ctcactggtg aaaagaaaac ccaccctggc ggcccaatac 60
gcaaccgcct ctccccggcg cgttggccga ttcattaatg cagctggcac gacaggtttc 120
ccgactgaaa agcgggcagt agcgcaacgc aattaatgtg agttagctca ctcattaggc 180
accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 240
acaatttcac acagaaacag ctatgaccat gattacgcca agctctaata cgactcacta 300
tagggaaagc tggtacgcct gcaggtaccg gtccggaatt cccgggtcga cccacgcgtc 360
cgcaaaatgt ctttgaatag agttcttcac attttcctta tcgcatatct cgcatgcact 420
gccctaaccc atgattttga tgacacgatt gccgttgtgg gagctggcta ctctggactg 480
agcgctgctt ttactctcgt caagaaaggg tacaccaacg ttgagattta cgaatcccag 540
ggcgaagttg gggggtatgt ctactctgtt gactataaca acgtcgcgca tgacctggcc 600
acgtacgctc tgactcctgc atactggaaa ttccaggagg ccatgaaaag tatcggcgtt 660
gggttttgtg agctcgatgt tgcaattgtg caaacgaatt ctacgcctgt ctcagtcccg 720
ttcgagaaat ggatggccgc ctactgggct gcgaaagtcc caaacccact caacctcgtg 780
aggaaggtct cgactcaagt ttcgacgtac gttgaagttt ggaagaagct cttcaatatg 840
gacttcattg acacgagcac gaagcgcact aatcgcctct ttccgttgaa gaccaacgac 900
gtcgacgtcc ttgcccaatt ttcaatgccc atgaaagatt ttgttgcatt gcataagctg 960
gacttgctcg agcctctttt tatccaggca accgactccc aggcgtacgg tccgtatgac 1020
acgacaccgg cactctacta catggtgtgg ttccctccga accttttcaa cggtgaggaa 1080
aataccgttc catgtggtac gtataactcg atgcagtcca tggccgagca catggccgaa 1140
tggttgaaga gcaaaggagt cacgttccac atgaatacga aggtgacgaa aatctctcgc 1200
gccaccgatg gatctagtcc atccctcttg gaagaaggtg tagctacgcc gaagctcttc 1260
gacaccataa tcagtacgaa caagctgccg tctgcgaacc gtgccgaagt tgtgacacct 1320
ctgcttccga aggagcggga ggccgccgat acgtacgagg agctacaaat gttctctgct 1380
cttctcgaga cgaatcgcag cgatgccatt ccgacgacag gcttcttgat ggtggatgcg 1440
gacgcaatta tagctcacga ccctaacacc gggttttggg gttgtttgaa tgctgagcgt 1500
cgcggaggct attcggatga gaatgctatt ctaagctcgg atactgtgac gcgcgtcagc 1560
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gccatctact actatacaga gcgtgcaaac aacgaacgca tcgacttttc tctcgacgag 1620
aagattcagc aggtgaagac caatcttgcg aacgtgggac tcggctacct gaccaatcta 1680
acctcccgta cgttcggtgg atatttccag aggtggagga cgccggatgt tatgggtcaa 1740
aagccgtgga atctggctga cattcaagga gaaggagatg tgtactacgt caactcggct 1800
gcatgcgggt tcgagtccgt cggccacgtt ttcgattgcg cggataattt gatcaaggat 1860
tttttctaga taaacacaac agaagtagac tgccgtcaaa gtctggttac ctcatggaaa 1920
<210> 27
<211> 1860
<212> DNA
<213> Ptyas mucosus
<400> 27
gcgcgttggc cgattcatta atcagctggc acgacaggtt tcccgcgaaa acggccatgg 60
agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct tttacacttt 120
atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 180
1 S agctatgacc atgattacgc caagctctaa tacgactcac tatagggaaa gctggtacgc 240
ctgcaggtac cggtccggaa ttcccgggtc gacccacgcg tccgctctcg cagaaaagct 300
gcatttccct cttctctcaa aatgtctttg aatagagttc ttcacatttc ccttatcgca 360
tatctcgcat gcactgccct aacccatgat tttgatgaca cgattgccgt tgtgggagct 420
ggctactctg gactgagcgc tgcttttact ctcgtcaaga aagggtacac caacgttgag 480
atttacgaat cccagggcga agttgggggg tatgtctact ctgttgacta taacaacgtc 540
gcgcatgacc tggccacgta cgctctgact cctgcatact ggaaattcca ggaggccatg 600
aaaagtatcg gcgttgggtt ttgtgagctc gatgttgcaa ttgtgcaaac gaattctacg 660
cctgtctcag tcccgttcga gaaatggatg gccgcctact gggctgcgaa agtcccaaac 720
ccactcaacc tcgtgaggaa ggtctcgact caagtttcga cgtacgttga agtttggaag 780
aagctcttca atatggactt cattgacacg agcacgaagc gcactaatcg cctctttccg 840
ttgaagacca acgacgtcga cgtccttgcc caattttcaa tgcccatgaa agattttgtt 900
gcattgcata agctggactt gctcgagcct ctttttatcc aggcaaccga ctcccaggcg 960
tacggtccgt atgacacgac accggcactc tactacatgg tgtggttccc tccgaacctt 1020
ttcaacggtg aggaaaatac cgttccatgt ggtacgtata actcgatgca gtccatggcc 1080
gagcacatgg ccgaatggtt gaagagcaag gagtcacgtt ccacatgaat tacgaaggtg 1140
acgaaaatct ctcgcgccac cgatggatct agtccatccc tcttggaaga aggtgtagct 1200
acgccgaagc tcttcgacac cataatcagt acgaacaagc tgccgtctgc gaaccgtgcc 1260
gaagttgtga cacctctgct tccgaaggag cgggaggcca ccaatacgta cgaggagcta 1320
caaatgttct ctgctcttct cgagacgaat cgcagcgatg ccattccgac gacaggcttc 1380
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ttgatggtgg atgcggacgc aattatagct cacgaccctg acaccgggtt ttggggttgt 1440
ttgaatgctg agcgtcgcgg aggctattcg gatgagaatg ctattctaag ctcggatact 1500
gtgacgcgcg tcagcgccat ctactactat acagagcgtg caaacaacga acgcatcgac 1560
ttttctctcg acgagaagat tcagcaggtg aagaccaatc ttgcgacgtg ggactcggct 1620
5 acctggacca atctaacttc ccgtacgttc ggtggatatt tccagaggtg gaggacgccg 1680
gatgttatgg gtcaaaagcc gtggaatctg gctgacattc aaggagaagg agatgtgtac 1740
tacgtcaacg cggctgcatg cgggttcgag tccgtcggcc acgtttcgat ttgcgcggat 1800
aatttgatca aggatttctt ctagataaac acaacagaag tagactgcgc ccaaagtctg 1860
